Molecular and evolutionary relationships among enteric bacteria

Classification of bacterial species into genera has traditionally relied upon variation in phenotypic characteristics. However, these phenotypes often have a multifactorial genetic basis, making unambiguous taxonomic placement of new species difficult. By designing evolutionarily conserved oligonucleotide primers, it is possible to amplify homologous regions of genes in diverse taxa using the polymerase chain reaction and determine their nucleotide sequences. We have constructed a phylogeny of some enteric bacteria, including five species classified as members of the genus Escherichia, based on nucleotide sequence variation at the loci encoding glyceraldehyde-3-phosphate dehydrogenase and outer membrane protein 3A, and compared this genealogy with the relationships inferred by biotyping. The DNA sequences of these genes defined congruent and robust phylogenetic trees indicating that they are an accurate reflection of the evolutionary history of the bacterial species. The five species of Escherichia were found to be distantly related and, contrary to their placement in the same genus, do not form a monophyletic group. These data provide a framework which allows the relationships of additional species of enteric bacteria to be inferred. These procedures have general applicability for analysis of the classification, evolution, and epidemiology of bacterial taxa.     

rpoB sequence analysis as a novel basis for bacterial identification

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta-subunit encoding gene ( rpoB  ) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra- and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.     

Conservation of the S10-spc-alpha locus within otherwise highly plastic genomes provides phylogenetic insight into the genus Leptospira

S10-spc-alpha is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-alpha locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-alpha locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-alpha locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously.     

Insights into the evolution of sorbitol metabolism: phylogenetic analysis of SDR196C family

ABSTRACT: BACKGROUND: Short chain dehydrogenases/reductases (SDR) are NAD(P)(H)-dependent oxidoreductases with a highly conserved 3D structure and of an early origin, which has allowed them to diverge into several families and enzymatic activities. The SDR196C family (http://www.sdr-enzymes.org) groups bacterial sorbitol dehydrogenases (SDH), which are of great industrial interest. In this study, we examine the phylogenetic relationship between the members of this family, and based on the findings and some sequence conserved blocks, a new and a more accurate classification is proposed. RESULTS: The distribution of the 66 bacterial SDH species analyzed was limited to Gram-negative bacteria. Six different bacterial families were found, encompassing alpha-, beta- and gamma-proteobacteria. This broad distribution in terms of bacteria and niches agrees with that of SDR, which are found in all forms of life. A cluster analysis of sorbitol dehydrogenase revealed different types of gene organization, although with a common pattern in which the SDH gene is surrounded by sugar ABC transporter proteins, another SDR, a kinase, and several gene regulators.According to the obtained trees, six different lineages and three sublineages can be discerned. The phylogenetic analysis also suggested two different origins for SDH in beta-proteobacteria and four origins for gamma-proteobacteria.Finally, this subdivision was further confirmed by the differences observed in the sequence of the conserved blocks described for SDR and some specific blocks of SDH, and by a functional divergence analysis, which made it possible to establish new consensus sequences and specific fingerprints for the lineages and sub lineages. CONCLUSION: SDH distribution agrees with that observed for SDR, indicating the importance of the polyol metabolism, as an alternative source of carbon and energy. The phylogenetic analysis pointed to six clearly defined lineages and three sub lineages, and great variability in the origin of this gene, despite its well conserved 3D structure. This suggests that SDH are very old and emerged early during the evolution. This study also opens up a new and more accurate classification of SDR196C family, introducing two numbers at the end of the family name, which indicate the lineage and the sublineage of each member, i.e, SDR196C6.3.     

头状链轮丝菌染色体复制起始区的克隆和对变铅青链霉菌的转化

头状链轮丝菌(Streptoverticillum caespitosus) ATCC27422是抗肿瘤药物——丝裂霉素A的产生菌,根据高GC含量革兰氏阳性菌染色体复制起始区两端基因序列的保守性,采用PCR方法从该菌中克隆了一段包含染色体复制起始区(oriC)的1.3 kb片段。序列分析发现,克隆片段与天蓝色链霉菌的oriC及邻近区域的同源性达80％以上;头状链轮丝菌oriC中含有22个DnaA-box,保守序列为TTGTCCACA。以克隆片段构建的质粒可以跨属转化变铅青链霉菌(S. lividans)ZX7,原生质体转化效率为3.2×10²个/μgDNA;质粒在S. lividans ZX7中能以低拷贝形式稳定存在;转化子的菌落和菌丝形态均正常。头状链轮丝菌oriC序列与几种链霉菌的oriC有较高的同源性,以及在变铅青链霉菌中仍具有复制起始活性等说明链轮丝菌属与链霉菌属在系统发生上关系较近;但采用最大似然法分析oriC序列构建的头状链轮丝菌与几种链霉菌的系统进化树表明,头状链轮丝菌与几种链霉菌之间的分化距离远大于链霉菌之间的分化距离。该证据支持链轮丝菌属的独立分属。     

Phylogeny and divergence time estimation of the genus Didymodon (Pottiaceae) based on nuclear and chloroplast markers

Didymodon Hedw., with approximately 140 species in the family Pottiaceae, is distributed nearly throughout the world, with the greatest diversity and important ecological functions in drought lands and alpine ecosystems. Several studies involving morphology, molecular systematics, and macro-systematic analysis have addressed the infrageneric classification of Didymodon, but controversy over the position of the infrageneric and species classification remains due to its high degree of morphological variation in micro-habitats and strong sensitivity to climate change at regional and global scale. To date, only a few phylogenetic studies have been conducted with an incomplete number of Didymodon species; further, there is no study published regarding the divergence time of Didymodon. Consequently, we conducted a comprehensive phylogenetic analysis of Didymodon species, sampling a total of 107 species, based on one nuclear (ITS) and five chloroplast DNA. Moreover, divergence time analysis was conducted to infer the age of origin and divergence of Didymodon species. Our results presented the largest scale phylogenetic relationship of Didymodon to date and resolved the phylogenetic status of some controversial taxa and the new species. The divergence time estimation showed that Didymodon species originated around the early Cretaceous, and the diversification was concentrated in the Cretaceous and Eocene. Paleoclimate and environmental change have a direct impact on the origin and divergence of Didymodon species by shaping their morphology, resource availability and ecological niche. Our study will help understand species origin and speciation of Didymodon as well as reflecting species adaptability and experience to historical events.     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.     

Recent Advances in the Identification of Replication Origins Based on the Z-curve Method

Precise DNA replication is critical for the maintenance of genetic integrity in all organisms. In all three domains of life, DNA replication starts at a specialized locus, termed as the replication origin, oriC or ORI, and its identification is vital to understanding the complex replication process. In bacteria and eukaryotes, replication initiates from single and multiple origins, respectively, while archaea can adopt either of the two modes. The Z-curve method has been successfully used to identify replication origins in genomes of various species, including multiple oriCs in some archaea. Based on the Z-curve method and comparative genomics analysis, we have developed a web-based system, Ori-Finder, for finding oriCs in bacterial genomes with high accuracy. Predicted oriC regions in bacterial genomes are organized into an online database, DoriC. Recently, archaeal oriC regions identified by both in vivo and in silico methods have also been included in the database. Here, we summarize the recent advances of in silico prediction of oriCs in bacterial and archaeal genomes using the Z-curve based method.     

尤马马杜拉放线菌染色体复制区的克隆及功能分析

尤马马杜拉放线菌(Actinomadura yumaensis)NRRL12515产生马杜拉霉素,用于防治禽类球虫病。试验以放线菌dnaA与dnaN基因保守区设计的简并引物进行PCR扩增,获得了包含尤马马杜拉放线菌的染色体复制区oriC的片段,并进行了序列分析和复制功能的研究。尤马马杜拉放线菌染色体的oriC全长为919碱基对,含有14个DnaA盒子和2个AT富含区,DnaA盒子的保守序列是(T/C)(T/C)GTCC(A/C)CA,与已发表的3个属的放线菌染色体oriC的序列特征不同。携带该oriC片段的大肠杆菌质粒可以在天蓝色链霉菌中复制并以低拷贝方式遗传,表明这是一段有复制功能的序列。比较来自放线菌4个属的oriC,发现以oriC序列和以16S rRNA基因序列构建的进化树十分相似,表明oriC序列也可以体现放线菌物种之间的关系。     

Divergence of the polytene chromosome banding sequences as a reflection of evolutionary rearrangements of the genome linear structure

Banding sequences of five chromosomal arms (A, C, D, E, and F), accounting for about 70% of the total genome size in 63 Chironomus species, were used as phylogenetic markers to analyze divergence patterns of the linear genome structure during the evolution. The number of chromosomal breakpoints between the pairs of banding sequences compared served as a measure of divergence. It was demonstrated that the greater the divergence between the species compared, the higher the number of chromosomal breakpoints and the smaller the size of the conserved chromosomal regions. A banding sequences comparison in sibling species demonstrated a lower number of chromosomal breakpoints; the breakpoint number was maximum in a comparison of the banding sequences in the subgenera Chironomus and Camptochironomus. The use of the number of chromosomal breakpoints as a divergence measure provided establishment of phylogenetic relationships between 63 Chironomus species and discrimination of sibling species groups and cytocomplexes on a phylogenetic tree.     

The phylogenetic origin of the bifunctional tyrosine-pathway protein in the enteric lineage of bacteria

Because bifunctional enzymes are distinctive and highly conserved products of relatively infrequent gene-fusion events, they are particularly useful markers to identify clusters of organisms at different hierarchical levels of a phylogenetic tree. Within the subdivision of gram-negative bacteria known as superfamily B, there are two distinctive types of tyrosine-pathway dehydrogenases: (1) a broad- specificity dehydrogenase (recently termed cyclohexadienyl dehydrogenase [CDH]) that can utilize either prephenate or L-arogenate as alternative substrates and (2) a bifunctional CDH that also posseses chorismate mutase activity. (T-proteins). The bifunctional T-protein, thought to be encoded by fused ancestral genes for chorismate mutase and CDH, was found to be present in enteric bacteria (Escherichia, Shigella, Salmonella, Citrobacter, Klebsiella, Erwinia, Serratia, Morganella, Cedecea, Kluyvera, Hafnia, Edwardsiella, Yersinia, and Proteus) and in Aeromonas and Alteromonas. Outside of the latter "enteric lineage," the T-protein is absent in other major superfamily-B genera, such as Pseudomonas (rRNA homology group I), Xanthomonas, Acinetobacter, and Oceanospirillum. Hence, the T-protein must have evolved after the divergence of the enteric and Oceanospirillum lineages. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase-phe, an early-pathway isozyme sensitive to feedback inhibition by L- phenylalanine, has been found in each member of the enteric lineage examined. The absence of both the T-protein and DAHP synthase-phe elsewhere in superfamily B indicates the emergence of these character states at approximately the same evolutionary time.      

Comparative Genomic Analysis Identifies Divergent Genomic Features of Pathogenic Enterococcus cecorum Including a Type IC CRISPR-Cas System,a Capsule Locus,an epa-Like Locus,and Putative Host Tissue Binding Proteins

Enterococcus cecorum (EC) is the dominant enteric commensal of adult chickens and contributes to the gut consortia of many avian and mammalian species. While EC infection is an uncommon zoonosis, like other enterococcal species it can cause life-threating nosocomial infection in people. In contrast to other enterococci which are considered opportunistic pathogens, emerging pathogenic strains of EC cause outbreaks of musculoskeletal disease in broiler chickens. Typical morbidity and mortality is comparable to other important infectious diseases of poultry. In molecular epidemiologic studies, pathogenic EC strains were found to be genetically clonal. These findings suggested acquisition of specific virulence determinants by pathogenic EC. To identify divergent genomic features and acquired virulence determinants in pathogenic EC; comparative genomic analysis was performed on genomes of 3 pathogenic and 3 commensal strains of EC. Pathogenic isolates had smaller genomes with a higher GC content, and they demonstrated large regions of synteny compared to commensal isolates. A molecular phylogenetic analysis demonstrated sequence divergence in pathogenic EC genomes. At a threshold of 98% identity, 414 predicted proteins were identified that were highly conserved in pathogenic EC but not in commensal EC. Among these, divergent CRISPR-cas defense loci were observed. In commensal EC, the type IIA arrangement typical for enterococci was present; however, pathogenic EC had a type IC locus, which is novel in enterococci but commonly observed in streptococci. Potential mediators of virulence identified in this analysis included a polysaccharide capsular locus similar to that recently described for E. faecium, an epa-like locus, and cell wall associated proteins which may bind host extracellular matrix. This analysis identified specific genomic regions, coding sequences, and predicted proteins which may be related to the divergent evolution and increased virulence of emerging pathogenic strains of EC.     

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.     

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

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

Homology of Escherichia coli R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria isolated from raw sewage and arsenic-enriched creek waters.

The occurrence and diversity of the Escherichia coli R773 ars operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters. Selected isolates from each creek location were screened for ars genes by colony hybridization and PCR. The occurrence of arsA, arsB, and arsC determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations. At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for arsA, arsB, and arsC, respectively. PCR results showed that the ars operon is conserved in some enteric bacteria isolated from creek waters and raw sewage. The occurrence of the arsBC genotype was about 50% in raw sewage enteric bacteria, while arsA was detected in only 9.4% of the isolates (n = 32). The arsABC and arsBC genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (n = 7), respectively. Average sequence divergence within arsB for six creek enteric bacteria was 20% compared to that of the E. coli R773 ars operon. Only 1 of 11 pseudomonads screened by PCR was positive for arsB. The results from this study suggest that significant divergence has occurred in the ars operon among As-resistant E. coli strains and in Pseudomonas spp.     

Klebsiella pneumoniae origin of replication (oriC) is not active in Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides.

A DNA fragment carrying genes encoding the conjugal transfer system of the broad host range plasmid RK2 was inserted into a plasmid carrying the chromosomal origin of replication (oriC) from Klebsiella pneumoniae. The resulting plasmid, pEON1, was readily transferred between gram-negative bacteria and carried two potential origins of replication: oriC and the replication origin from pBR322 (oriPBR). Although pEON1 could be transferred to Caulobacter crescentus, Pseudomonas putida, and Rhodobacter sphaeroides, pEON1 was not maintained in these strains. However, an oriC-containing plasmid was maintained in these nonenteric bacteria when an RK2 origin of replication was present on the plasmid. Thus, the inability of pEON1 to be established in a nonenteric bacterium represents a failure of oriC to function as an origin of replication rather than a toxic effect of oriC. The initiation potential of the chromosomal origin of replication from K. pneumoniae appears to be realized only in enteric bacteria.     

Phylogenetic analysis and evolution of aromatic amino acid hydroxylase

A study was performed to investigate the phylogenetic relationship among AAAH members and to statistically evaluate sequence conservation and functional divergence. In total, 161 genes were identified from 103 species. Phylogenetic analysis showed that well-conserved subfamilies exist. Exon-intron structure analysis showed that the gene structures of AAAH were highly conserved across some different lineage species, while some species-specific introns were also found. The dynamic distribution of ACT domain suggested one gene fusion event has occurred in eukaryota. Significant functional divergence was found between some subgroups. Analysis of the site-specific profiles revealed critical amino acid residues for functional divergence. This study highlights the molecular evolution of this family and may provide a starting point for further experimental verifications.     

Comparative genomics in the Brassicaceae: a family-wide perspective

Comparative genomics of Arabidopsis relatives has great potential to improve our understanding of molecular function and evolutionary processes. Recent studies of phylogenetic relationships within Brassicaceae and the publication of a new tribal classification scheme provide an important framework for comparative genomics research. Comparative linkage mapping and chromosome painting in the close relatives of Arabidopsis have inferred an ancestral karyotype of these species. In addition, comparative mapping to Brassica has identified genomic blocks that have been maintained since the divergence of the Arabidopsis and Brassica lineages. Several analyses of conserved non-coding regions have identified putative cis-regulatory sequences, and have highlighted the need for comparative sequencing at greater evolutionary distances. The development of new model species with novel physiological and ecological traits allows analysis of phenotypes that are not available in A. thaliana. Looking towards the future, we suggest a prioritized research agenda for comparative genomics in the Brassicaceae.     

Phylogenetic analysis and evolutionary studies of plant carotenoid cleavage dioxygenase gene

The oxidative breakdown of carotenoid evidences the formation of apocarotenoids through carotenoid cleavage dioxygenases (CCDs). Numerous CCDs and apocarotenoids have been identified and characterized in plants. Using available sequence data, a study was performed to investigate the phylogenetic relationship among CCD genes and to statistically estimate the sequence conservation and functional divergence. In total, 77 genes were identified from 39 species belonging to 21 families. Our result of phylogenetic analysis indicated the existence of well-conserved subfamilies. Moreover, comparative genomic analysis showed that the gene structures of the CCDs were highly conserved across some different lineage species. Through functional divergence analysis, a substantial divergence was found between CCD subfamilies. In addition, examination of the site-specific profile revealed the critical amino acid residues accounting for functional divergence. This study mainly focused on the evolution of CCD genes and their functional divergence which may deliver an initial step for further experimental verifications.