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 phylogenetics to phylogenomics: the evolutionary relationships of insect endosymbiotic gamma-Proteobacteria as a test case

The increasing availability of complete genome sequences and the development of new, faster methods for phylogenetic reconstruction allow the exploration of the set of evolutionary trees for each gene in the genome of any species. This has led to the development of new phylogenomic methods. Here, we have compared different phylogenetic and phylogenomic methods in the analysis of the monophyletic origin of insect endosymbionts from the gamma-Proteobacteria, a hotly debated issue with several recent, conflicting reports. We have obtained the phylogenetic tree for each of the 579 identified protein-coding genes in the genome of the primary endosymbiont of carpenter ants, Blochmannia floridanus, after determining their presumed orthologs in 20 additional Proteobacteria genomes. A reference phylogeny reflecting the monophyletic origin of insect endosymbionts was further confirmed with different approaches, which led us to consider it as the presumed species tree. Remarkably, only 43 individual genes produced exactly the same topology as this presumed species tree. Most discrepancies between this tree and those obtained from individual genes or by concatenation of different genes were due to the grouping of Xanthomonadales with beta-Proteobacteria and not to uncertainties over the monophyly of insect endosymbionts. As previously noted, operational genes were more prone to reject the presumed species tree than those included in information-processing categories, but caution should be exerted when selecting genes for phylogenetic inference on the basis of their functional category assignment. We have obtained strong evidence in support of the monophyletic origin of gamma-Proteobacteria insect endosymbionts by a combination of phylogenetic and phylogenomic methods. In our analysis, the use of concatenated genes has shown to be a valuable tool for analyzing primary phylogenetic signals coded in the genomes. Nevertheless, other phylogenomic methods such as supertree approaches were useful in revealing alternative phylogenetic signals and should be included in comprehensive phylogenomic studies.     

Multiple genome alignments facilitate development of NPCL markers: a case study of tetrapod phylogeny focusing on the position of turtles

In recent years, the increasing availability of genomic resources has provided an opportunity to develop phylogenetic markers for phylogenomics. Efficient methods to search for candidate markers from the huge number of genes within genomic data are particularly needed in the era of phylogenomics. Here, rather than using the traditional approach of comparing genomes of two distantly related taxa to develop conserved primers, we take advantage of the multiple genome alignment resources from the the University of California-San Cruz Genome Browser and present a simple and straightforward bioinformatic approach to automatically screen for candidate nuclear protein-coding locus (NPCL) markers. We tested our protocol in tetrapods and successfully obtained 21 new NPCL markers with high success rates of polymerase chain reaction amplification (mostly over 80%) in 16 diverse tetrapod taxa. These 21 newly developed markers together with two reference genes (RAG1 and mitochondrial 12S-16S) are used to infer the higher level relationships of tetrapods, with emphasis on the debated position of turtles. Both maximum likelihood (ML) and Bayesian analyses on the concatenated data combining the 23 markers (21,137 bp) yield the same tree, with ML bootstrap values over 95% and Bayesian posterior probability equaling 1.0 for most nodes. Species tree estimation using the program BEST without data concatenation produces similar results. In all analyses, turtles are robustly recovered as the sister group of Archosauria (birds and crocodilians). The jackknife analysis on the concatenated data showed that the minimum sequence length needed to robustly resolve the position of turtles is 13-14 kb. Based on the large 23-gene data set and the well-resolved tree, we also estimated evolutionary timescales for tetrapods with the popular Bayesian method MultiDivTime. Most of the estimated ages among tetrapods are similar to the average estimates of the previous dating studies summarized by the book The Timetree of Life.     

Systematic reverse genetic screening of T-DNA tagged genes in rice for functional genomic analyses: MADS-box genes as a test case

We have generated 47 DNA pools and 235 subpools from 21,049 T-DNA insertion lines of rice. DNA pools of 500-1,000 lines were adequate for screening a T-DNA insertion within a 2-kb region. To examine the efficacy of the DNA pools, we selected MADS-box genes, which play an important role in controlling various aspects of plant development. A total of 34 MIKC-type MADS-box genes have now been identified from rice sequence databases. Our PCR screening for T-DNA insertions within 12 MADS-box genes resulted in the identification of five insertions in four different genes. These DNA pools will be valuable when isolating T-DNA insertional mutants in various rice genes. The DNA pool screening service and the mutant seeds are available upon request to genean@postech.ac.kr.     

Rooting a phylogeny with homologous genes on opposite sex chromosomes (gametologs): a case study using avian CHD

We describe a previously unrecognized form of gene homology using the term "gametology," which we define as homology arising through lack of recombination and subsequent differentiation of sex chromosomes. We demonstrate use of gametologous genes to root each other in phylogenetic analyses of sex-specific avian Chromo-helicase-DNA binding gene (CHD) sequences. Phylogenetic analyses of a set of neognath bird sequences yield monophyletic groups for CHD-W and CHD-Z gametologs, as well as congruent relationships between these two clades and between them and current views of avian taxonomy. Phylogenetic analyses including paleognath bird CHD sequences and rooting with crocodilian CHD sequences, suggest an early divergence for paleognath CHD within the avian CHD clade. Based on our CHD analyses calibrated with avian fossil dates, we estimate the divergence between CHD-W and CHD-Z at 123 MYA, suggesting an early differentiation of sex chromosomes that predates most extant avian orders. In agreement with the notion of male-driven evolution, we find a faster rate of change in male-linked CHD-Z sequences.     

Linguistic analysis in phylogeny estimation: a case study of mtDNAs of Bovidae

Molecular phylogenetic studies are executed by the alignment of protein or nucleotide sequences, followed by the construction of trees according either to distance, parsimony or maximum likelihood methods. Linguistic analysis was investigated here as an alternative method to aligning sequences. In an empirical study, we inferred trees for a variable number of Bovidae and sister taxa based on three different mitochondrial orthologous sequences. Comparison of our results with existing phylogenies indicated that the method, except for some still disputable points, was able to establish sensible systematic relationships, similar to patterns of radiation of the family found in recent studies.     

Resolution of the laurasiatherian phylogeny: evidence from genomic data

Despite great progress over the past decade, some portions of the mammalian tree of life remain unresolved. In particular, relationships among the different orders included within the supraordinal group Laurasiatheria have been proven difficult to determine, and have received poor support in the vast majority of phylogenomic studies of mammalian systematics. We estimated interordinal relationships within Laurasiatheria using sequence data from 3733 protein-coding genes. Our study included data from from 11 placental mammals, corresponding to five of the six orders of Laurasiatheria, plus five outgroup species. Ingroup and outgroup species were chosen to maximize the number single-copy ortholog genes for which sequence data was available for all species in our study. Phylogenetic analyses of the concatenated dataset using maximum likelihood and Bayesian methods resulted on an identical and well supported topology in all alignment strategies compared. Our analyses provide high support for the sister relationship between Chiroptera and Cetartiodactyla and also provide support for placing Perissodactyla as sister to Carnivora. We obtained maximal estimates of bootstrap support (100%) and posterior probability (1.00) for all nodes within Laurasiatheria. Our study provides a further demonstration of the utility of very large and conserved genomic dataset to clarify our understanding of the evolutionary relationships among mammals.     

Novel intron markers to study the phylogeny of closely related mammalian species

Background  

Multilocus phylogenies can be used to infer the species tree of a group of closely related species. In species trees, the nodes represent the actual separation between species, thus providing essential information about their evolutionary history. In addition, multilocus phylogenies can help in analyses of species delimitation, gene flow and genetic differentiation within species. However, few adequate markers are available for such studies.     

Genomics as a means to understand bacterial phylogeny and ecological adaptation: the case of bifidobacteria

The field of microbiology has in recent years been transformed by the ever increasing number of publicly available whole-genome sequences. This sequence information has significantly enhanced our understanding of the physiology, genetics and evolutionary development of bacteria. Among the latter group of microorganisms, bifidobacteria represent important human commensals because of their perceived contribution to maintaining a balanced gastrointestinal tract microbiota. In recent years bifidobacteria have drawn much scientific attention because of their use as live bacteria in numerous food preparations with various health-related claims. For this reason, these bacteria constitute a growing area of interest with respect to genomics, molecular biology and genetics. Recent genome sequencing of a number of bifidobacterial species has allowed access to the complete genetic make-up of these bacteria. In this review we will discuss how genomic data has allowed us to understand bifidobacterial evolution, while also revealing genetic functions that explains their presence in the particular ecological environment of the gastrointestinal tract. An erratum to this article can be found at     

Non-clonability correlates with genomic instability: a case study of a unique DNA region

Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon. With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known. The intergenic spacer located between the pi and alpha(D) chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids. Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene). Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of "unclonable" DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins.     

Evaluating gene flow using selected markers: a case study.

The extent to which an organism is locally adapted in an environmental pocket depends on the selection intensities inside and outside the pocket, on migration, and on the size of the pocket. When two or more loci are involved in this local adaptation, measuring their frequency gradients and their linkage disequilbria allows one to disentangle the forces-migration and selection-acting on the system. We apply this method to the case of a local adaptation to organophosphate insecticides in the mosquito Culex pipiens pipiens in southern France. The study of two different resistance loci allowed us to estimate with support limits gene flow as well as selection pressure on insecticide resistance and the fitness costs associated with each locus. These estimates permit us to pinpoint the conditions for the maintenance of this pocket of adaptation as well as the effect of the interaction between the two resistance loci.     

Diversity, abundance or rare species as a target for the conservation of mammalian carnivores: a case study in Southern Spain

Management goals in protected areas and/or communities usually include diversity as one of the most valuable and confident criteria. Nevertheless, the use of diversity and related indices as a means of evaluating successful management practices could produce conflicting results. Here we report a case study in one of the most important European protected areas. After 6 years of intensive conservation management of the Don~ana National Park, the general abundance and numbers of the target single-species conservation plan (the Iberian lynx) increased, although carnivore community diversity and evenness decreased. This was a result of a disproportionate increase of an oportunistic native species, the red fox. We propose the combined use of diversity, richness and evenness indices when monitoring management practices such as those reported here.     

Recapitulating the evolution of Afrotheria: 57 genes and rare genomic changes (RGCs) consolidate their history

The decipherment of higher level relationships among the orders of Afrotheria – an extraordinary assumption in mammalian evolution – constitutes one of the major disputes in the evolutionary history of mammals. Recent comprehensive studies of various genomic data, including mitochondrial and nuclear DNA sequences, chromosomal syntenic associations and retroposon insertions support strongly the monophyly of Afrotheria. However, the relationships within Afrotheria have remained ambiguous and there is a necessity for a more sophisticated analysis (i.e. combination of gene phylogeny and Rare Genomic Changes (RGCs)), which could aid in the comprehension of the evolutionary history of this old group of mammals. The present study investigated the phylogenetic relationships within Afrotheria by analysing a data set of coding and non-coding sequences (～32 000 bp) comprising 57 orthologous genes and 31 RGCs, such as chromosomal associations and retroposon insertions, and re-evaluated a molecular timescale for afrotherian mammals using a Bayesian relaxed clock approach. The interordinal afrotherians phylogeny presented here contributed to the elucidation of the evolutionary history of this ancient clade of mammals, which is one of the most unorthodox proposals in mammalian biology. This is critical not only for understanding how Afrotheria evolved in Africa, but also to comprehend the early biogeographical history of placental mammals.     

The human hyaluronan synthase genes: genomic structures,proximal promoters and polymorphic microsatellite markers

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.     

Conservation of rare species with island-like distributions: A case study of Lasthenia conjugens (Asteraceae) using population genetic structure and the distribution of rare markers

Californian vernal pools, a patchy, island-like habitat, are endangered as a result of habitat destruction. Conservation of the remaining vernal pool habitat is essential for the persistence of several endangered species. We present the first study examining DNA-level genetic diversity within and among populations of a vernal pool plant species. We investigated genetic variation across eight populations of the US federally endangered vernal pool endemic Lasthenia conjugens (Asteraceae) using intersimple sequence repeat (ISSR) markers. Genetic diversity within the species was high (Nei's gene diversity estimate was 0.37), with moderate differentiation among populations (Bayesian F _ST analog of 0.124). Using an amova analysis, we found that the majority of the genetic variation (84%) was distributed within populations. There is a significant relationship between geographical distance and pairwise genetic differentiation as measured by the Bayesian estimate θ^B. The alternative hypotheses of historic geological processes within the Central Valley and contemporary gene flow are discussed as explanations of the data. Because of the vulnerability of the populations, we calculated a probability of loss for rare alleles (fragments) in the populations. Calculations show that sampling only one of the eight populations for ex-situ conservation or restoration will capture approximately 54% of the sampled rare fragments. We believe that one of the sampled populations has become extinct since it was sampled. When removing this population from the above-mentioned calculations, sampling one population will capture only 41.3% of the sampled rare fragments. We recommend sampling strategies for future conservation and restoration efforts of L. conjugens.