The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Background

Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens.

Results

The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation.

Conclusions

Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1777-9) contains supplementary material, which is available to authorized users.     

Acrophiarin (antibiotic S31794/F-1) from Penicillium arenicola shares biosynthetic features with both Aspergillus- and Leotiomycete-type echinocandins

The antifungal echinocandin lipopeptide, acrophiarin, was circumscribed in a patent in 1979. We confirmed that the producing strain NRRL 8095 is Penicillium arenicola and other strains of P. arenicola produced acrophiarin and acrophiarin analogues. Genome sequencing of NRRL 8095 identified the acrophiarin gene cluster. Penicillium arenicola and echinocandin-producing Aspergillus species belong to the family Aspergillaceae of the Eurotiomycetes, but several features of acrophiarin and its gene cluster suggest a closer relationship with echinocandins from Leotiomycete fungi. These features include hydroxy-glutamine in the peptide core instead of a serine or threonine residue, the inclusion of a non-heme iron, α-ketoglutarate-dependent oxygenase for hydroxylation of the C3 of the glutamine, and a thioesterase. In addition, P. arenicola bears similarity to Leotiomycete echinocandin-producing species because it exhibits self-resistance to exogenous echinocandins. Phylogenetic analysis of the genes of the echinocandin biosynthetic family indicated that most of the predicted proteins of acrophiarin gene cluster exhibited higher similarity to the predicted proteins of the pneumocandin gene cluster of the Leotiomycete Glarea lozoyensis than to those of the echinocandin B gene cluster from A. pachycristatus. The fellutamide gene cluster and related gene clusters are recognized as relatives of the echinocandins. Inclusion of the acrophiarin gene cluster into a comprehensive phylogenetic analysis of echinocandin gene clusters indicated the divergent evolutionary lineages of echinocandin gene clusters are descendants from a common ancestral progenitor. The minimal 10-gene cluster may have undergone multiple gene acquisitions or losses and possibly horizontal gene transfer after the ancestral separation of the two lineages.     

The Evolution of Fungal Metabolic Pathways

Fungi contain a remarkable range of metabolic pathways, sometimes encoded by gene clusters, enabling them to digest most organic matter and synthesize an array of potent small molecules. Although metabolism is fundamental to the fungal lifestyle, we still know little about how major evolutionary processes, such as gene duplication (GD) and horizontal gene transfer (HGT), have interacted with clustered and non-clustered fungal metabolic pathways to give rise to this metabolic versatility. We examined the synteny and evolutionary history of 247,202 fungal genes encoding enzymes that catalyze 875 distinct metabolic reactions from 130 pathways in 208 diverse genomes. We found that gene clustering varied greatly with respect to metabolic category and lineage; for example, clustered genes in Saccharomycotina yeasts were overrepresented in nucleotide metabolism, whereas clustered genes in Pezizomycotina were more common in lipid and amino acid metabolism. The effects of both GD and HGT were more pronounced in clustered genes than in their non-clustered counterparts and were differentially distributed across fungal lineages; specifically, GD, which was an order of magnitude more abundant than HGT, was most frequently observed in Agaricomycetes, whereas HGT was much more prevalent in Pezizomycotina. The effect of HGT in some Pezizomycotina was particularly strong; for example, we identified 111 HGT events associated with the 15 Aspergillus genomes, which sharply contrasts with the 60 HGT events detected for the 48 genomes from the entire Saccharomycotina subphylum. Finally, the impact of GD within a metabolic category was typically consistent across all fungal lineages, whereas the impact of HGT was variable. These results indicate that GD is the dominant process underlying fungal metabolic diversity, whereas HGT is episodic and acts in a category- or lineage-specific manner. Both processes have a greater impact on clustered genes, suggesting that metabolic gene clusters represent hotspots for the generation of fungal metabolic diversity.     

Phylum-wide comparative genomics unravel the diversity of secondary metabolism in Cyanobacteria

Background

Cyanobacteria are an ancient lineage of photosynthetic bacteria from which hundreds of natural products have been described, including many notorious toxins but also potent natural products of interest to the pharmaceutical and biotechnological industries. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. However, current understanding of the diversification of these pathways is largely based on the chemical structure of the bioactive compounds, while the evolutionary forces driving their remarkable chemical diversity are poorly understood.

Results

We carried out a phylum-wide investigation of genetic diversification of the cyanobacterial NRPS and PKS pathways for the production of bioactive compounds. 452 NRPS and PKS gene clusters were identified from 89 cyanobacterial genomes, revealing a clear burst in late-branching lineages. Our genomic analysis further grouped the clusters into 286 highly diversified cluster families (CF) of pathways. Some CFs appeared vertically inherited, while others presented a more complex evolutionary history. Only a few horizontal gene transfers were evidenced amongst strongly conserved CFs in the phylum, while several others have undergone drastic gene shuffling events, which could result in the observed diversification of the pathways.

Conclusions

Therefore, in addition to toxin production, several NRPS and PKS gene clusters are devoted to important cellular processes of these bacteria such as nitrogen fixation and iron uptake. The majority of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new natural products.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-977) contains supplementary material, which is available to authorized users.     

Degree of Functional Divergence in Duplicates Is Associated with Distinct Roles in Plant Evolution

Gene duplication is a major mechanism to create new genes. After gene duplication, some duplicated genes undergo functionalization, whereas others largely maintain redundant functions. Duplicated genes comprise various degrees of functional diversification in plants. However, the evolutionary fate of high and low diversified duplicates is unclear at genomic scale. To infer high and low diversified duplicates in Arabidopsis thaliana genome, we generated a prediction method for predicting whether a pair of duplicate genes was subjected to high or low diversification based on the phenotypes of knock-out mutants. Among 4,017 pairs of recently duplicated A. thaliana genes, 1,052 and 600 are high and low diversified duplicate pairs, respectively. The predictions were validated based on the phenotypes of generated knock-down transgenic plants. We determined that the high diversified duplicates resulting from tandem duplications tend to have lineage-specific functions, whereas the low diversified duplicates produced by whole-genome duplications are related to essential signaling pathways. To assess the evolutionary impact of high and low diversified duplicates in closely related species, we compared the retention rates and selection pressures on the orthologs of A. thaliana duplicates in two closely related species. Interestingly, high diversified duplicates resulting from tandem duplications tend to be retained in multiple lineages under positive selection. Low diversified duplicates by whole-genome duplications tend to be retained in multiple lineages under purifying selection. Taken together, the functional diversities determined by different duplication mechanisms had distinct effects on plant evolution.     

Characterization of the locus of genes encoding enzymes producing heptadepsipeptide micropeptin in the unicellular cyanobacterium Microcystis

The gene cluster involved in producing the cyclic heptadepsipeptide micropeptin was cloned from the genome of the unicellular cyanobacterium Microcystis aeruginosa K-139. Sequencing revealed four genes encoding non-ribosomal peptide synthetases (NRPSs) that are highly similar to the gene cluster involved in cyanopeptolins biosynthesis. According to predictions based on the non-ribosomal consensus code, the order of the mcnABCE NPRS modules was well consistent with that of the biosynthetic assembly of cyclic peptides. The biochemical analysis of a McnB(K-139) adenylation domain and the knock-out of mcnC in a micropeptin-producing strain, M. viridis S-70, revealed that the mcn gene clusters were responsible for the production of heptadepsipeptide micropeptins. A detailed comparison of nucleotide sequences also showed that the regions between the mcnC and mcnE genes of M. aeruginosa K-139 retained short stretches of DNA homologous to halogenase genes involved in the synthesis of halogenated cyclic peptides of the cyanopeptolin class including anabaenopeptilides. This suggests that the mcn clusters of M. aeruginosa K-139 have lost the halogenase genes during evolution. Finally, a comparative bioinformatics analysis of the congenial gene cluster for depsipetide biosynthesis suggested the diversification and propagation of the NRPS genes in cyanobacteria.     

Module evolution and substrate specificity of fungal nonribosomal peptide synthetases involved in siderophore biosynthesis

Background  

Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches.     

Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.     

Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

Background  

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.     

Two different secondary metabolism gene clusters occupied the same ancestral locus in fungal dermatophytes of the arthrodermataceae

Background

Dermatophyte fungi of the family Arthrodermataceae (Eurotiomycetes) colonize keratinized tissue, such as skin, frequently causing superficial mycoses in humans and other mammals, reptiles, and birds. Competition with native microflora likely underlies the propensity of these dermatophytes to produce a diversity of antibiotics and compounds for scavenging iron, which is extremely scarce, as well as the presence of an unusually large number of putative secondary metabolism gene clusters, most of which contain non-ribosomal peptide synthetases (NRPS), in their genomes. To better understand the historical origins and diversification of NRPS-containing gene clusters we examined the evolution of a variable locus (VL) that exists in one of three alternative conformations among the genomes of seven dermatophyte species.

Results

The first conformation of the VL (termed VLA) contains only 539 base pairs of sequence and lacks protein-coding genes, whereas the other two conformations (termed VLB and VLC) span 36 Kb and 27 Kb and contain 12 and 10 genes, respectively. Interestingly, both VLB and VLC appear to contain distinct secondary metabolism gene clusters; VLB contains a NRPS gene as well as four porphyrin metabolism genes never found to be physically linked in the genomes of 128 other fungal species, whereas VLC also contains a NRPS gene as well as several others typically found associated with secondary metabolism gene clusters. Phylogenetic evidence suggests that the VL locus was present in the ancestor of all seven species achieving its present distribution through subsequent differential losses or retentions of specific conformations.

Conclusions

We propose that the existence of variable loci, similar to the one we studied, in fungal genomes could potentially explain the dramatic differences in secondary metabolic diversity between closely related species of filamentous fungi, and contribute to host adaptation and the generation of metabolic diversity.     

Distribution of NRPS gene families within the Neotyphodium/Epichloë complex

Neotyphodium and Epichloë spp are closely related asexual and sexual endophytic fungi, respectively, that form mutualistic associations with cool season grasses of the subfamily Pooideae. The endophytes confer a number of advantages to their hosts, but also can cause animal toxicoses and these effects are, in many cases, due to the production of fungal secondary metabolites. In filamentous fungi, secondary metabolite genes are commonly clustered and, for those pathways involved in non-ribosomal peptide synthesis, a non-ribosomal peptide synthetase (NRPS) gene is always found as a key component of the cluster. Members of this gene family encode large multifunctional enzymes that synthesize a diverse range of bioactive compounds and in numerous cases have been shown to serve as pathogenicity or virulence factors, in addition to suggested roles in niche adaptation. We have used a degenerate PCR approach to identify members of the NRPS gene family from symbiotic fungi of the Neotyphodium/Epichloë complex, and have shown that collectively, at least 12 NRPS genes exist within the genomes examined. This suggests that secondary metabolites are important during the life cycles of these fungi with their hosts. Indeed, both the ergovaline and peramine biosynthetic pathways, which confer competitive abilities to Neotyphodium and Epichloë symbioses, contain NRPS genes at their core. The distribution of these genes among different Neotyphodium/Epichloë lineages suggests that a common ancestor contributed most of the complement of NRPS genes, which have been either retained or lost during the evolution of these fungi.     

Halogenase genes in nonribosomal peptide synthetase gene clusters of Microcystis (cyanobacteria): sporadic distribution and evolution

Cyanobacteria of the genus Microcystis are known to produce secondary metabolites of large structural diversity by nonribosomal peptide synthetase (NRPS) pathways. For a number of such compounds, halogenated congeners have been reported along with nonhalogenated ones. In the present study, chlorinated cyanopeptolin- and/or aeruginosin-type peptides were detected by mass spectrometry in 17 out of 28 axenic strains of Microcystis. In these strains, a halogenase gene was identified between 2 genes coding for NRPS modules in respective gene clusters, whereas it was consistently absent when the strains produced only nonchlorinated corresponding congeners. Nucleotide sequences were obtained for 12 complete halogenase genes and 14 intermodule regions of gene clusters lacking a halogenase gene or containing only fragments of it. When a halogenase gene was found absent, a specific, identical excision pattern was observed for both synthetase gene clusters in most strains. A phylogenetic analysis including other bacterial halogenases showed that the NRPS-related halogenases of Microcystis form a monophyletic group divided into 2 subgroups, corresponding to either the cyanopeptolin or the aeruginosin peptide synthetases. The distribution of these peptide synthetase gene clusters, among the tested Microcystis strains, was found in relative agreement with their phylogeny reconstructed from 16S-23S rDNA intergenic spacer sequences, whereas the distribution of the associated halogenase genes appears to be sporadic. The presented data suggest that in cyanobacteria these prevalent halogenase genes originated from an ancient horizontal gene transfer followed by duplication in the cyanobacterial lineage. We propose an evolutionary scenario implying repeated gene losses to explain the distribution of halogenase genes in 2 NRPS gene clusters that subsequently defines the seemingly erratic production of halogenated and nonhalogenated aeruginosins and cyanopeptolins among Microcystis strains.     

Unusual evolutionary conservation and further species-specific adaptations of a large family of nonclassical MHC class Ib genes across different degrees of genome ploidy in the amphibian subfamily Xenopodinae

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.     

Host conservatism,geography, and elevation in the evolution of a Neotropical moth radiation

The origins of evolutionary radiations are often traced to the colonization of novel adaptive zones, including unoccupied habitats or unutilized resources. For herbivorous insects, the predominant mechanism of diversification is typically assumed to be a shift onto a novel lineage of host plants. However, other drivers of diversification are important in shaping evolutionary history, especially for groups residing in regions with complex geological histories. We evaluated the contributions of shifts in host plant clade, bioregion, and elevation to diversification in Eois (Lepidoptera: Geometridae), a hyper‐diverse genus of moths found throughout the Neotropics. Relationships among 107 taxa were reconstructed using one mitochondrial and two nuclear genes. In addition, we used a genotyping‐by‐sequencing approach to generate 4641 SNPs for 137 taxa. Both datasets yielded similar phylogenetic histories, with relationships structured by host plant clade, bioregion, and elevation. While diversification of basal lineages often coincided with host clade shifts, more recent speciation events were more typically associated with shifts across bioregions or elevational gradients. Overall, patterns of diversification in Eois are consistent with the perspective that shifts across multiple adaptive zones synergistically drive diversification in hyper‐diverse lineages.     

Historical Divergence and Gene Flow in the Genus Zea

Gene flow plays a fundamental role in plant evolutionary history, yet its role in population divergence—and ultimately speciation—remains poorly understood. We investigated gene flow and the modalities of divergence in the domesticate Zea mays ssp. mays and three wild Zea taxa using sequence polymorphism data from 26 nuclear loci. We described diversity across loci and assessed evidence for adaptive and purifying selection at nonsynonymous sites. For each of three divergence events in the history of these taxa, we used approximate Bayesian simulation to estimate population sizes and divergence times and explicitly compare among alternative models of divergence. Our estimates of divergence times are surprisingly consistent with previous data from other markers and suggest rapid diversification of lineages within Zea in the last ~150,000 years. We found widespread evidence of historical gene flow, including evidence for divergence in the face of gene flow. We speculate that cultivated maize may serve as a bridge for gene flow among otherwise allopatric wild taxa.     

Duplication and adaptive evolution of the COR15 genes within the highly cold-tolerant Draba lineage (Brassicaceae)

Plants have evolved diverse adaptive mechanisms that enable them to tolerate abiotic stresses, to varying degrees, and such stresses may have strongly influenced evolutionary changes at levels ranging from molecular to morphological. Previous studies on these phenomena have focused on the adaptive evolution of stress-related orthologous genes in specific lineages. However, heterogenetic evolution of the paralogous genes following duplication has only been examined in a very limited number of stress-response gene families. The COR15 gene encodes a low molecular weight protein that plays an important role in protecting plants from cold stresses. Although two different copies of this gene have been found in the model species, Arabidopsis thaliana, evolutionary patterns of this small gene family in plants have not been previously explored. In this study, we cloned COR15-like sequences and performed evolutionary analyses of these sequences (including those previously reported) in the highly cold-tolerant Draba lineage and related lineages of Brassicaceae. Our phylogenetic analyses indicate that all COR15-like sequences clustered into four clades that corresponded well to the morphological lineages. Gene conversions were found to have probably occurred before/during the divergence of Brassica and Draba lineage. However, repeated, independent duplications of this gene have occurred in different lineages of Brassicaceae. Further comparisons of all sequences suggest that there have been significant inter-lineage differences in evolutionary rates between the duplicated and original genes. We assessed the likelihood that the differences between two well-supported gene subfamilies that appear to have originated from a single duplication, COR15a and COR15b, within the Draba lineage have been driven by adaptive evolution. Comparisons of their non-synonymous/synonymous substitution ratios and rates of predicted amino acid changes indicate that these two gene groups are evolving under different selective pressures and may be functionally divergent. This functional divergence was confirmed by comparing site-specific shifts in evolution indexes of the two groups of predicted proteins. The evidence of differential selection and possible functional divergence suggests that the duplication may be of adaptive significance, with possible implications for the explosive diversification of the Draba lineage during the cooling Quaternary stages and the following worldwide colonization of arid alpine and artic regions.     

牛樟芝非核糖体多肽合成酶基因(ＡcNRPS)的克隆与鉴定

胶霉毒素属于真菌天然次生代谢产物epipolythiodioxopiperazine (ETP)家族,具有免疫抑制剂、抗真菌等多种生物活性,是由非核糖体多肽合成酶(NRPSs)催化合成。从牛樟芝(Antrodia camphorata)基因组中挖掘出非核糖体多肽合成酶基因(AcNRPS,NCBI登录号为KX430967),克隆获取其全长cDNA,并对其进行生物信息学分析和表达谱分析。结果显示AcNRPS基因cDNA全长6 687 bp;与其DNA序列比对发现AcNRPS基因含有12 个内含子;其开放阅读框编码2 229 个氨基酸残基,BLAST比对发现其含有2 个A-C-T结构域,底物需2个氨基酸;系统发育树结果显示AcNRPS与其他合成产物为胶霉毒素的NRPS基因聚为一类,其可合成胶霉毒素类化合物;表达谱分析显示,以葡萄糖和土豆蛋白胨作为碳、氮源的培养基能够有效促进牛樟芝NRPS基因的表达。     

Rate and polarity of gene fusion and fission in Oryza sativa and Arabidopsis thaliana

Eukaryotic gene fusion and fission events are mechanistically more complicated than in prokaryotes, and their quantitative contributions to genome evolution are still poorly understood. We have identified all differentially composite or split genes in 2 fully sequenced plant genomes, Oryza sativa and Arabidopsis thaliana. Out of 10,172 orthologous gene pairs, 60 (0.6% of the total) revealed a verified fusion or fission event in either lineage after the divergence of O. sativa and A. thaliana. Polarizing these events by outgroup comparison revealed differences in the rate of gene fission but not of gene fusion in the rice and Arabidopsis lineages. Gene fission occurred at a higher rate than gene fusion in the O. sativa lineage and was furthermore more common in rice than in Arabidopsis. Nucleotide insertion bias has promoted gene fission in the O. sativa lineage, consistent with its generally longer nucleotide sequences than A. thaliana in selectively neutral regions, and with the abundance of transposable elements in rice. The divergence time of monocots and dicots (140-200 Myr) indicates that gene fusion/fission events occur at an average rate of 1x10(-11) to 2x10(-11) events per gene per year, approximately 100-fold slower than the average per site nuclear nucleotide substitution rate in these lineages. Gene fusion and fission are thus rare and slow processes in higher plant genomes; they should be of utility to address deeper evolutionary relationships among plants--and the relationship of plants to other eukaryotic lineages--where sequence-based phylogenies provide equivocal or conflicting results.