Detection of recombination among Salmonella enterica strains using the incongruence length difference test

Particular serovars of Salmonella enterica have emerged as significant foodborne pathogens in humans. At the chromosomal level, discrete regions in the Salmonella genome have been identified that are known to play important roles in the maintenance, survival, and virulence of S. enterica within the host. Interestingly, several of these loci appear to have been acquired by horizontal transfer of DNA among and between bacterial species. The profound importance of recombination in pathogen emergence is just now being realized, perhaps explaining the sudden interest in developing novel and facile ways for detecting putative horizontal transfer events in bacteria. The incongruence length difference (ILD) test offers one such means. ILD uses phylogeny to trace sequences that may have been acquired promiscuously by exchange of DNA during chromosome evolution. We show here that the ILD test readily detects recombinations that have taken place in several housekeeping genes in Salmonella as well as genes composing the type 1 pilin complex (14 min) and the inv-spa invasion gene complex (63 min). Moreover, the ILD test indicated that the mutS gene (64 min), whose product helps protect the bacterial genome from invasion by foreign DNA, appears to have undergone intragenic recombination within S. enterica subspecies I. ILD findings were supported using additional tests known to be independent of the ILD approach (e.g., split decomposition analysis and compatibility of sites). Taken together, these data affirm the application of the ILD test as one approach for identifying recombined sequences in the Salmonella chromosome. Furthermore, horizontally acquired sequences within mutS support a model whereby evolutionarily important recombinants of S. enterica are rescued from strains carrying defective mutS alleles via horizontal transfer.     

Escherichia coli molecular phylogeny using the incongruence length difference test

Molecular phylogeny of the species Escherichia coli using the E. coli reference (ECOR) collection strains has been hampered by (1) the absence of rooting in the commonly used phenogram obtained from multilocus enzyme electrophoresis (MLEE) data and (2) the existence of recombination events between strains that scramble phylogenetic trees reconstructed from the nucleotide sequences of genes. We attempted to determine the phylogeny for E. coli based on the ECOR strain data by extracting from GenBank the nucleotide sequences of 11 chromosomal structural and 2 plasmid genes for which the Salmonella enterica homologous gene sequences were available. For each of the 13 DNA data sets studied, incongruence with a nonnucleotide whole-genome data set including MLEE, random amplified polymorphic DNA, and rrn restriction fragment length polymorphism data was measured using the incongruence length difference (ILD) test of Farris et al. As previously reported, the incongruence observed between the gnd and plasmid gene data and the whole-genome data was multiple, indicating numerous horizontal transfer and/or recombination events. In five cases, the incongruence detected by the ILD test was punctual, and the donor group was identified. Congruence was not rejected for the remaining data sets. The strains responsible for incongruences with the whole-genome data set were removed, leading to a "prior-agreement" approach, i.e., the determination of a phylogeny for E. coli based on several genes, excluding (1) the genes with multiple incongruences with the whole genome data, (2) the strains responsible for punctual incongruences, and (3) the genes incongruent with each other. The obtained phylogeny shows that the most basal group of E. coli strains is the B2 group rather than the A group, as generally thought. The D group then emerges as the sister group of the rest. Finally, the A and B1 groups are sister groups. Interestingly, the most primitive taxon within E. coli in terms of branching pattern, i.e., the B2 group, includes highly virulent extraintestinal strains with derived characters (extraintestinal virulence determinants) occurring on its own branch.      

Mitochondrial DNA,morphology, and the phylogenetic relationships of Antarctic icefishes (Notothenioidei: Channichthyidae)

The Channichthyidae is a lineage of 16 species in the Notothenioidei, a clade of fishes that dominate Antarctic near-shore marine ecosystems with respect to both diversity and biomass. Among four published studies investigating channichthyid phylogeny, no two have produced the same tree topology, and no published study has investigated the degree of phylogenetic incongruence between existing molecular and morphological datasets. In this investigation we present an analysis of channichthyid phylogeny using complete gene sequences from two mitochondrial genes (ND2 and 16S) sampled from all recognized species in the clade. In addition, we have scored all 58 unique morphological characters used in three previous analyses of channichthyid phylogenetic relationships. Data partitions were analyzed separately to assess the amount of phylogenetic resolution provided by each dataset, and phylogenetic incongruence among data partitions was investigated using incongruence length difference (ILD) tests. We utilized a parsimony-based version of the Shimodaira-Hasegawa test to determine if alternative tree topologies are significantly different from trees resulting from maximum parsimony analysis of the combined partition dataset. Our results demonstrate that the greatest phylogenetic resolution is achieved when all molecular and morphological data partitions are combined into a single maximum parsimony analysis. Also, marginal to insignificant incongruence was detected among data partitions using the ILD. Maximum parsimony analysis of all data partitions combined results in a single tree, and is a unique hypothesis of phylogenetic relationships in the Channichthyidae. In particular, this hypothesis resolves the phylogenetic relationships of at least two species (Channichthys rhinoceratus and Chaenocephalus aceratus), for which there was no consensus among the previous phylogenetic hypotheses. The combined data partition dataset provides substantial statistical power to discriminate among alternative hypotheses of channichthyid relationships. These findings suggest the optimal strategy for investigating the phylogenetic relationships of channichthyids is one that uses all available phylogenetic data in analyses of combined data partitions.     

Phylogenetic analyses of the rhipicephaline ticks indicate that the genus Rhipicephalus is paraphyletic

We inferred the phylogeny of 21 species and subspecies of ticks from the subfamilies Rhipicephalinae and Hyalomminae using cytochrome c oxidase subunit I (COI) and 12S rRNA mitochondrial gene sequences. Two members of the subfamily Haemaphysalinae were used for outgroup reference. The largest rhipicephaline genus, Rhipicephalus, was represented by ticks from six of the species groups, the second largest genus, Dermacentor, by species from two of three of its subgenera, and the genus Boophilus by 3 of its 5 species. We analyzed the 12S and COI sequences separately and together; statistically significant incongruence between the 12S rDNA and the COI sequences was not detected in the combined dataset using the incongruence length difference test. The combined dataset provided greater phylogenetic resolution than the individual datasets, and although the 12S rDNA data had only 25% of the parsimony-informative characters, it provided half of the total partitioned Bremer support for the combined dataset. We present the first hypothesis of phylogenetic relationships among some species groups of Rhipicephalus but our most controversial result was that the genus Rhipicephalus is apparently paraphyletic, unless species of Boophilus are included in it. The species of Rhipicephalus most closely related to Boophilus spp. were from the R. pravus and R. evertsi species groups, which may implicate an African origin for this important group of ticks.     

Can three incongruence tests predict when data should be combined?

Advocates of conditional combination have argued that testing for incongruence between data partitions is an important step in data exploration. Unless the partitions have had distinct histories, as in horizontal gene transfer, incongruence means that one or more data support the wrong phylogeny. This study examines the relationship between incongruence and phylogenetic accuracy using three tests of incongruence. These tests were applied to pairs of mitochondrial DNA data partitions from two well-corroborated vertebrate phylogenies. Of the three tests, the most useful was the incongruence length difference test (ILD, also called the partition homogeneity test). This test distinguished between cases in which combining the data generally improved phylogenetic accuracy (P > 0.01) and cases in which accuracy of the combined data suffered relative to the individual partitions (P < 0.001). In contrast, in several cases, the Templeton and Rodrigo tests detected highly significant incongruence (P < 0.001) even though combining the incongruent partitions actually increased phylogenetic accuracy. All three tests identified cases in which improving the reconstruction model would improve the phylogenetic accuracy of the individual partitions.      

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.     

Lack of evidence for horizontal transfer of the lac operon into Escherichia coli

The idea that Escherichia coli gained the lac operon via horizontal transfer, allowing it to invade a new niche and form a new species, has become a paradigmatic example of bacterial nonpathogenic adaptation and speciation catalyzed by horizontal transfer. Surprisingly, empirical evidence for this event is essentially nonexistent. To see whether horizontal transfer occurred, I compared a phylogeny of 14 Enterobacteriaceae based on two housekeeping genes to a phylogeny of a part of their lac operon. Although several species in this clade appear to have acquired some or all of the operon via horizontal transfer, there is no evidence of horizontal transfer into E. coli. It is not clear whether the horizontal transfer events for which there is evidence were adaptive because those species which have acquired the operon are not thought to live in high lactose environments. I propose that vertical transmission from the common ancestor of the Enterobacteriaceae, with subsequent loss of these genes in many species can explain much of the patchy distribution of lactose use in this clade. Finally, I argue that we need new, well-supported examples of horizontal transfer spurring niche expansion and speciation, particularly in nonpathogenic cases, before we can accept claims that horizontal transfer is a hallmark of bacterial adaptation.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.      

Detecting phylogenetic incongruence using BIONJ: an improvement of the ILD test

The problem of testing for congruence between phylogenetic data has long been debated among phylogeneticists, but reaches a critical point with the availability of large amount of biological sequences. Notably in prokaryotes, where the amount of lateral transfers is believed to be important, the inference of phylogenies using multiple genes requires testing for incongruence before concatenating the genes. On another scale, incongruence tests can be used to detect recombination points within single gene alignments. The incongruence length difference test (ILD), based on parsimony, has been proved to be useful for finding incongruent data sets, but its application remains limited to small data sets for computational time reasons. Here, we have adapted the principle of ILD to the BIONJ algorithm. This algorithm is based on a tree length minimisation criterion and is suitable to replace parsimony in this test when used with uncorrected distance (model-free approach). We show that this new test, ILD-BIONJ, while being much faster, is often more accurate than the ILD test, especially when the alignments compared are simulated under different evolutionary models.     

mILD: a tool for constructing and analyzing matrices of pairwise phylogenetic character incongruence tests

SUMMARY: Pairwise comparisons of disagreement in phylogenetic datasets offer a powerful tool for isolating historical incongruence for closer analysis. Statistically significant phylogenetic character incongruence may reflect important differences in evolutionary history, such as horizontal gene transfer. Such testing can also be used to specify possible combinations of datasets for further phylogenetic analysis. The process of comparing multiple datasets can be very time consuming, and it is sometimes unclear how to combine data partitions given the observed patterns of incongruence. Here we present an application that automates the process of making pairwise comparisons between large numbers of phylogenetic datasets using the Incongruence Length Difference (ILD) test. The application also implements strategies for data combination based on the patterns of incongruence observed in pairwise comparisons.     

A scalable and flexible approach for investigating the genomic landscapes of phylogenetic incongruence

Analyses of DNA sequence datasets have repeatedly revealed inconsistencies in phylogenetic trees derived with different data. This is termed phylogenetic incongruence, and may arise from a methodological failure of the inference process or from biological processes, such as horizontal gene transfer, incomplete lineage sorting, and introgression. To better understand patterns of incongruence, we developed a method (PartFinder) that uses likelihood ratios applied to sliding windows for visualizing tree-support changes across genome-sequence alignments, allowing the comparative examination of complex phylogenetic scenarios among many species. As a pilot, we used PartFinder to investigate incongruence in the Homo-Pan-Gorilla group as well as Platyrrhini using high-quality bacterial artificial chromosome (BAC)-derived sequences as well as assembled whole-genome shotgun sequences. Our simulations verified the sensitivity of PartFinder, and our results were comparable to other studies of the Homo-Pan-Gorilla group. Analyses of the whole-genome alignments reveal significant associations between support for the accepted species relationship and specific characteristics of the genomic regions, such as GC-content, alignment score, exon content, and conservation. Finally, we analyzed sequence data generated for five platyrrhine species, and found incongruence that suggests a polytomy within Cebidae, in particular. Together, these studies demonstrate the utility of PartFinder for investigating the patterns of phylogenetic incongruence.     

Phylogeny and distribution of the soxB gene among thiosulfate-oxidizing bacteria

A PCR protocol for the detection of sulfur-oxidizing bacteria based on soxB genes that are essential for thiosulfate oxidation by sulfur-oxidizing bacteria of various phylogenetic groups which use the 'Paracoccus sulfur oxidation' pathway was developed. Five degenerate primers were used to specifically amplify fragments of soxB genes from different sulfur-oxidizing bacteria previously shown to oxidize thiosulfate. The PCR yielded a soxB fragment of approximately 1000 bp from most of the bacteria. Amino acid and nucleotide sequences of soxB from reference strains as well as from new isolates and environmental DNA from a hydrothermal vent habitat in the North Fiji Basin were compared and used to infer relationships of soxB between sulfur-oxidizing bacteria belonging to various 16S rDNA-based phylogenetic groups. Major phylogenetic lines derived from 16S rDNA were confirmed by soxB phylogeny. Thiosulfate-oxidizing green sulfur bacteria formed a coherent group by their soxB sequences. Likewise, clearly separated branches demonstrated the distant relationship of representatives of alpha-, beta-, and gamma-Proteobacteria including representative species of the former genus Thiobacillus (now Halothiobacillus - gamma-Proteobacteria, Thiobacillus - beta-Proteobacteria and Starkeya - alpha-Proteobacteria). This general picture emerged although apparent evidence for lateral transfer of the soxB gene is indicated and comparison of soxB phylogeny and 16S rDNA phylogeny points to the significance of this gene transfer in hydrothermal vent bacterial communities of the North Fiji Basin.     

Phylogenetic analysis supports horizontal transfer of P transposable elements

Nucleotide sequence comparisons were used to investigate the evolution of P transposable elements and the possibility that horizontal transfer has played a role in their occurrence in natural populations of Drosophila and other Diptera. The phylogeny of P elements was examined using published sequences from eight dipteran taxa and a new, partial sequence from Scaptomyza elmoi. The results from a number of different analyses are highly consistent and reveal a P-element phylogeny that contradicts the phylogeny of the species. At least three instances of horizontal transfer are necessary to explain this incongruence, but other explanations cannot be ruled out at this time.      

Is a robust phylogeny of the enterobacterial plant pathogens attainable?

Sequences from gapA, gyrA and ompA were used to evaluate the relationships of the enterobacterial plant pathogens, and assess whether a robust phylogeny can be ascertained using this group of housekeeping genes. Up to 48 taxa were included in a combined phylogenetic analysis to explore the evolutionary distribution of plant pathogenic species across the family Enterobacteriaceae. Phylogenies were reconstructed from gapA, gyrA and ompA gene sequences using maximum parsimony and maximum likelihood algorithms, and phylogenetic congruence was evaluated by the incongruence length difference test and the partition addition bootstrap alteration approach. The resulting gene trees were found to be incongruent, with gapA supporting a monophyletic origin for the plant pathogenic species. In contrast, gyrA and ompA supported multiple polyphyletic origins of Erwinia, Brenneria, Pectobacterium and Pantoea in conjunction with a previously published 16S rDNA phylogeny. However, none of the trees (not even the published 16S rDNA gene tree) supports the current taxonomic classification of these genera into four clades, with Pantoea forming the only monophyletic group in the gapA, gyrA and 16S rDNA trees. Finally, the gapA, gyrA and previously published 16S rDNA phylogenies differ in the taxonomic placement of several bacterial strains which are separated in the three trees. The observed incongruence among the four gene histories is likely to be the result of horizontal transfer events, confounding the search for a robust set of housekeeping genes with a shared evolutionary history that could be used to confidently characterize the relationships of the plant pathogenic enterobacteria. © The Willi Hennig Society 2010.     

Free from mitochondrial DNA: Nuclear genes and the inference of species trees among closely related darter lineages (Teleostei: Percidae: Etheostomatinae)

Investigations into the phylogenetics of closely related animal species are dominated by the use of mitochondrial DNA (mtDNA) sequence data. However, the near-ubiquitous use of mtDNA to infer phylogeny among closely related animal lineages is tempered by an increasing number of studies that document high rates of transfer of mtDNA genomes among closely related species through hybridization, leading to substantial discordance between phylogenies inferred from mtDNA and nuclear gene sequences. In addition, the recent development of methods that simultaneously infer a species phylogeny and estimate divergence times, while accounting for incongruence among individual gene trees, has ushered in a new era in the investigation of phylogeny among closely related species. In this study we assess if DNA sequence data sampled from a modest number of nuclear genes can resolve relationships of a species-rich clade of North American freshwater teleost fishes, the darters. We articulate and expand on a recently introduced method to infer a time-calibrated multi-species coalescent phylogeny using the computer program ^*BEAST. Our analyses result in well-resolved and strongly supported time-calibrated darter species tree. Contrary to the expectation that mtDNA will provide greater phylogenetic resolution than nuclear gene data; the darter species tree inferred exclusively from nuclear genes exhibits a higher frequency of strongly supported nodes than the mtDNA time-calibrated gene tree.     

Partial combination applied to phylogeny of European cyprinids using the mitochondrial control region

Previous molecular phylogenies of European cyprinids led to some solid facts and some uncertainties. This study is based on a stretch of more than 1 kb in the mitochondrial control region newly sequenced for 35 European cyprinids and on previous cytochrome b and 16S rDNA data. The trees based on the control region are more accurate and robust than those obtained from the two other genes. Character incongruence among the three genes was tested using the incongruence length difference (ILD) test. Iterative removals of individual sequences followed by new ILD tests identified two sequences responsible for statistically significant incongruence. A partial combination was conducted, that is, a combination of the three data sets, removing the two sequences previously identified. The phylogenetic analysis of this partial combination gives a more robust and resolved picture of subfamilial interrelationships. The Rasborinae are the sister group of all other cyprinids. The monophyletic Cyprininae emerges next. Tinca tinca first and then Rhodeus are the sister groups of all the remaining nonrasborine and noncyprinine species. Gobio is the sister group of the Leuciscinae, in which the Phoxinini are the sister group of the Leuciscini. Within the Leuciscini, the genus Leuciscus and the subfamily Alburninae are both paraphyletic. The Rasborinae are the most basal cyprinid subfamily and the Tincinae are not the sister group of the Cyprininae. These two results challenge only two anatomical characters, which need to be reinterpreted or considered as homoplastic in cyprinid evolution: the modification of the first pleural rib and its parapophysis and the bony composition of the interorbital septum.     

Possible role of horizontal gene transfer in the colonization of sea ice by algae

Diatoms and other algae not only survive, but thrive in sea ice. Among sea ice diatoms, all species examined so far produce ice-binding proteins (IBPs), whereas no such proteins are found in non-ice-associated diatoms, which strongly suggests that IBPs are essential for survival in ice. The restricted occurrence also raises the question of how the IBP genes were acquired. Proteins with similar sequences and ice-binding activities are produced by ice-associated bacteria, and so it has previously been speculated that the genes were acquired by horizontal transfer (HGT) from bacteria. Here we report several new IBP sequences from three types of ice algae, which together with previously determined sequences reveal a phylogeny that is completely incongruent with algal phylogeny, and that can be most easily explained by HGT. HGT is also supported by the finding that the closest matches to the algal IBP genes are all bacterial genes and that the algal IBP genes lack introns. We also describe a highly freeze-tolerant bacterium from the bottom layer of Antarctic sea ice that produces an IBP with 47% amino acid identity to a diatom IBP from the same layer, demonstrating at least an opportunity for gene transfer. Together, these results suggest that the success of diatoms and other algae in sea ice can be at least partly attributed to their acquisition of prokaryotic IBP genes.     

Evidence for Recombination in the Microcystin Synthetase (<Emphasis Type="Italic">mcy</Emphasis>) Genes ofToxic Cyanobacteria <Emphasis Type="Italic">Microcystis</Emphasis><Emphasis Type="Bold">spp</Emphasis>

Recombination has been suggested to be an important factor for the genetic variation of bacterial genes, but few studies have dealt with intragenic recombination between the same or closely related species of cyanobacteria. Here we provide strong evidence for recombination in the microcystin synthetase (mcy) gene cluster of the toxic cyanobacteria Microcystis spp. This gene cluster contains 10 genes (mcyA to J) that encode a mixed polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) complex. mcy gene sequences were determined for four selected regions (within mcyA, D, G, and J) within the mcy gene cluster from 1 Canadian and 10 Asian toxic Microcystis and compared with previously published mcy sequences. Split decomposition analysis indicated a reticulate phylogeny of mcyA, and several potential recombination tracts of mcyA were identified by the RDP analysis and a runs test implemented in GENECONV. In contrast, no recombination was detected in the mcyD, G, and J sequences. However, discrepancies among the four mcy gene genealogies were evident from the results of independent split decomposition analyses, which were further supported by incongruence length difference (ILD) tests. Taken together, these findings suggest that both intragenic and intergenic recombination within the mcy gene cluster contributes to the genetic diversity of the mcy genes of Microcystis spp.This article contains online supplementary material.     

Multilocus sequence analysis of the genus Bradyrhizobium

The use of multilocus sequence analysis (MLSA) for the taxonomy of Bradyrhizobium was assessed. We compared partial sequences for atpD, recA, gyrB, rpoB and dnaK for a set of reference strains representing named species and genospecies, and a number of new isolates from Lupinus albus, Arachis hypogaea and Ornithopus compressus from Spain. The phylogenies of the individual genes were compared with previous DNA–DNA hybridization results. High hybridization values were well reflected, but intermediary hybridization values were less clearly apparent. However, the phylogeny of a concatenated dataset of the five genes did reflect all values and thus is more informative of overall genome similarity. Our results indicate that only for the genes gyrB, rpoB and dnaK there is a small gap between the interspecies sequence similarities and the intraspecies similarity, and therefore cut-off levels for species delineation cannot be set, although high sequence similarity (>99%) does permit identification. In a few instances, a reference strain did not group as expected for one of the five genes tested. This may be a result of horizontal gene transfer and recombination events occasionally involving housekeeping genes. This observation indicates it is best to consider more than one gene for taxonomic inferences. The majority of the new isolates from the three host species was identified as Bradyrhizobium canariense. Four strains from L. albus from León, Spain, formed a separate group close to Bradyrhizobium japonicum.     

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.