Phylogenetic analysis reveals gene conversions in multigene families of rhizobia

Gene families are an important and intrinsic trait of rhizobial species. These gene copies can participate in non-reciprocal recombination events, also called gene conversions. Gene conversion has diverse roles, but it is usually implicated in the evolution of multigene families. Here, we searched for gene conversions in multigene families of six representative rhizobial genomes. We identified 11 gene families with different numbers of copies, genome location and function in CFN42 and CIAT652 strains of Rhizobium etli, Rhizobium sp NGR234, Mesorhizobium loti MAFF303099, Sinorhizobium meliloti 1021, and Bradyrhizobium japonicum USDA110. Gene conversions were detected by phylogenetic inference in the nifD and nifK gene families in R. etli. Sequence analysis confirmed multiple gene conversions in these two gene families. We suggest that gene conversion events have an important role in homogenizing multigene families in rhizobia.     

Characterization of the gene conversions between the multigene family members of the yeast genome

Stanley Sawyer's gene conversion detection method, implemented in his GENECONV computer program, was used to detect and characterize the gene conversions between the multigene family members of the yeast genome. This method gave different gene conversion frequencies and size distribution for gene families with two members and multigene families with more than two members. The 69 gene conversions detected in multigene families with more than two members occur at a frequency of 7.8% gene conversion/pair of genes compared and have an average size of 173+/-220 nucleotides. Larger gene conversions are found only between more similar genes, the genes involved in gene conversions are distributed almost randomly among the 16 yeast chromosomes, and the frequency of gene conversions increases as the distance between repeated genes decreases. In contrast to previous studies, no relationship was observed between the level of expression of a gene and its involvement in gene conversions. These analyses also suggest that gene conversions might occur by different mechanisms in closely linked genes and unlinked genes. The excess of converted regions at the 3? end of unlinked genes suggests that recombination with incomplete cDNA molecules is the main mechanism responsible for gene conversions between such genes.     

Perils of paralogy: using HSP70 genes for inferring organismal phylogenies

Conserved genes have found their way into the mainstream of molecular systematics. Many of these genes are members of multigene families. A difficulty with using single genes of multigene families for phylogenetic inference is that genes from one species may be paralogous to those from another taxon. We focus attention on this problem using heat shock 70 (HSP70) genes. Using polymerase chain reaction techniques with genomic DNA, we isolated and sequenced 123 distinct sequences from 12 species of sharks. Phylogenetic analysis indicated that the sequences cluster with constituitively expressed cytoplasmic heat shock-like genes. Three highly divergent gene clades were sampled. A number of similar sequences were sampled from each species within each distinct gene clade. Comparison of published species trees with an HSP70 gene tree inferred using Bayesian phylogenetic analysis revealed several cases of gene duplication and differential sorting of gene lineages within this group of sharks. Gene tree parsimony based on the objective criteria of duplication and losses showed that previously published hypotheses of species relationships and two novel hypothesis based on Bayesian phylogenetics were concordant with the history of HSP70 gene duplication and loss. By contrast, two published hypotheses based on morphological data were not significantly different from the null hypothesis of a random association between species relatedness and the HSP70 gene tree. These results suggest that gene tree parsimony using data from multigene families can be used for inferring species relationships or testing published alternative hypotheses. More importantly, the results suggest that systematic studies relying on phylogenetic inferences from HSP70 genes may by plagued by unrecognized paralogy of sampled genes. Our results underscore the distinction between gene and species trees and highlight an underappreciated source of discordance between gene trees and organismal phylogeny, i.e., unrecognized paralogy of sampled genes.     

Identification of members of gene families in Arabidopsis thaliana by contig construction from partial cDNA sequences: 106 genes encoding 50 cytoplasmic ribosomal proteins

Partial cDNA sequencing to obtain expressed sequence tags (ESTs) has led to the identification of tags to about 8000 of the estimated 20 000 genes in Arabidopsis thaliana . This figure represents four to five times the number of complete coding sequences from this organism available in international databases. In contrast to mammals, many proteins are encoded by multigene families in A. thaliana . Using ribosomal protein gene families as an example, it is possible to construct relatively long sequences from overlapping ESTs which are of sufficiently high quality to be able to unambiguously identify tags to individual members of multigene families, even when the sequences are highly conserved. A total of 106 genes encoding 50 different cytoplasmic ribosomal protein types have been identified, most proteins being encoded by at least two and up to four genes. Coding sequences of members of individual gene families are almost always very highly conserved and derived amino acid sequences are almost, if not completely, identical in the vast majority of cases. Sequence divergence is observed in untranslated regions which allows the definition of gene-specific probes. The method can be used to construct high-quality tags to any protein.     

Ectopic gene conversions in bacterial genomes.

We characterized the gene conversions found between the duplicated genes of 75 bacterial genomes from five species groups (archaea, nonpathogenic and pathogenic firmicutes, and nonpathogenic and pathogenic proteobacteria). The number of gene conversions is positively correlated with the size of multigene families and the size of multigene families is not significantly different between pathogenic and nonpathogenic taxa. However, gene conversions occur twice as frequently in pathogenic species as in nonpathogenic species. Comparisons between closely related species also indicate a trend towards increased gene conversion in pathogenic species. Whereas the length of the conversions is positively correlated with flanking sequence similarity in all five groups, these correlations are smaller for pathogenic firmicutes and proteobacteria than for nonpathogenic firmicutes and proteobacteria. These results are consistent with our previous work on E. coli genomes and suggest that pathogenic bacteria allow recombination between more divergent gene sequences. This higher permissiveness is likely adaptive because it allows them to generate more genetic variability.     

Molecular history of gene conversions in the primate fetal gamma-globin genes. Nucleotide sequences from the common gibbon, Hylobates lar

Comparative and phylogenetic analyses of homologous sequences from closely related species reveal genetic events which have happened in the past and thus provide considerable insight into molecular genetic processes. One such process which has been especially important in the evolution of multigene families is gene conversion. The fetal gamma 1 and gamma 2-globin genes of catarrhine primates (humans, apes, and Old World monkeys) underwent numerous gene conversion events after they arose from a gene duplication event 25-35 million years ago. By including the gamma 1- and gamma 2-globin gene sequences from the common gibbon, Hylobates lar, the present work expands the gamma-globin data set to represent all major groups of hominoid primates. A computer-assisted algorithm is introduced which reveals converted DNA segments and provides results very similar to those obtained by site-by-site evolutionary reconstruction. Both methods provide strong evidence for at least 14 different converted stretches in catarrhine primates as well as five conversions in ancestral lineages. Features of gene conversions generalized from this molecular history are 1) conversions are restricted to regions maintaining high degrees of sequence similarity, 2) one gene may dominate in converting another gene, 3) sequences involved in conversions may accumulate changes more rapidly than expected, and 4) certain elements, such as polypurine/polypyrimidine [Y)n) and (TG)n elements, appear to be hotspots for initiating or terminating conversion events.     

Independent gene evolution in the potato actin gene family demonstrated by phylogenetic procedures for resolving gene conversions and the phylogeny of angiosperm actin genes

Summary Nine different actin DNA sequences were isolated from the common potato,Solanum tuberosum, and the nucleotide sequence of five actin loci and of two allelic variants are presented. Unlike the wide variation in intron position among animal actin genes, the potato actin genes have three introns situated in the same positions as reported for all other angiosperm actin genes. Using a novel combination of analytical procedures (G-test and compatibility analysis), we could not find evidence of frequent large or small nonreciprocal exchanges of genetic material between the sequenced loci, although there were a few candidates. Resolution of such gene conversion events and the quantification of independence of gene evolution in multigene families is critical to the inference of phylogenetic relationships. Comparison with actin genes in other angiosperm species suggests that the actin multigene family can be divided into a number of subfamilies, evolved by descent rather than gene conversion, which are of possible functional origin, with one major subfamily diversification occurring before the divergence of monocots and dicots. The silent rate of nucleotide substitution was estimated to be similar to that suggested for a number of other plant nuclear genes, whereas the replacement rate was extremely slow, suggestive of selective constraints.     

An integrated approach for the comparative analysis of a multigene family: The nicotianamine synthase genes of barley

Recent genomic projects reveal that about half of the gene repertoire in plant genomes is made up by multigene families. In this paper, a set of structural and phylogenetic analyses have been applied to compare the differently sized nicotianamine synthase (NAS) gene families in barley and rice. Nicotianamine acts as a chelator of iron and other heavy metals and plays a key role in uptake, phloem transport and cytoplasmic distribution of iron, challenging efforts for the breeding of iron-efficient crop plants. Nine barley NAS genes have been mapped, and co-linearity of flanking genes in barley and rice was determined. The combined analyses reveal that the NAS multigene family members in barley originated through at least one duplication event that occurred before the divergence of rice and barley. Additional duplications appear to have occurred within each of the species. Although we detected no evidence for positive selection of recently duplicated genes within species, codon-based tests revealed evidence for positive selection having contributed to the divergence of some amino acids. The integrated comparative and phylogenetic analysis improved our current view of NAS gene family evolution, might facilitate the functional characterization of individual members and is applicable to other multigene families. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

From gene trees to organismal phylogeny in prokaryotes: the case of the gamma-Proteobacteria

The rapid increase in published genomic sequences for bacteria presents the first opportunity to reconstruct evolutionary events on the scale of entire genomes. However, extensive lateral gene transfer (LGT) may thwart this goal by preventing the establishment of organismal relationships based on individual gene phylogenies. The group for which cases of LGT are most frequently documented and for which the greatest density of complete genome sequences is available is the gamma-Proteobacteria, an ecologically diverse and ancient group including free-living species as well as pathogens and intracellular symbionts of plants and animals. We propose an approach to multigene phylogeny using complete genomes and apply it to the case of the gamma-Proteobacteria. We first applied stringent criteria to identify a set of likely gene orthologs and then tested the compatibilities of the resulting protein alignments with several phylogenetic hypotheses. Our results demonstrate phylogenetic concordance among virtually all (203 of 205) of the selected gene families, with each of the exceptions consistent with a single LGT event. The concatenated sequences of the concordant families yield a fully resolved phylogeny. This topology also received strong support in analyses aimed at excluding effects of heterogeneity in nucleotide base composition across lineages. Our analysis indicates that single-copy orthologous genes are resistant to horizontal transfer, even in ancient bacterial groups subject to high rates of LGT. This gene set can be identified and used to yield robust hypotheses for organismal phylogenies, thus establishing a foundation for reconstructing the evolutionary transitions, such as gene transfer, that underlie diversity in genome content and organization.     

From Gene Trees to Organismal Phylogeny in Prokaryotes:The Case of the γ-Proteobacteria

The rapid increase in published genomic sequences for bacteria presents the first opportunity to reconstruct evolutionary events on the scale of entire genomes. However, extensive lateral gene transfer (LGT) may thwart this goal by preventing the establishment of organismal relationships based on individual gene phylogenies. The group for which cases of LGT are most frequently documented and for which the greatest density of complete genome sequences is available is the γ-Proteobacteria, an ecologically diverse and ancient group including free-living species as well as pathogens and intracellular symbionts of plants and animals. We propose an approach to multigene phylogeny using complete genomes and apply it to the case of the γ-Proteobacteria. We first applied stringent criteria to identify a set of likely gene orthologs and then tested the compatibilities of the resulting protein alignments with several phylogenetic hypotheses. Our results demonstrate phylogenetic concordance among virtually all (203 of 205) of the selected gene families, with each of the exceptions consistent with a single LGT event. The concatenated sequences of the concordant families yield a fully resolved phylogeny. This topology also received strong support in analyses aimed at excluding effects of heterogeneity in nucleotide base composition across lineages. Our analysis indicates that single-copy orthologous genes are resistant to horizontal transfer, even in ancient bacterial groups subject to high rates of LGT. This gene set can be identified and used to yield robust hypotheses for organismal phylogenies, thus establishing a foundation for reconstructing the evolutionary transitions, such as gene transfer, that underlie diversity in genome content and organization.     

From Gene Trees to Organismal Phylogeny in Prokaryotes:The Case of the γ-Proteobacteria

The rapid increase in published genomic sequences for bacteria presents the first opportunity to reconstruct evolutionary events on the scale of entire genomes. However, extensive lateral gene transfer (LGT) may thwart this goal by preventing the establishment of organismal relationships based on individual gene phylogenies. The group for which cases of LGT are most frequently documented and for which the greatest density of complete genome sequences is available is the γ-Proteobacteria, an ecologically diverse and ancient group including free-living species as well as pathogens and intracellular symbionts of plants and animals. We propose an approach to multigene phylogeny using complete genomes and apply it to the case of the γ-Proteobacteria. We first applied stringent criteria to identify a set of likely gene orthologs and then tested the compatibilities of the resulting protein alignments with several phylogenetic hypotheses. Our results demonstrate phylogenetic concordance among virtually all (203 of 205) of the selected gene families, with each of the exceptions consistent with a single LGT event. The concatenated sequences of the concordant families yield a fully resolved phylogeny. This topology also received strong support in analyses aimed at excluding effects of heterogeneity in nucleotide base composition across lineages. Our analysis indicates that single-copy orthologous genes are resistant to horizontal transfer, even in ancient bacterial groups subject to high rates of LGT. This gene set can be identified and used to yield robust hypotheses for organismal phylogenies, thus establishing a foundation for reconstructing the evolutionary transitions, such as gene transfer, that underlie diversity in genome content and organization.     

The vestigial olfactory receptor subgenome of odontocete whales: phylogenetic congruence between gene-tree reconciliation and supermatrix methods

The macroevolutionary transition of whales (cetaceans) from a terrestrial quadruped to an obligate aquatic form involved major changes in sensory abilities. Compared to terrestrial mammals, the olfactory system of baleen whales is dramatically reduced, and in toothed whales is completely absent. We sampled the olfactory receptor (OR) subgenomes of eight cetacean species from four families. A multigene tree of 115 newly characterized OR sequences from these eight species and published data for Bos taurus revealed a diverse array of class II OR paralogues in Cetacea. Evolution of the OR gene superfamily in toothed whales (Odontoceti) featured a multitude of independent pseudogenization events, supporting anatomical evidence that odontocetes have lost their olfactory sense. We explored the phylogenetic utility of OR pseudogenes in Cetacea, concentrating on delphinids (oceanic dolphins), the product of a rapid evolutionary radiation that has been difficult to resolve in previous studies of mitochondrial DNA sequences. Phylogenetic analyses of OR pseudogenes using both gene-tree reconciliation and supermatrix methods yielded fully resolved, consistently supported relationships among members of four delphinid subfamilies. Alternative minimizations of gene duplications, gene duplications plus gene losses, deep coalescence events, and nucleotide substitutions plus indels returned highly congruent phylogenetic hypotheses. Novel DNA sequence data for six single-copy nuclear loci and three mitochondrial genes (> 5000 aligned nucleotides) provided an independent test of the OR trees. Nucleotide substitutions and indels in OR pseudogenes showed a very low degree of homoplasy in comparison to mitochondrial DNA and, on average, provided more variation than single-copy nuclear DNA. Our results suggest that phylogenetic analysis of the large OR superfamily will be effective for resolving relationships within Cetacea whether supermatrix or gene-tree reconciliation procedures are used.     

Evolution of substrate recognition across a multigene family of glycosyltransferases in Arabidopsis

The complete sequence of the Arabidopsis genome enables definitive characterization of multigene families and analysis of their phylogenetic relationships. Using a consensus sequence previously defined for glycosyltransferases that use small-molecular-weight acceptors, 107 gene sequences were identified in the Arabidopsis genome and used to construct a phylogenetic tree. Screening recombinant proteins for their catalytic activities in vitro has revealed enzymes active toward physiologically important substrates, including hormones and secondary metabolites. The aim of this study has been to use the phylogenetic relationships across the entire family to explore the evolution of substrate recognition and regioselectivity of glucosylation. Hydroxycoumarins have been used as the model substrates for the analysis in which 90 sequences have been assayed and 48 sequences shown to recognize these compounds. The study has revealed activity in 6 of the 14 phylogenetic groups of the multigene family, suggesting that basic features of substrate recognition are retained across substantial evolutionary periods.     

Sequence heterogeneities among 16S ribosomal RNA sequences, and their effect on phylogenetic analyses at the species level

We have analyzed what phylogenetic signal can be derived by small subunit rRNA comparison for bacteria of different but closely related genera (enterobacteria) and for different species or strains within a single genus (Escherichia or Salmonella), and finally how similar are the ribosomal operons within a single organism (Escherichia coli). These sequences have been analyzed by neighbor-joining, maximum likelihood, and parsimony. The robustness of each topology was assessed by bootstrap. Sequences were obtained for the seven rrn operons of E. coli strain PK3. These data demonstrated differences located in three highly variable domains. Their nature and localization suggest that since the divergence of E. coli and Salmonella typhimurium, most point mutations that occurred within each gene have been propagated among the gene family by conversions involving short domains, and that homogenization by conversions may not have affected the entire sequence of each gene. We show that the differences that exist between the different operons are ignored when sequences are obtained either after cloning of a single operon or directly from polymerase chain reaction (PCR) products. Direct sequencing of PCR products produces a mean sequence in which mutations present in the most variable domains become hidden. Cloning a single operon results in a sequence that differs from that of the other operons and of the mean sequence by several point mutations. For identification of unknown bacteria at the species level or below, a mean sequence or the sequence of a single nonidentified operon should therefore be avoided. Taking into account the seven operons and therefore mutations that accumulate in the most variable domains would perhaps increase tree resolution. However, if gene conversions that homogenize the rRNA multigene family are rare events, some nodes in phylogenetic trees will reflect these recombination events and these trees may therefore be gene trees rather than organismal trees.      

Systematic Detection of Large-Scale Multigene Horizontal Transfer in Prokaryotes

Horizontal gene transfer (HGT) is central to prokaryotic evolution. However, little is known about the “scale” of individual HGT events. In this work, we introduce the first computational framework to help answer the following fundamental question: How often does more than one gene get horizontally transferred in a single HGT event? Our method, called HoMer, uses phylogenetic reconciliation to infer single-gene HGT events across a given set of species/strains, employs several techniques to account for inference error and uncertainty, combines that information with gene order information from extant genomes, and uses statistical analysis to identify candidate horizontal multigene transfers (HMGTs) in both extant and ancestral species/strains. HoMer is highly scalable and can be easily used to infer HMGTs across hundreds of genomes. We apply HoMer to a genome-scale data set of over 22,000 gene families from 103 Aeromonas genomes and identify a large number of plausible HMGTs of various scales at both small and large phylogenetic distances. Analysis of these HMGTs reveals interesting relationships between gene function, phylogenetic distance, and frequency of multigene transfer. Among other insights, we find that 1) the observed relative frequency of HMGT increases as divergence between genomes increases, 2) HMGTs often have conserved gene functions, and 3) rare genes are frequently acquired through HMGT. We also analyze in detail HMGTs involving the zonula occludens toxin and type III secretion systems. By enabling the systematic inference of HMGTs on a large scale, HoMer will facilitate a more accurate and more complete understanding of HGT and microbial evolution.     

Tarsius delta- and beta-globin genes: conversions, evolution, and systematic implications

Comparisons between duplicated genes have shown that gene conversions play an important role in the evolution of multigene families. Previous comparisons have documented in the recently duplicated gamma-fetal globin genes of catarrhine primates, over 15 separate conversions affecting extensive stretches of coding and noncoding sequences. In the present study, delta- and beta- globin genes from a lower primate Tarsius syrichta, and the delta-globin gene of the Asian great ape, Pongo pygmaeus, have been isolated and sequenced. Comparisons of these sequences with other primate delta and beta sequences confirmed a previously reported conversion in an anthropoid ancestor and revealed additional conversions in basal primate, stem haplorhine, tarsier, and early lemur lineages. Conversions found between primate delta- and beta-globin genes contrast with those found in the gamma-genes in that delta-beta conversions appear much less frequently and are more restricted to regions conserved by selection (i.e. coding and 5'-regulatory sequences). These differences indicate that soon after a duplication occurs, conversions can be quite frequent and encompass extensive portions of the duplicated region. With time, sequence differences accumulate, particularly in noncoding regions, and limit both the frequency and size of the conversions. Sequences conserved by selection accumulate differences more slowly and are therefore subject to gene conversions for a longer period of time. Both unconverted and converted sequences were consistent in supporting the placement of tarsier with anthropoids.     

Allopolyploid evolution in Geinae (Colurieae: Rosaceae) – building reticulate species trees from bifurcating gene trees

A previous phylogenetic study of paralogous nuclear low-copy granule-bound starch synthase (GBSSI) gene sequences from polyploid and diploid species in Geinae indicated that the clade has experienced two major allopolyploid events in its history. These were estimated to have occurred several million years ago. In this extended study we test if the reticulate phylogenetic hypothesis for Geinae can be maintained when additional sequences are added. The results are compatible with the hypothesis and strengthen it in minor aspects. We also attempt to identify extant members of one of the inferred ancestral lineages of the allopolyploids. On the basis of previous molecular phylogenies, one specific group has been proposed to be the descendants of this taxon. However, none of the additional paralogues belong to this ancestral lineage. A general method is proposed for converting a bifurcating gene tree, with multiple paralogous low-copy gene sequences from allopolyploid taxa, into a reticulate species tree.     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.