Relative Branching Order Research Articles

Plastid phylogenomics of the cool-season grass subfamily: clarification of relationships among early-diverging tribes.

Whole plastid genomes are being sequenced rapidly from across the green plant tree of life, and phylogenetic analyses of these are increasing resolution and support for relationships that have varied among or been unresolved in earlier single- and multi-gene studies. Pooideae, the cool-season grass lineage, is the largest of the 12 grass subfamilies and includes important temperate cereals, turf grasses and forage species. Although numerous studies of the phylogeny of the subfamily have been undertaken, relationships among some 'early-diverging' tribes conflict among studies, and some relationships among subtribes of Poeae have not yet been resolved. To address these issues, we newly sequenced 25 whole plastomes, which showed rearrangements typical of Poaceae. These plastomes represent 9 tribes and 11 subtribes of Pooideae, and were analysed with 20 existing plastomes for the subfamily. Maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) robustly resolve most deep relationships in the subfamily. Complete plastome data provide increased nodal support compared with protein-coding data alone at nodes that are not maximally supported. Following the divergence of Brachyelytrum, Phaenospermateae, Brylkinieae-Meliceae and Ampelodesmeae-Stipeae are the successive sister groups of the rest of the subfamily. Ampelodesmeae are nested within Stipeae in the plastome trees, consistent with its hybrid origin between a phaenospermatoid and a stipoid grass (the maternal parent). The core Pooideae are strongly supported and include Brachypodieae, a Bromeae-Triticeae clade and Poeae. Within Poeae, a novel sister group relationship between Phalaridinae and Torreyochloinae is found, and the relative branching order of this clade and Aveninae, with respect to an Agrostidinae-Brizinae clade, are discordant between MP and ML/BI trees. Maximum likelihood and Bayesian analyses strongly support Airinae and Holcinae as the successive sister groups of a Dactylidinae-Loliinae clade.

The mangotoxin biosynthetic operon (mbo) is specifically distributed within Pseudomonas syringae genomospecies 1 and was acquired only once during evolution.

Mangotoxin production was first described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for the mbo operon to examine its distribution within the P. syringae complex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of the mbo operon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex.

Of woods and webs: possible alternatives to the tree of life for studying genomic fluidity in E. coli

BackgroundWe introduce several forest-based and network-based methods for exploring microbial evolution, and apply them to the study of thousands of genes from 30 strains of E. coli. This case study illustrates how additional analyses could offer fast heuristic alternatives to standard tree of life (TOL) approaches.ResultsWe use gene networks to identify genes with atypical modes of evolution, and genome networks to characterize the evolution of genetic partnerships between E. coli and mobile genetic elements. We develop a novel polychromatic quartet method to capture patterns of recombination within E. coli, to update the clanistic toolkit, and to search for the impact of lateral gene transfer and of pathogenicity on gene evolution in two large forests of trees bearing E. coli. We unravel high rates of lateral gene transfer involving E. coli (about 40% of the trees under study), and show that both core genes and shell genes of E. coli are affected by non-tree-like evolutionary processes. We show that pathogenic lifestyle impacted the structure of 30% of the gene trees, and that pathogenic strains are more likely to transfer genes with one another than with non-pathogenic strains. In addition, we propose five groups of genes as candidate mobile modules of pathogenicity. We also present strong evidence for recent lateral gene transfer between E. coli and mobile genetic elements.ConclusionsDepending on which evolutionary questions biologists want to address (i.e. the identification of modules, genetic partnerships, recombination, lateral gene transfer, or genes with atypical evolutionary modes, etc.), forest-based and network-based methods are preferable to the reconstruction of a single tree, because they provide insights and produce hypotheses about the dynamics of genome evolution, rather than the relative branching order of species and lineages. Such a methodological pluralism - the use of woods and webs - is to be encouraged to analyse the evolutionary processes at play in microbial evolution.This manuscript was reviewed by: Ford Doolittle, Tal Pupko, Richard Burian, James McInerney, Didier Raoult, and Yan Boucher

Resolving the question of trypanosome monophyly: A comparative genomics approach using whole genome data sets with low taxon sampling

Since the first attempts to classify the evolutionary history of trypanosomes, there have been conflicting reports regarding their true phylogenetic relationships and, in particular, their relationships with other vertebrate trypanosomatids, e.g. Leishmania sp., as well as with the many insect parasitising trypanosomatids. Perhaps the issue that has provided most debate is that concerning the monophyly (or otherwise) of genus Trypanosoma and, even with the advent of molecular methods, the findings of numerous studies have varied significantly depending on the gene sequences analysed, number of taxa included, choice of outgroup and phylogenetic methodology. While of arguably limited applied importance, resolution of the question as to whether or not trypanosomes are monophyletic is critical to accurate evaluation of competing, mutually exclusive evolutionary scenarios for these parasites, namely the ‘vertebrate-first’ or ‘insect-first’ hypotheses. Therefore, a new approach, which could overcome previous limitations was needed. At its most simple, the problem can be defined within the framework of a trifurcated tree with three hypothetical positions at which the root can be placed. Using BLASTp and whole-genome gene-by-gene phylogenetic analyses of Trypanosoma brucei, Trypanosoma cruzi, Leishmania major and Naegleria gruberi, we have identified 599 gene markers – putative homologues – that were shared between the genomes of these four taxa. Of these, 75 homologous gene families that demonstrate monophyly of the kinetoplastids were identified. We then used these data sets in combination with an additional outgroup, Euglena gracilis, coupled with large-scale gene concatenation and diverse phylogenetic techniques to investigate the relative branching order of T. brucei, T. cruzi and L. major. Our findings confirm the monophyly of genus Trypanosoma and demonstrate that <1% of the analysed gene markers shared between the genomes of T. brucei, T. cruzi and L. major reject the hypothesis that the trypanosomes form a monophyletic group.

Phylogenomic Analysis of Kinetoplastids Supports That Trypanosomatids Arose from within Bodonids

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.

Phylogenomics and protein signatures elucidating the evolutionary relationships among the Gammaproteobacteria

The class Gammaproteobacteria, which forms one of the largest groups within bacteria, is currently distinguished from other bacteria solely on the basis of its branching in phylogenetic trees. No molecular or biochemical characteristic is known that is unique to the class Gammaproteobacteria or its different subgroups (orders). The relationship among different orders of gammaproteobacteria is also not clear. In this study, we present detailed phylogenomic and comparative genomic analyses on gammaproteobacteria that clarify some of these issues. Phylogenetic trees based on concatenated sequences for 13 and 36 universally distributed proteins were constructed for 45 members of the class Gammaproteobacteria covering 13 of its 14 orders. In these trees, species from a number of the subgroups formed distinct clades and their relative branching order was indicated as follows (from the most recent to the earliest diverging): Enterobacteriales >Pasteurellales >Vibrionales, Aeromonadales >Alteromonadales >Oceanospirillales, Pseudomonadales >Chromatiales, Legionellales, Methylococcales, Xanthomonadales, Cardiobacteriales, Thiotrichales. Four conserved indels in four widely distributed proteins that are specific for gammaproteobacteria are also described. A 2 aa deletion in 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide transformylase (AICAR transformylase; PurH) was a distinctive characteristic of all gammaproteobacteria (except Francisella tularensis). Two other conserved indels (a 4 aa deletion in RNA polymerase beta-subunit and a 1 aa deletion in ribosomal protein L16) were found uniquely in various species of the orders Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales, but were not found in other gammaproteobacteria. Lastly, a 2 aa deletion in leucyl-tRNA synthetase was commonly present in the above orders of the class Gammaproteobacteria and also in some members of the order Oceanospirillales. The presence of the conserved indels in these gammaproteobacterial orders indicates that species from these orders shared a common ancestor that was separate from other bacteria, a suggestion that is supported by phylogenetic studies. Systematic blastp searches were also conducted on various open reading frames (ORFs) in the genome of Escherichia coli K-12. These analyses identified 75 proteins that were unique to most members of the class Gammaproteobacteria or were restricted to species from some of its main orders (Enterobacteriales; Enterobacteriales and Pasteurellales; Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales and Alteromonadales; and the Enterobacteriales, Pasteurellales, Vibrionales, Aeromonadales, Alteromonadales, Oceanospirillales and Pseudomonadales etc.). The genes for these proteins have evolved at various stages during the evolution of gammaproteobacteria and their species distribution pattern, in conjunction with other results presented here, provide valuable information regarding the evolutionary relationships among these bacteria.

The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga-Flavobacterium-Bacteroides division.

The phylogenetic placement of the rumen bacterium Fibrobacter succinogenes was determined using a signature sequence approach that allows determination of the relative branching order of the major divisions among Bacteria [Gupta, R. S. (2000) FEMS Microbiol Rev 24, 367-402]. For this purpose, segments of the Hsp60 (groEL), Hsp70 (dnaK), CTP synthase and alanyl-tRNA synthetase genes, which are known to contain signature sequences that are useful for phylogenetic deterministic purposes, were cloned. Using degenerate oligonucleotide primers for highly conserved regions in these proteins, 1.4 kb, 0.75 kb, 401 bp and 171 bp fragments of the Hsp70, Hsp60, CTP synthase and alanyl-tRNA synthetase genes respectively were amplified by PCR, and these fragments were cloned and sequenced. These primers, because of their high degree of conservation, could also be used for cloning these genes from other bacterial species. The Hsp70 homologues from different Gram-negative bacteria contain a 21-23 aa insert that is not found in any Gram-positive bacteria. The presence of this insert in the F. succinogenes Hsp70 supports its placement within the Gram-negative group of bacteria. A conserved insert in F. succinogenes Hsp60 that is commonly present in all bacterial species, except various Gram-positive bacteria, Deinococcus-Thermus groups and green non-sulphur bacteria, provides evidence that F. succinogenes does not belong to these taxa. A particularly useful signature consisting of a 4 aa insert is found in Ala-tRNA synthetase. This insert is present in all proteobacterial homologues as well as in homologues from species belonging to the Chlamydia and Cytophaga-Flavobacterium- Bacteroides (CFB) groups, but it is not found in homologues from any other groups of bacteria. The presence of this insert in F. succinogenes Ala-tRNA synthetase provides evidence that this species is related to these groups. However, two other signatures in CTP synthase and Hsp70 proteins, that are distinctive of the proteobacterial species, are not present in the F. succinogenes homologues. These results provide evidence that F. succinogenes does not belong to the proteobacterial division and thus should be placed in a similar position as the Chlamydia and CFB groups of species.

Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily.

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.

Small subunit ribosomal RNA + of Hexamita inflata and the quest for the first branch in the eukaryotic tree

A phylogenetic analysis of the small subunit ribosomal RNA (16S-like rRNA) coding region from Hexamita inflata demonstrates that parasitism alone cannot explain early diverging eukaryotic lineages. Parasitic and free-living diplomonads, as well as trichomonads and microsporidia, diverge at the base of the eukaryotic tree. The relative branching order of diplomonads, trichomonads and microsporidia is influenced by outlying prokaryotic taxa with different G + C compositions in their rRNA coding regions. The high G + C prokaryotes position Giardia lamblia at the base of the eukaryotic tree but split diplomonads into a paraphyletic group. When the outlying groups are restricted to rRNAs with nominal G + C compositions, diplomonads form a monophyletic group that diverged after the microsporidia and trichomonads. This unstable branching pattern correlates with unusual nucleotide compositions in the rRNAs of G. lamblia (75% G + C) and Vairimorpha necatrix (35% G + C). In contrast, the 51% G + C composition of the H. inflata rRNA is typical of other eukaryotic rRNAs. Its divergence after trichomonads is strongly supported by bootstrap replicates in distance analyses that do not include G. lamblia. Because of a low G + C composition in its rRNA coding region, the phylogenetic placement of V. necatrix is uncertain and the identity of the deepest branching eukaryotic lineage is ambiguous.

ALGAE CONTAINING CHLOROPHYLLS a + c ARE PARAPHYLETIC: MOLECULAR EVOLUTIONARY ANALYSIS OF THE CHROMOPHYTA.

Sequence comparisons of small subunit ribosomal RNA coding regions from 12 chlorophylls a + c-containing algae were used to infer phylogenetic relationships within the Chromophyta. Three chromophyte lines of descent, delineated by the Bacillariophyceae, the Phaeophyceae/Xanthophyceae, and the Chrysophyceae/Eustigmatophyceae/Synurophyceae are members of a complex evolutionary assemblage, which also includes representatives of the Oomycota ("lower" fungi). Maximum parsimony and distance matrix methods demonstrate a common evolutionary history for these lineages but their relative branching order could not be determined. Other algal species with chlorophylls a + c, including dinoflagellates and prymnesiophytes, are not members of this complex assemblage. Dinoflagellates are specifically related to apicomplexans and ciliates, and the prymnesiophyte, Emiliania huxleyi, represents an independent photosynthetic lineage that separated from other eukaryotes during the nearly simultaneous divergence of plants, animals, fungi, and a number of other protist lineages. The small subunit rRNA phylogenies of chromophytes/oomycetes were compared to those derived from comparisons of ultrastructural characters. Only tubular, tripartite mastigonemes (flagellar hairs) characterized all studied taxa of chromophytes/oomycetes as a monophyletic assemblage.

Evolution of DNA sequences has been retarded in Malagasy primates

It is generally accepted that there are six major groups of living primates: (1) lemurs (including all the primates of Madagascar), (2) lorises (including galago and potto), (3) tarsiers, (4) New World monkeys, (5) Old World monkeys and (6) apes (including man). Tree shrews, once considered to be primates, are now generally recognized as not significantly more closely related to the six groups than other mammals. The first surviving primate lines to diverge from the common primate ancestor are believed to have given rise to one or more of the first three groups. However, the fossil record is insufficient to determine their relative branching order. Furthermore, neither morphological considerations nor studies of protein evolution produce unanimity as to whether tarsiers are more closely related to the prosimians (the lemurs plus lorises) or the simians (the monkeys and apes). In an attempt to resolve these discrepancies, we have measured the DNA sequence difference between several primates. We report here that the evolution of DNA of primates from Madagascar is significantly less than that of all other groups of living primates. This is not expected in the simplest form of the theory of neutral selection and may be important for our understanding of evolution at the molecular level.