Increased Microbial Diversity and Decreased Prevalence of Common Pathogens in the Gut Microbiomes of Wild Turkeys Compared to Domestic Turkeys.

Turkeys (Meleagris gallopavo) provide a globally important source of protein and constitute the second most important source of poultry meat in the world. Bacterial diseases are common in commercial poultry production, causing significant production losses for farmers. Due to the increasingly recognized problems associated with large-scale/indiscriminate antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study, we compared the cecal microbiota of wild and domestic turkeys, hypothesizing that environmental pressures faced by wild birds may select for a disease-resistant microbial community. Sequence analyses of 16S rRNA genes amplified from cecal samples indicate that free-roaming wild turkeys carry a rich and variable microbiota compared to domestic turkeys raised on large-scale poultry farms. Wild turkeys also had very low levels of Staphylococcus, Salmonella, and Escherichia coli compared to domestic turkeys. E. coli strains isolated from wild and domestic turkey cecal samples also belong to distinct phylogenetic backgrounds and differ in their propensity to carry virulence genes. E. coli strains isolated from factory-raised turkeys were far more likely to carry genes for capsule (kpsII and kpsIII) or siderophore (iroN and fyuA) synthesis than were those isolated from wild turkeys. These results suggest that the microbiota of wild turkeys may provide colonization resistance against common poultry pathogens. IMPORTANCE Due to the increasingly recognized problems associated with antibiotic use in agricultural settings, poultry producers need alternative methods to control common bacterial pathogens. In this study, we compare the microbiota of wild and domestic turkeys. The results suggest that free-ranging wild turkeys carry a distinct microbiome compared to farm-raised turkeys. The microbiome of wild birds contains very low levels of poultry pathogens compared to that of farm-raised birds. The microbiomes of wild turkeys may be used to guide the development of new ways to control disease in large-scale poultry production.

Polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads to improve genomic analyses.

Oxford Nanopore sequencing has been widely used to achieve complete genomes of bacterial pathogens. However, the error rates of Oxford Nanopore long reads are high. Various polishing algorithms using Illumina short reads to correct the errors in Oxford Nanopore long-read assemblies have been developed. The impact of polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads on improving genomic analyses was evaluated using both simulated and real reads. Ten species (10 strains) were selected for simulated reads, while real reads were tested on 11 species (11 strains). Oxford Nanopore long reads were assembled with Unicycler to produce a draft assembly, followed by three rounds of polishing with Illumina short reads using two polishing tools, Pilon and NextPolish. One round of NextPolish polishing generated genome completeness and accuracy parameters similar to the reference genomes, whereas two or three rounds of Pilon polishing were needed, though contiguity remained unchanged after polishing. The polished assemblies of Escherichia coli O157:H7, Salmonella Typhimurium, and Cronobacter sakazakii with simulated reads did not provide accurate plasmid identifications. One round of NextPolish polishing was needed for accurately identifying plasmids in Staphylococcus aureus and E. coli O26:H11 with real reads, whereas one and two rounds of Pilon polishing were necessary for these two strains, respectively. Polishing failed to provide an accurate antimicrobial resistance (AMR) genotype for S. aureus with real reads. One round of polishing recovered an accurate AMR genotype for Klebsiella pneumoniae with real reads. The reference genome and draft assembly of Citrobacter braakii with real reads differed, which carried blaCMY-83 and fosA6, respectively, while both genes were present after one round of polishing. However, polishing did not improve the assembly of E. coli O26:H11 with real reads to achieve numbers of virulence genes similar to the reference genome. The draft and polished assemblies showed a phylogenetic tree topology comparable with the reference genomes. For multilocus sequence typing and pan-genome analyses, one round of NextPolish polishing was sufficient to obtain accurate results, while two or three rounds of Pilon polishing were needed. Overall, NextPolish outperformed Pilon for polishing the Oxford Nanopore long-read assemblies of bacterial pathogens, though both polishing strategies improved genomic analyses compared to the draft assemblies.

Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

HPLC-UV, Metabarcoding and Genome Skims of Botanical Dietary Supplements: A Case Study in Echinacea.

The use of DNA-based methods to authenticate botanical dietary supplements has been vigorously debated for a variety of reasons. More comparisons of DNA-based and chemical methods are needed, and concordant evaluation of orthogonal approaches on the same products will provide data to better understand the strengths and weaknesses of both approaches. The overall application of DNA-based methods is already firmly integrated into a wide array of continually modernizing stand alone and complementary authentication protocols. Recently, the use of full-length chloroplast genome sequences provided enhanced discriminatory capacity for closely related species of Echinacea compared to traditional DNA barcoding approaches (matK and rbcL). Here, two next-generation sequencing approaches were used: (1) genome skimming and (2) PCR amplicon (metabarcoding). The two genetic approaches were then combined with HPLC-UV to evaluate 20 commercially available dietary supplements of Echinacea representing "finished" products. The trade-offs involved in different DNA approaches were discussed, with a focus on how DNA methods support existing, accepted chemical methods. In most of the products (19/20), HPLC-UV suggested the presence of Echinacea spp. While metabarcoding was not useful with this genus and instead only resolved 7 products to the family level, genome skimming was able to resolve to species (9) or genus (1) with the 10/20 products where it was successful. Additional ingredients that HPLC-UV was unable to identify were also found in four products along with the relative sequence proportion of the constituents. Additionally, genome skimming was able to identify one product that was a different Echinacea species entirely.

Whole-genome sequencing analysis of uncommon Shiga toxin-producing Escherichia coli from cattle: Virulence gene profiles, antimicrobial resistance predictions, and identification of novel O-serogroups.

Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the "top-7 STEC", little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p < 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.

A Trimeric Autotransporter Enhances Biofilm Cohesiveness in Yersinia pseudotuberculosis but Not in Yersinia pestis.

Cohesion of biofilms made by Yersinia pestis and Yersinia pseudotuberculosis has been attributed solely to an extracellular polysaccharide matrix encoded by the hms genes (Hms-dependent extracellular matrix [Hms-ECM]). However, mutations in the Y. pseudotuberculosis BarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilized Y. pseudotuberculosiscsrB mutant biofilms, suggesting that cell-cell interactions might be mediated by protein adhesins or extracellular matrix proteins. We identified an uncharacterized trimeric autotransporter lipoprotein (YPTB2394), repressed by csrB, which has been referred to as YadE. Biofilms made by a ΔyadE mutant strain were extremely sensitive to mechanical disruption. Overexpression of yadE in wild-type Y. pseudotuberculosis increased biofilm cohesion, similar to biofilms made by csrB or uvrY mutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression of yadE The yadE gene appears to be functional in Y. pseudotuberculosis but is a pseudogene in modern Y. pestis strains. Expression of functional yadE in Y. pestis KIM6+ weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increases Y. pseudotuberculosis biofilm stability, it may be incompatible with the Hms-ECM production that is essential for Y. pestis biofilm production in fleas. Inactivation of yadE in Y. pestis may be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCE The evolution of Yersinia pestis from its Y. pseudotuberculosis ancestor involved gene acquisition and gene losses, leading to differences in biofilm production. Characterizing the unique biofilm features of both species may provide better understanding of how each adapts to its specific niches. This study identifies a trimeric autotransporter, YadE, that promotes biofilm stability of Y. pseudotuberculosis but which has been inactivated in Y. pestis, perhaps because it is not compatible with the Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression, activates YadE in Y. pseudotuberculosis The ability of Y. pseudotuberculosis to use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

Benchmarking hybrid assembly approaches for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

BACKGROUND: We benchmarked the hybrid assembly approaches of MaSuRCA, SPAdes, and Unicycler for bacterial pathogens using Illumina and Oxford Nanopore sequencing by determining genome completeness and accuracy, antimicrobial resistance (AMR), virulence potential, multilocus sequence typing (MLST), phylogeny, and pan genome. Ten bacterial species (10 strains) were tested for simulated reads of both mediocre- and low-quality, whereas 11 bacterial species (12 strains) were tested for real reads. RESULTS: Unicycler performed the best for achieving contiguous genomes, closely followed by MaSuRCA, while all SPAdes assemblies were incomplete. MaSuRCA was less tolerant of low-quality long reads than SPAdes and Unicycler. The hybrid assemblies of five antimicrobial-resistant strains with simulated reads provided consistent AMR genotypes with the reference genomes. The MaSuRCA assembly of Staphylococcus aureus with real reads contained msr(A) and tet(K), while the reference genome and SPAdes and Unicycler assemblies harbored blaZ. The AMR genotypes of the reference genomes and hybrid assemblies were consistent for the other five antimicrobial-resistant strains with real reads. The numbers of virulence genes in all hybrid assemblies were similar to those of the reference genomes, irrespective of simulated or real reads. Only one exception existed that the reference genome and hybrid assemblies of Pseudomonas aeruginosa with mediocre-quality long reads carried 241 virulence genes, whereas 184 virulence genes were identified in the hybrid assemblies of low-quality long reads. The MaSuRCA assemblies of Escherichia coli O157:H7 and Salmonella Typhimurium with mediocre-quality long reads contained 126 and 118 virulence genes, respectively, while 110 and 107 virulence genes were detected in their MaSuRCA assemblies of low-quality long reads, respectively. All approaches performed well in our MLST and phylogenetic analyses. The pan genomes of the hybrid assemblies of S. Typhimurium with mediocre-quality long reads were similar to that of the reference genome, while SPAdes and Unicycler were more tolerant of low-quality long reads than MaSuRCA for the pan-genome analysis. All approaches functioned well in the pan-genome analysis of Campylobacter jejuni with real reads. CONCLUSIONS: Our research demonstrates the hybrid assembly pipeline of Unicycler as a superior approach for genomic analyses of bacterial pathogens using Illumina and Oxford Nanopore sequencing.

Staphylococcus aureus metal acquisition in the mastitic mammary gland.

Mastitis, resulting from mammary gland infection, is a common and painful disease associated with lactation. In addition to the impact on human and animal health, mastitis causes substantial economic losses in the dairy industry. Staphylococcus aureus is a frequent cause of mastitis worldwide. Despite significant progress in understanding S. aureus pathogenesis in general, much remains to be learned regarding virulence factors relevant in the context of mastitis. This review outlines the molecular mechanisms by which S. aureus acquires essential metals such as iron, zinc, manganese, copper, cobalt and nickel within lactating mammary glands, while exposing areas where our current knowledge is deficient. Increased understanding of how these factors facilitate bacterial survival in the lactating mammary gland can provide therapeutic targets for more effective mastitis prevention and treatment.

Benchmarking Long-Read Assemblers for Genomic Analyses of Bacterial Pathogens Using Oxford Nanopore Sequencing.

Oxford Nanopore sequencing can be used to achieve complete bacterial genomes. However, the error rates of Oxford Nanopore long reads are greater compared to Illumina short reads. Long-read assemblers using a variety of assembly algorithms have been developed to overcome this deficiency, which have not been benchmarked for genomic analyses of bacterial pathogens using Oxford Nanopore long reads. In this study, long-read assemblers, namely Canu, Flye, Miniasm/Racon, Raven, Redbean, and Shasta, were thus benchmarked using Oxford Nanopore long reads of bacterial pathogens. Ten species were tested for mediocre- and low-quality simulated reads, and 10 species were tested for real reads. Raven was the most robust assembler, obtaining complete and accurate genomes. All Miniasm/Racon and Raven assemblies of mediocre-quality reads provided accurate antimicrobial resistance (AMR) profiles, while the Raven assembly of Klebsiella variicola with low-quality reads was the only assembly with an accurate AMR profile among all assemblers and species. All assemblers functioned well for predicting virulence genes using mediocre-quality and real reads, whereas only the Raven assemblies of low-quality reads had accurate numbers of virulence genes. Regarding multilocus sequence typing (MLST), Miniasm/Racon was the most effective assembler for mediocre-quality reads, while only the Raven assemblies of Escherichia coli O157:H7 and K. variicola with low-quality reads showed positive MLST results. Miniasm/Racon and Raven were the best performers for MLST using real reads. The Miniasm/Racon and Raven assemblies showed accurate phylogenetic inference. For the pan-genome analyses, Raven was the strongest assembler for simulated reads, whereas Miniasm/Racon and Raven performed the best for real reads. Overall, the most robust and accurate assembler was Raven, closely followed by Miniasm/Racon.

Alignment-free genome comparison enables accurate geographic sourcing of white oak DNA.

BACKGROUND: The application of genomic data and bioinformatics for the identification of restricted or illegally-sourced natural products is urgently needed. The taxonomic identity and geographic provenance of raw and processed materials have implications in sustainable-use commercial practices, and relevance to the enforcement of laws that regulate or restrict illegally harvested materials, such as timber. Improvements in genomics make it possible to capture and sequence partial-to-complete genomes from challenging tissues, such as wood and wood products. RESULTS: In this paper, we report the success of an alignment-free genome comparison method, [Formula: see text] that differentiates different geographic sources of white oak (Quercus) species with a high level of accuracy with very small amount of genomic data. The method is robust to sequencing errors, different sequencing laboratories and sequencing platforms. CONCLUSIONS: This method offers an approach based on genome-scale data, rather than panels of pre-selected markers for specific taxa. The method provides a generalizable platform for the identification and sourcing of materials using a unified next generation sequencing and analysis framework.

Genome-Wide Identification of Fitness Factors in Mastitis-Associated Escherichia coli.

Virulence factors of mammary pathogenic Escherichia coli (MPEC) have not been identified, and it is not known how bacterial gene content influences the severity of mastitis. Here, we report a genome-wide identification of genes that contribute to fitness of MPEC under conditions relevant to the natural history of the disease. A highly virulent clinical isolate (M12) was identified that killed Galleria mellonella at low infectious doses and that replicated to high numbers in mouse mammary glands and spread to spleens. Genome sequencing was combined with transposon insertion site sequencing to identify MPEC genes that contribute to growth in unpasteurized whole milk, as well as during G. mellonella and mouse mastitis infections. These analyses show that strain M12 possesses a unique genomic island encoding a group III polysaccharide capsule that greatly enhances virulence in G. mellonella Several genes appear critical for MPEC survival in both G. mellonella and in mice, including those for nutrient-scavenging systems and resistance to cellular stress. Insertions in the ferric dicitrate receptor gene fecA caused significant fitness defects under all conditions (in milk, G. mellonella, and mice). This gene was highly expressed during growth in milk. Targeted deletion of fecA from strain M12 caused attenuation in G. mellonella larvae and reduced growth in unpasteurized cow's milk and lactating mouse mammary glands. Our results confirm that iron scavenging by the ferric dicitrate receptor, which is strongly associated with MPEC strains, is required for MPEC growth and may influence disease severity in mastitis infections.IMPORTANCE Mastitis caused by E. coli inflicts substantial burdens on the health and productivity of dairy animals. Strains causing mastitis may express genes that distinguish them from other E. coli strains and promote infection of mammary glands, but these have not been identified. Using a highly virulent strain, we employed genome-wide mutagenesis and sequencing to discover genes that contribute to mastitis. This extensive data set represents a screen for mastitis-associated E. coli fitness factors and provides the following contributions to the field: (i) global comparison of genes required for different aspects of mastitis infection, (ii) discovery of a unique capsule that contributes to virulence, and (iii) conclusive evidence for the crucial role of iron-scavenging systems in mastitis, particularly the ferric dicitrate transport system. Similar approaches applied to other mastitis-associated strains will uncover conserved targets for prevention or treatment and provide a better understanding of their relationship to other E. coli pathogens.

DNA metabarcoding illuminates dietary niche partitioning by African large herbivores.

Niche partitioning facilitates species coexistence in a world of limited resources, thereby enriching biodiversity. For decades, biologists have sought to understand how diverse assemblages of large mammalian herbivores (LMH) partition food resources. Several complementary mechanisms have been identified, including differential consumption of grasses versus nongrasses and spatiotemporal stratification in use of different parts of the same plant. However, the extent to which LMH partition food-plant species is largely unknown because comprehensive species-level identification is prohibitively difficult with traditional methods. We used DNA metabarcoding to quantify diet breadth, composition, and overlap for seven abundant LMH species (six wild, one domestic) in semiarid African savanna. These species ranged from almost-exclusive grazers to almost-exclusive browsers: Grass consumption inferred from mean sequence relative read abundance (RRA) ranged from >99% (plains zebra) to <1% (dik-dik). Grass RRA was highly correlated with isotopic estimates of % grass consumption, indicating that RRA conveys reliable quantitative information about consumption. Dietary overlap was greatest between species that were similar in body size and proportional grass consumption. Nonetheless, diet composition differed between all species-even pairs of grazers matched in size, digestive physiology, and location-and dietary similarity was sometimes greater across grazing and browsing guilds than within them. Such taxonomically fine-grained diet partitioning suggests that coarse trophic categorizations may generate misleading conclusions about competition and coexistence in LMH assemblages, and that LMH diversity may be more tightly linked to plant diversity than is currently recognized.

The relationship between tree biodiversity and biomass dynamics changes with tropical forest succession.

Theory predicts shifts in the magnitude and direction of biodiversity effects on ecosystem function (BEF) over succession, but this theory remains largely untested. We studied the relationship between aboveground tree biomass dynamics (Δbiomass) and multiple dimensions of biodiversity over 8-16 years in eight successional rainforests. We tested whether successional changes in diversity-Δbiomass correlations reflect predictions of niche theories. Diversity-Δbiomass correlations were positive early but weak later in succession, suggesting saturation of niche space with increasing diversity. Early in succession, phylogenetic diversity and functional diversity in two leaf traits exhibited the strongest positive correlations with Δbiomass, indicating complementarity or positive selection effects. In mid-successional stands, high biodiversity was associated with greater mortality-driven biomass loss, i.e. negative selection effects, suggesting successional niche trade-offs and loss of fast-growing pioneer species. Our results demonstrate that BEF relationships are dynamic across succession, thus successional context is essential to understanding BEF in a given system.

Wood nitrogen concentrations in tropical trees: phylogenetic patterns and ecological correlates.

In tropical and temperate trees, wood chemical traits are hypothesized to covary with species' life-history strategy along a 'wood economics spectrum' (WES), but evidence supporting these expected patterns remains scarce. Due to its role in nutrient storage, we hypothesize that wood nitrogen (N) concentration will covary along the WES, being higher in slow-growing species with high wood density (WD), and lower in fast-growing species with low WD. In order to test this hypothesis we quantified wood N concentrations in 59 Panamanian hardwood species, and used this dataset to examine ecological correlates and phylogenetic patterns of wood N. Wood N varied > 14-fold among species between 0.04 and 0.59%; closely related species were more similar in wood N than expected by chance. Wood N was positively correlated with WD, and negatively correlated with log-transformed relative growth rates, although these relationships were relatively weak. We found evidence for co-evolution between wood N and both WD and log-transformed mortality rates. Our study provides evidence that wood N covaries with tree life-history parameters, and that these patterns consistently co-evolve in tropical hardwoods. These results provide some support for the hypothesized WES, and suggest that wood is an increasingly important N pool through tropical forest succession.

PhyloPlus: a Universal Tool for Phylogenetic Interrogation of Metagenomic Communities.

Phylogeny is a powerful tool that can be incorporated into quantitative descriptions of community diversity, yet its use has been limited largely due to the difficulty in constructing phylogenies which incorporate the wide genomic diversity of microbial communities. Here, we describe the development of a web portal, PhyloPlus, which enables users to generate customized phylogenies that may be applied to any bacterial or archaeal communities. We demonstrate the power of phylogeny by comparing metrics that employ phylogeny with those that do not when applied to data sets from two metagenomic studies (fermented food, n = 58; human microbiome, n = 60). This example shows how inclusion of all bacterial species identified by taxonomic classifiers (Kraken2 and Kaiju) made the phylogeny perfectly congruent to the corresponding classification outputs. Our phylogeny-based approach also enabled the construction of more constrained null models which (i) shed light into community structure and (ii) minimize potential inflation of type I errors. Construction of such null models allowed for the observation of under-dispersion in 44 (75.86%) food samples, with the metacommunity defined as bacteria that were found in different food matrices. We also observed that closely related species with high abundance and uneven distribution across different sites could potentially exaggerate the dissimilarity between phylogenetically similar communities if they were measured using traditional species-based metrics (Padj. = 0.003), whereas this effect was mitigated by incorporating phylogeny (Padj. = 1). In summary, our tool can provide additional insights into microbial communities of interest and facilitate the use of phylogeny-based approaches in metagenomic analyses. IMPORTANCE There has been an explosion of interest in how microbial diversity affects human health, food safety, and environmental functions among many other processes. Accurately measuring the diversity and structure of those communities is central to understanding their effects. Here, we describe the development of a freely available online tool, PhyloPlus, which allows users to generate custom phylogenies that may be applied to any data set, thereby removing a major obstacle to the application of phylogeny to metagenomic data analysis. We demonstrate that the genetic relatedness of the organisms within those communities is a critical feature of their overall diversity, and that using a phylogeny which captures and quantifies this diversity allows for much more accurate descriptions while preventing misleading conclusions based on estimates that ignore evolutionary relationships.

The contribution of rare species to community phylogenetic diversity across a global network of forest plots.

Niche differentiation has been proposed as an explanation for rarity in species assemblages. To test this hypothesis requires quantifying the ecological similarity of species. This similarity can potentially be estimated by using phylogenetic relatedness. In this study, we predicted that if niche differentiation does explain the co-occurrence of rare and common species, then rare species should contribute greatly to the overall community phylogenetic diversity (PD), abundance will have phylogenetic signal, and common and rare species will be phylogenetically dissimilar. We tested these predictions by developing a novel method that integrates species rank abundance distributions with phylogenetic trees and trend analyses, to examine the relative contribution of individual species to the overall community PD. We then supplement this approach with analyses of phylogenetic signal in abundances and measures of phylogenetic similarity within and between rare and common species groups. We applied this analytical approach to 15 long-term temperate and tropical forest dynamics plots from around the world. We show that the niche differentiation hypothesis is supported in six of the nine gap-dominated forests but is rejected in the six disturbance-dominated and three gap-dominated forests. We also show that the three metrics utilized in this study each provide unique but corroborating information regarding the phylogenetic distribution of rarity in communities.

Temporal turnover in the composition of tropical tree communities: functional determinism and phylogenetic stochasticity.

The degree to which turnover in biological communities is structured by deterministic or stochastic factors and the identities of influential deterministic factors are fundamental, yet unresolved, questions in ecology. Answers to these questions are particularly important for projecting the fate of forests with diverse disturbance histories worldwide. To uncover the processes governing turnover we use species-level molecular phylogenies and functional trait data sets for two long-term tropical forest plots with contrasting disturbance histories: one forest is older-growth, and one was recently disturbed. Having both phylogenetic and functional information further allows us to parse out the deterministic influences of different ecological filters. With the use of null models we find that compositional turnover was random with respect to phylogeny on average, but highly nonrandom with respect to measured functional traits. Furthermore, as predicted by a deterministic assembly process, the older-growth and disturbed forests were characterized by less than and greater than expected functional turnover, respectively. These results suggest that the abiotic environment, which changes due to succession in the disturbed forest, strongly governs the temporal dynamics of disturbed and undisturbed tropical forests. Predicting future changes in the composition of disturbed and undisturbed forests may therefore be tractable when using a functional-trait-based approach.

Development and characterization of microsatellite loci for two Caribbean Heliconia (Heliconiaceae: H. bihai and H. caribaea).

PREMISE OF THE STUDY: Microsatellite loci were developed to characterize genetic variation and population subdivision in Heliconia bihai and H. caribaea from the Caribbean Islands. METHODS AND RESULTS: A total of 13 new microsatellite markers were developed and characterized in the two Caribbean heliconias. Di-, tri-, and tetranucleotide repeats were identified with one to 17 alleles per locus, and the observed heterozygosity ranged from 0.13 to 0.87. Additionally, cross-species amplification was successful in eight out of 13 loci. CONCLUSIONS: The microsatellite loci developed have discriminatory potential to be used in genetic characterizations of Caribbean Heliconia. Both H. bihai and H. caribaea are known to have adaptive interactions with their hummingbird pollinators, and the characterized microsatellite markers will be used to study mating system, genetic structure, and phylogeographic patterns in Caribbean Heliconia.

Gene expression analysis of Xenopsylla cheopis (Siphonaptera: Pulicidae) suggests a role for reactive oxygen species in response to Yersinia pestis infection.

Fleas are vectors for a number of pathogens including Yersinia pestis, yet factors that govern interactions between fleas and Y. pestis are not well understood. Examining gene expression changes in infected fleas could reveal pathways that affect Y. pestis survival in fleas and subsequent transmission. We used suppression subtractive hybridization to identify genes that are induced in Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae) in response to oral or hemocoel infection with Y. pestis. Overall, the transcriptional changes we detected were very limited. We identified several genes that are likely involved in the production or removal of reactive oxygen species (ROS). Midgut ROS levels were higher in infected fleas and antioxidant treatment before infection reduced ROS levels and resulted in higher bacterial loads. An ROS-sensitive mutant strain of Y. pestis lacking the OxyR transcriptional regulator showed reduced growth early after infection. Our results indicate that ROS may limit Y. pestis early colonization of fleas and that bacterial strategies to overcome ROS may enhance transmission.

Plant DNA barcodes and a community phylogeny of a tropical forest dynamics plot in Panama.

The assembly of DNA barcode libraries is particularly relevant within species-rich natural communities for which accurate species identifications will enable detailed ecological forensic studies. In addition, well-resolved molecular phylogenies derived from these DNA barcode sequences have the potential to improve investigations of the mechanisms underlying community assembly and functional trait evolution. To date, no studies have effectively applied DNA barcodes sensu strictu in this manner. In this report, we demonstrate that a three-locus DNA barcode when applied to 296 species of woody trees, shrubs, and palms found within the 50-ha Forest Dynamics Plot on Barro Colorado Island (BCI), Panama, resulted in >98% correct identifications. These DNA barcode sequences are also used to reconstruct a robust community phylogeny employing a supermatrix method for 281 of the 296 plant species in the plot. The three-locus barcode data were sufficient to reliably reconstruct evolutionary relationships among the plant taxa in the plot that are congruent with the broadly accepted phylogeny of flowering plants (APG II). Earlier work on the phylogenetic structure of the BCI forest dynamics plot employing less resolved phylogenies reveals significant differences in evolutionary and ecological inferences compared with our data and suggests that unresolved community phylogenies may have increased type I and type II errors. These results illustrate how highly resolved phylogenies based on DNA barcode sequence data will enhance research focused on the interface between community ecology and evolution.