Molecular Genetics and Phylogeny of Bigmouth Shiner, Notropis dorsalis

The study of North American freshwater ichthyofauna reveals a diversity shaped by the continent\u27s geologic past and the extensive effects of Pleistocene glaciation on river systems. Ericymba dorsalis (Bigmouth 69 Shiner) is a minnow species with a broad distribution in the Mississippi River basin from Wyoming to New York. Despite exhibiting consistent morphological features across its range, the species\u27 widespread northern distribution suggests the potential existence of multiple, genetically distinct lineages. This research tested the hypothesis that separate drainage systems harbor genetically distinct groups of E. dorsalis. Our results identified five clades, each associated with major drainages. Populations of the Missouri River, upper Mississippi River, and Illinois River, respectively, grouped into three clades with divergence of 1.3-1.6%, indicating a Pleistocene origin. Three disjunct populations located in Great Lakes tributaries in western Michigan, Ohio, and New York, respectively, formed a fourth clade, and population samples from the Allegheny River in Pennsylvania formed a fifth clade. The divergence of the Great Lakes and Allegheny clades from the western clades ranged 4.3-5.3%, suggesting an early Pleistocene or Pliocene origin. This pattern of distribution underscores the role of vicariance biogeography during Pleistocene glaciation, on the current distribution of genetic diversity in E. dorsalis

Multivariate analyses in microbial ecology

Environmental microbiology is undergoing a dramatic revolution due to the increasing accumulation of biological information and contextual environmental parameters. This will not only enable a better identification of diversity patterns, but will also shed more light on the associated environmental conditions, spatial locations, and seasonal fluctuations, which could explain such patterns. Complex ecological questions may now be addressed using multivariate statistical analyses, which represent a vast potential of techniques that are still underexploited. Here, well-established exploratory and hypothesis-driven approaches are reviewed, so as to foster their addition to the microbial ecologist toolbox. Because such tools aim at reducing data set complexity, at identifying major patterns and putative causal factors, they will certainly find many applications in microbial ecology

Functional abilities of cultivable plant growth promoting bacteria associated with wheat (Triticum aestivum L.) crops

Abstract In the pursuit of sustainable agriculture, bioinoculants usage as providers of a crop's needs is a method to limit environmental damage. In this study, a collection of cultivable putative plant growth promoting (PGP) bacteria associated with wheat crops was obtained and this bacterial sample was characterized in relation to the functional diversity of certain PGP features. The isolates were obtained through classical cultivation methods, identified by partial 16S rRNA gene sequencing and characterized for PGP traits of interest. Functional diversity characterization was performed using Categorical Principal Component Analysis (CatPCA) and Multiple Correspondence Analysis (MCA). The most abundant genera found among the 346 isolates were Pseudomonas, Burkholderia, and Enterobacter. Occurrence of PGP traits was affected by genus, niche, and sampling site. A large number of genera grouped together with the ability to produce indolic compounds; phosphate solubilization and siderophores production formed a second group related to fewer genera, in which the genus Burkholderia has a great importance. The results obtained may help future studies aiming prospection of putative plant growth promoting bacteria regarding the desired organism and PGP trait

Substrate Type Determines Metagenomic Profiles from Diverse Chemical Habitats

Environmental parameters drive phenotypic and genotypic frequency variations in microbial communities and thus control the extent and structure of microbial diversity. We tested the extent to which microbial community composition changes are controlled by shifting physiochemical properties within a hypersaline lagoon. We sequenced four sediment metagenomes from the Coorong, South Australia from samples which varied in salinity by 99 Practical Salinity Units (PSU), an order of magnitude in ammonia concentration and two orders of magnitude in microbial abundance. Despite the marked divergence in environmental parameters observed between samples, hierarchical clustering of taxonomic and metabolic profiles of these metagenomes showed striking similarity between the samples (>89%). Comparison of these profiles to those derived from a wide variety of publically available datasets demonstrated that the Coorong sediment metagenomes were similar to other sediment, soil, biofilm and microbial mat samples regardless of salinity (>85% similarity). Overall, clustering of solid substrate and water metagenomes into discrete similarity groups based on functional potential indicated that the dichotomy between water and solid matrices is a fundamental determinant of community microbial metabolism that is not masked by salinity, nutrient concentration or microbial abundance

Toward a More Robust Assessment of Intraspecies Diversity, Using Fewer Genetic Markers

Phylogenetic sequence analysis of single or multiple genes has dominated the study and census of the genetic diversity among closely related bacteria. It remains unclear, however, how the results based on a few genes in the genome correlate with whole-genome-based relatedness and what genes (if any) best reflect whole-genome-level relatedness and hence should be preferentially used to economize on cost and to improve accuracy. We show here that phylogenies of closely related organisms based on the average nucleotide identity (ANI) of their shared genes correspond accurately to phylogenies based on state-of-the-art analysis of their whole-genome sequences. We use ANI to evaluate the phylogenetic robustness of every gene in the genome and show that almost all core genes, regardless of their functions and positions in the genome, offer robust phylogenetic reconstruction among strains that show 80 to 95% ANI (16S rRNA identity, >98.5%). Lack of elapsed time and, to a lesser extent, horizontal transfer and recombination make the selection of genes more critical for applications that target the intraspecies level, i.e., strains that show >95% ANI according to current standards. A much more accurate phylogeny for the Escherichia coli group was obtained based on just three best-performing genes according to our analysis compared to the concatenated alignment of eight genes that are commonly employed for phylogenetic purposes in this group. Our results are reproducible within the Salmonella, Burkholderia, and Shewanella groups and therefore are expected to have general applicability for microevolution studies, including metagenomic surveys

Species Abundance and Diversity of Burkholderia cepacia Complex in the Environment

Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a B. cepacia complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting B. cepacia complex-specific recA and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of recA for a total of 285 confirmed isolates of the B. cepacia complex, up to seven B. cepacia complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of B. cepacia complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants

Identification and Onion Pathogenicity of Burkholderia cepacia Complex Isolates from the Onion Rhizosphere and Onion Field Soil▿

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia

The bacterial species definition in the genomic era

The bacterial species definition, despite its eminent practical significance for identification, diagnosis, quarantine and diversity surveys, remains a very difficult issue to advance. Genomics now offers novel insights into intra-species diversity and the potential for emergence of a more soundly based system. Although we share the excitement, we argue that it is premature for a universal change to the definition because current knowledge is based on too few phylogenetic groups and too few samples of natural populations. Our analysis of five important bacterial groups suggests, however, that more stringent standards for species may be justifiable when a solid understanding of gene content and ecological distinctiveness becomes available. Our analysis also reveals what is actually encompassed in a species according to the current standards, in terms of whole-genome sequence and gene-content diversity, and shows that this does not correspond to coherent clusters for the environmental Burkholderia and Shewanella genera examined. In contrast, the obligatory pathogens, which have a very restricted ecological niche, do exhibit clusters. Therefore, the idea of biologically meaningful clusters of diversity that applies to most eukaryotes may not be universally applicable in the microbial world, or if such clusters exist, they may be found at different levels of distinction

Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species

Burkholderia species are widely distributed in the natural environment. We evaluated the use of the recA gene in a cultivation-independent approach to examine the Burkholderia diversity associated with the maize rhizosphere. Two types of recA gene library were constructed, one with broad-specificity recA primers (BUR1 and BUR2) and a second from the products of nested PCRs using Burkholderia-specific primers (BUR3 and BUR4). The broad-specificity primer set provided near full-length recA sequences (869 bp) suitable for the creation of robust environmental sequence data sets; however, the nested PCR approach demonstrated the greatest specificity (84%) for detection of Burkholderia species recA genes. In addition, the screening approach was able to identify recA phylotypes matching Burkholderia cepacia complex species previously cultivated from the maize samples and discriminate these from other Burkholderia. The ecological benefit of Burkholderia species cultivated from maize rhizosphere is well documented, however, the fact that the majority of Burkholderia recA genes detected in this study (90%) were suggestive of novel taxa indicates that a wealth of potentially important interactions with uncultivated Burkholderia species remain unstudied in this habitat