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Madagascar reports more human plague (causative agent: Yersinia pestis) cases annually than any other country (1), often several hundred each season (typically September-March) (2). Y. pestis persists in multiple rural [...]

Burkholderia thailandensis, a gram-negative bacterium found in the environment, poses a public health threat both because of its ability to cause infections as an opportunistic pathogen and potential misidentification as [...]

Francisella tularensis, a Tier 1 select agent (7), has 3 subspecies: tularensis (type A), holarctica (type B), and mediasiatica (Appendix 1 Figure, https://wwwnc. cdc.gov/EID/article/25/5/18-0363-App1.pdf). In humans, disease is caused by [...]

Tularemia is a disease caused primarily by 2 subspecies of Francisella tularensis: F. tularensis subsp. tularensis, which is restricted to North America; and F. tularensis subsp. holarctica, which is found [...]

Leptospirosis, caused by spirochetes of the genus Leptospira, is a neglected and potentially fatal disease that burdens impoverished communities of developing nations in tropical regions (1-4). The bacteria cause 1.7 [...]

Tularemia is a disease caused by distinct subspecies and phylogenetic groups within the bacterial species Francisella tularensis (1,2). These groups exhibit distinct phylogeographic patterns; F. tularensis subsp. tularensis (type A) [...]

Tularemia, caused by the bacterium Francisella tularensis, is a potentially severe disease that often causes unspecific symptoms; because of its low infectious dose and ease of dissemination, F. tularensis is [...]

Bacillus anthracis is a gram-positive endosporeforming bacterium that causes the disease anthrax in livestock, wildlife, and humans. Because of its hardy spores, B. anthracis can survive for extended periods in [...]

Fraancisella tularensis is the etiologic agent of tularemia and a highly virulent category A biothreat agent (1,2). The most widely distributed subspecies is F. tularensis subsp. holarctica, which is found [...]

Genotyping of microorganisms typically relies on comparison of genomic features (e.g., fragment size, repeats, single-nucleotide polymorphisms [SNPs]) between strains and/or against a database of feature profiles (e.g., PulseNET and mlst.net) [...]

Glanders is an equine disease that was recognized by Hippocrates and Aristotle (1). It is caused by the bacterium Burkholderia mallei, an obligate pathogen of horses, donkeys, and mules (Equidae), [...]

Brucellae are worldwide bacterial pathogens of livestock and wildlife, but phylogenetic reconstructions have been challenging due to limited genetic diversity. We assessed the taxonomic and evolutionary relationships of five Brucella species--Brucella abortus, B. melitensis, B. suis, B. canis, and B. ovis--using whole-genome comparisons. We developed a phylogeny using single nucleotide polymorphisms (SNPs) from 13 genomes and rooted the tree using the closely related soil bacterium and opportunistic human pathogen, Ochrobactrum anthropi. Whole-genome sequencing and a SNP-based approach provided the requisite level of genetic detail to resolve species in the highly conserved brucellae. Comparisons among the Brucella genomes revealed 20,154 orthologous SNPs that were shared in all genomes. Rooting with Ochrobactrum anthropi reveals that the B. ovis lineage is basal to the rest of the Brucella lineage. We found that B. suis is a highly divergent clade with extensive intraspecific genetic diversity. Furthermore, B. suis was determined to be paraphyletic in our analyses, only forming a monophyletic clade when the B. canis genome was included. Using a molecular clock with these data suggests that most Brucella species diverged from their common B. ovis ancestor in the past 86,000 to 296,000 years, which precedes the domestication of their livestock hosts. Detailed knowledge of the Brucella phylogeny will lead to an improved understanding of the ecology, evolutionary history, and host relationships for this genus and can be used for determining appropriate genotyping approaches for rapid detection and diagnostic assays for molecular epidemiological and clinical studies.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

Endemic melioidosis is caused by genetically diverse Burkholderia pseudomallei strains. However, clonal outbreaks (multiple cases caused by 1 strain) have occurred, such as from contaminated potable water. B. pseudomallei is [...]

A small number of conserved canonical single nucleotide polymorphisms (canSNP) that define major phylogenetic branches for Bacillus anthracis were used to place a Sverdlovsk patient's B. anthracis genotype into 1 [...]

Burkholderia pseudomallei is the etiologic agent of melioidosis. Many disease manifestations are associated with melioidosis, and the mechanisms causing this variation are unknown; genomic differences among strains offer one explanation. We compared the genome sequences of two strains of B. pseudomallei: the original reference strain K96243 from Thailand and strain MSHR305 from Australia. We identified a variable homologous region between the two strains. This region was previously identified in comparisons of the genome of B. pseudomallei strain K96243 with the genome of strain E264 from the closely related B. thailandensis. In that comparison, K96243 was shown to possess a horizontally acquired Yersinia-like fimbrial (YLF) gene cluster. Here, we show that the homologous genomic region in B. pseudomallei strain 305 is similar to that previously identified in B. thailandensis strain E264. We have named this region in B. pseudomallei strain 305 the B. thailandensis-like flagellum and chemotaxis (BTFC) gene cluster. We screened for these different genomic components across additional genome sequences and 571 B. pseudomallei DNA extracts obtained from regions of endemicity. These alternate genomic states define two distinct groups within B. pseudomallei: all strains contained either the BTFC gene cluster (group BTFC) or the YLF gene cluster (group YLF). These two groups have distinct geographic distributions: group BTFC is dominant in Australia, and group YLF is dominant in Thailand and elsewhere. In addition, clinical isolates are more likely to belong to group YLF, whereas environmental isolates are more likely to belong to group BTFC. These groups should be further characterized in an animal model.

Burkholderia pseudomallei soil isolates from northeast Thailand are genotyped using multiple-locus variable number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST). MLVA identified 19 genotypes within three clades, while MLST revealed two genotypes.

Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.

Although polygyny can potentially increase male reproductive success, the benefits of this strategy could be offset by losses to extra-pair paternity or reduced offspring survival. We developed microsatellite markers to assess the influence of extra-pair offspring (EPO) on reproductive success and paternity in monogamous and polygynous pairs of the facultatively polygynous Southwestern Willow Flycatcher (Empidonax traillii extimus). Based on genotypes of 140 offspring from 56 clutches over six years, 14% of nestlings in our study population were extra-pair offspring, with 23% of all successful nests containing at least one EPO. We found that polygynous males produced 2.11 [+ or -] 0.35 offspring per season, compared to 1.15 [+ or -] 0.18 for monogamous males. This increased reproductive success was due primarily to the increased number of nests of polygynous males, as the number of offspring per pair did not differ between monogamous and polygynous males. Twenty of the 140 genotyped nestlings were extrapair offspring. Sires could be assigned to 16 of these; one polygynous male sired two EPO in one nest, two monogamous males sired eight EPO in two nests, and four nonterritorial males sired six EPO in four nests. Overall, these results indicate that in this population, females of polygynous males did not raise a disproportionate number of EPO as a result of the polygynous mating strategy of their mate, and that both territorial and nonterritorial males sired EPO. Key words: extra-pair offspring, microsatellite, polygyny, reproductive success, Willow Flycatcher. A pesar de que la poliginia puede potencialmente aumentar el exito reproductivo de los machos, el beneficio de esta estrategia puede ser contrarestado por perdidas ocasionadas por la paternidad extra pareja o pot la reduccion de la supervivencia de la progenie. Desarrollamos marcadores microsatelitales para determinar la influencia de la progenie extra pareja (PEP) sobre el exito reproductivo y la paternidad en parejas monogamas y poliginas de la especie facultativamente poligina Empidonax traillii extimus. Con base en genotipos de 140 crias provenientes de 56 nidadas en seis anos, determinamos que el 14% de los polluelos en nuestra poblacion de estudio fueron PEP y que el 23% de todos los nidos exitosos contuvo por lo menos una PEP. Encontramos que los machos poliginos produjeron 2.11 [+ or -] 0.35 crias por estacion, en comparacion con 1.15 [+ or -] 0.18 crias producidas por machos monogamos. Este exito reproductivo mayor se debio principalmente al mayor numero de nidos de los machos poliginos, ya que el numero de crias por pareja no difirio entre machos monogamos y poliginos. De los 140 polluelos analizados genotipicamente, 20 fueron PEP. Los padres progenitores pudieron ser identificados para 16 de estos polluelos. Un macho poligino engendro dos PEP en un nido, dos machos monogamos engendraron ocho PEP en dos nidos y cuatro machos no territoriales engendraron seis PEP en cuatro nidos. En general, estos resultados indican que en esta poblacion las hembras de machos poliginos no criaron un numero desproporcionado de PEP como resultado de la estrategia de apareamiento poligina de sus parejas y que tanto los machos territoriales como los no territoriales engendraron PEP.

Variable-number tandem repeat (VNTR) loci have shown a remarkable ability to discriminate among isolates of the recently emerged clonal pathogen Escherichia coli O157:H7, making them a very useful molecular epidemiological tool. However, little is known about the rates at which these sequences mutate, the factors that affect mutation rates, or the mechanisms by which mutations occur at these loci. Here, we measure mutation rates for 28 VNTR loci and investigate the effects of repeat copy number and mismatch repair on mutation rate using in vitro-generated populations for 10 E. coli O157:H7 strains. We find single-locus rates as high as 7.0 x [10.sup.-4] mutations/generation and a combined 28-locus rate of 6.4 x [10.sup.-4] mutations/generation. We observed single-and multirepeat mutations that were consistent with a slipped-strand mispairing mutation model, as well as a smaller number of large repeat copy number mutations that were consistent with recombination-mediated events. Repeat copy number within an array was strongly correlated with mutation rate both at the most mutable locus, O157-10 ([r.sub.2] = 0.565, P = 0.0196), and across all mutating loci. The combined locus model was significant whether locus O157-10 was included ([r.sup.2] = 0.833, P < 0.0001) or excluded ([r.sup.2] = 0.452, P < 0.0001) from the analysis. Deficient mismatch repair did not affect mutation rate at any of the 28 VNTRs with repeat unit sizes of >5 bp, although a poly(G) homomeric tract was destabilized in the mutS strain. Finally, we describe a general model for VNTR mutations that encompasses insertions and deletions, single-and multiple-repeat mutations, and their relative frequencies based upon our empirical mutation rate data.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp, konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27 and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.

Although it is understood that the composition of riparian trees can affect stream function through leaf litter fall, the potential effects of genetic variation within species are less understood. Using a naturally hybridizing cottonwood system, we examined the hypothesis that genetic differences among two parental species (PopuIus fremontii and P. angustifolia) and two groups of their hybrids ([F.sub.1] and backcrosses to P. angustifolia) would affect litter decomposition rates and the composition of the aquatic invertebrate community that colonizes leaves. Three major findings emerged: (1) parental and hybrid types differ in litter quality, (2) decomposition differs between two groups, a fast group (P. fremontii and [F.sub.1] hybrid), and a slow group (P. angustifolia and backcross hybrids), and (3) aquatic invertebrate communities colonizing P. fremontii litter differed significantly in composition from all other cross types, even though P. fremontii and the [F.sub.1] hybrid decomposed at similar rates. These findings are in agreement with terrestrial arthropod studies in the same cottonwood system. However, the effects are less pronounced aquatically than those observed in the adjacent terrestrial community, which supports a genetic diffusion hypothesis. Importantly, these findings argue that genetic interactions link terrestrial and aquatic communities and may have significant evolutionary and conservation implications. Key words: aquatic decomposition; aquatic-terrestrial interaction; cottonwood hybridization; ecological genetics; genetic introgression; genetic variation; hybrids; leaf litter decomposition; macroinvertebrate communities; Populus.

The causative agent of tularemia, Francisella tularensis, is a formidable biologic agent that occurs naturally throughout North America. We examined genetic and spatial diversity patterns among 161 US F. tularensis [...]

The association of historical plague pandemics with Yersinia pestis remains controversial, partly because the evolutionary history of this largely monomorphic bacterium was unknown. The microevolution of Y. pestis was therefore investigated by three different multilocus molecular methods, targeting genomewide synonymous SNPs, variation in number of tandem repeats, and insertion of IS100 insertion elements. Eight populations were recognized by the three methods, and we propose an evolutionary tree for these populations, rooted on Yersinia pseudotuberculosis. The tree invokes microevolution over millennia, during which enzootic pestoides isolates evolved. This initial phase was followed by a binary split 6,500 years ago, which led to populations that are more frequently associated with human disease. These populations do not correspond directly to classical biovars that are based on phenotypic properties. Thus, we recommend that henceforth groupings should be based on molecular signatures. The age of Y. pestis inferred here is compatible with the dates of historical pandemic plague. However, it is premature to infer an association between any modern molecular grouping and a particular pandemic wave that occurred before the 20th century. insertion element | SNP | variable number tandem repeats | pandemic | molecular clock

Phylogenetic reconstruction using molecular data is often subject to homoplasy, leading to inaccurate conclusions about phylogenetic relationships among operational taxonomic units. Compared with other molecular markers, single-nucleotide polymorphisms (SNPs) exhibit extremely low mutation rates, making them rare in recently emerged pathogens, but they are less prone to homoplasy and thus extremely valuable for phylogenetic analyses. Despite their phylogenetic potential, ascertainment bias occurs when SNP characters are discovered through biased taxonomic sampling; by using whole-genome comparisons of five diverse strains of Bacillus anthracis to facilitate SNP discovery, we show that only polymorphisms lying along the evolutionary pathway between reference strains will be observed. We illustrate this in theoretical and simulated data sets in which complex phylogenetic topologies are reduced to linear evolutionary models. Using a set of 990 SNP markers, we also show how divergent branches in our topologies collapse to single points but provide accurate information on internodal distances and points of origin for ancestral clades. These data allowed us to determine the ancestral root of B. anthracis, showing that it lies closer to a newly described 'C' branch than to either of two previously described 'A' or 'B' branches. In addition, subclade rooting of the C branch revealed unequal evolutionary rates that seem to be correlated with ecological parameters and strain attributes. Our use of nonhomoplastic whole-genome SNP characters allows branch points and clade membership to be estimated with great precision, providing greater insight into epidemiological, ecological, and forensic questions.

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp, tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp, tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp, holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU $4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.

Three selection experiments were used to identify chromosome regions that contain QTL affecting late-life and early-life fitness in Drosophila melanogaster. The selection experiments were initiated by crossing pairs of inbred lines that had been derived from outbred laboratory populations that had different mean life spans. QTL regions were located by association with microsatellite markers that showed significant selection responses. Regions between recombination map positions 54 and 81 on chromosome 2, between 0 and 30 on chromosome 3, and near locations 49 and 81 on chromosome 3 had the strongest support as locations of life-span QTL. There was good general agreement between the life-span QTL regions that were identified by selection and those that were identified in a companion recombination mapping experiment that used the same fly stocks. Many marker loci responded in opposite directions to selection for late- and early-life fitness, indicating negative genetic correlations or trade-offs between those traits. Indirect evidence suggested that some negative genetic correlations were due to antagonistic pleiotropy.

Composite interval mapping was used to identify life-span QTL in [F.sub.2] progeny of three crosses between different pairs of inbred lines. Each inbred line was derived from a different outbred population that had undergone long-term selection for either long or short life span. Microsatellite loci were used as genetic markers, and confidence intervals for QTL location were estimated by bootstrapping. A minimum of 10 QTL were detected, nine of which were located on the two major autosomes. Five QTL were present in at least two crosses and five were present in both sexes. Observation of the same QTL in more than one cross was consistent with the hypothesis that genetic variation for life span is maintained by balancing selection. For all QTL except one, allelic effects were in the direction predicted on the basis of outbred source population. Alleles that conferred longer life were always at least partially dominant.

Plague, the disease caused by the bacterium Yersinia pestis, has greatly impacted human civilization. Y. pestis is a successful global pathogen, with active foci on all continents except Australia and Antarctica. Because the Y. pestis genome is highly monomorphic, previous attempts to characterize the population genetic structure within a single focus have been largely unsuccessful. Here we report that highly mutable marker loci allow determination of Y. pestis population genetic structure and tracking of transmission patterns at two spatial scales within a single focus. In addition, we found that in vitro mutation rates for these loci are similar to those observed in vivo, which allowed us to develop a mutation-rate-based model to examine transmission mechanisms. Our model suggests there are two primary components of plague ecology: a rapid expansion phase for population growth and dispersal followed by a slower persistence phase. This pattern seems consistent across local, regional, and even global scales.

Bacillus thuringiensis, Bacillus cereus and Bacillus anthracis are three closely related species of the B.cereus group with each of them being economically important and their DNA isolates were analyzed using fluorescent amplified fragment length polymorphism (AFLP). Phytogenetic analysis of AFLP data showed diversity with B.thuringinesis and B.cereus compared to the monomorphic nature of B.anthracis.

In July 1993, a liquid suspension of Bacillus anthracis was aerosolized from the roof of an eight-story building in Kameido, Tokyo, Japan, by the religious group Aum Shinrikyo. During 1999 [...]

To the Editor: Tularemia is a zoonosis caused by the bacterium Francisella tularensis; the main forms of disease that occur in humans are ulceroglandular/glandular, oculoglandular, oropharyngeal, and respiratory. In Turkey, [...]

Using molecular-genetic techniques, we determined the gender of 202 Southwestern Willow Flycatcher (Empidonax traillii extimus) nestlings from 95 nests sampled over a five-year period. Overall nestling sex ratio did not vary significantly from 50:50 among years, by clutch order, or by mating strategy (monogamous vs. polygamous pairings). However, we did observe significant differences among the four sites sampled, with sex ratios biased either toward males or females at the different sites. Given the small population sizes and geographic isolation of many of the endangered subspecies' breeding populations, sex-ratio differences may have localized negative impacts. Key words: Empidonax traillii, endangered species, nestling sex ratio, Willow Flycatcher.

Comparison of the whole-genome sequence of Bacillus anthracis isolated from a victim of a recent bioterrorist anthrax attack with a reference reveals 60 new markers that include single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats. Genome comparison detected four high-quality SNPs between the two sequenced B. anthracis chromosomes and seven differences among different preparations of the reference genome. These markers have been tested on a collection of anthrax isolates and were found to divide these samples into distinct families. These results demonstrate that genome-based analysis of microbial pathogens will provide a powerful new tool for investigation of infectious disease outbreaks., On 4 October 2001, the Centers for Disease Control reported a highly unusual case of inhalational anthrax in a photo editor at a West Palm Beach, Florida, media organization (1). [...]

Researchers used fluorescent amplified fragment length polymorphism to analyze 154 Bacillus cereus and Bacillus thuringiensis isolates taken from five different locations and compare them to reference strains. This method can be automated, allowing scientists to process very large samples.

Three separate genetic markers were used to determine whether wild turkeys in Arizona's Huachuca Mountains originate from translocations of Gould's turkeys from the 1980s, or if they resulted from interbreeding with Merriam's turkeys from a 1950 translocation. Findings show that the current turkey population descends from the Gould's turkey; researchers will now watch for warnings of inbreeding depression.

Research demonstrates that introgression between genomes is selective and gene flow filtering between species occurs in hybrid populations as evidenced by genetically mapped restriction fragment length polymorphism of cottonwood species and their hybrids. Data show that natural hybridization has the potential to offer genetic variation that impacts evolutionary processes.

The Southwestern Willow Flycatcher (Empidonax trailtii extimus) is an endangered Neotropical migrant that breeds in isolated remnants of dense riparian habitat in the southwestern United States. We estimated genetic variation at 20 breeding sites of the Southwestern Willow Flycatcher (290 individuals) using 38 amplified fragment length polymorphisms (AFLPs). Our results suggest that considerable genetic diversity exists within the subspecies and within local breeding sites. Statistical analyses of genetic variation revealed only slight, although significant, differentiation among breeding sites (Mantel's r = 0.0705, P < 0.0005; (theta) = 0.0816, 95% CI = 0.0608 to 0.1034; (phi)(sub ST) = 0.0458, P < 0.001). UPGMA cluster analysis of the AFLP markers indicates that extensive gene flow has occurred among breeding sites. No one site stood out as being genetically unique or isolated. Therefore, the small level of genetic structure that we detected may not be biologically significant. Ongoing field studies are consistent with this conclusion. Of the banded birds that were resighted or recaptured in Arizona during the 1996 to 1998 breeding seasons, one-third moved between breeding sites and two-thirds were philopatric. Low differentiation may be the result of historically high rangewide diversity followed by recent geographic isolation of breeding sites, although observational data indicate that gene flow is a current phenomenon. Our data suggest that breeding groups of E. t. extimus act as a metapopulation.

Research has been conducted on the homogeneous bacteria Bacillus anthracis. The discovery of the ca. 750-nucleotide open reading frame containing in-frame variable-number tandem-repeat sequences is described.

The variation in the anthrax toxin in Bacillus anthracis was investigated by sequencing the 2,294-nucleotide protective antigen gene from 26 of the most diverse B. anthracis strains. Five different alleles were found among the strains, including two not seen in the 26-sample survey. One of the two alleles is a new missense mutation that is located adjacent to the highly antigenic region. Results also suggested that plasmid evolution of B. anthracis took place with few or no horizontal transfer between different strains.

We used molecular techniques to examine the ectomycorrhizal fungal community associated with pinyon pine (Pinus edulis) growing in two soil types in a semiarid region of northern Arizona: cinder soils low in nutrients and moisture, and sandy-loam soils with higher moisture and nutrient levels. Pinyon performance (e.g., growth, reproduction, water stress) has been shown to be markedly lower in cinder than in sandy-loam environments. Fungal community composition and richness were determined using RFLP (restriction fragment length polymorphism) analysis of ectomycorrhizal root tips collected from three sites within each soil type. Several patterns emerged from these analyses. First, communities in both cinder and sandy-loam soils were dominated by one or a few abundant ectomycorrhizal types, a species abundance pattern common to many plant and animal communities. Second, unlike the pattern for many other organisms, ectomycorrhizal fungal type (species) richness was not correlated with measures of ecosystem productivity such as soil nutrient and moisture levels; cinder and sandy-loam soils had similar numbers of ectomycorrhizal fungal types (range of 15-19 fungal types for both soil types). Third, soil type and fungal community composition were linked, as cluster analysis demonstrated greater similarity of fungal communities from sites within soil types than between them. Differential amplification using primers with enhanced specificity for basidiomycetes indicated that an average of 85% of the ectomycorrhiza found at the sandy-loam sites were members of the subphylum Basidiomycotina, whereas over half (mean = 52%) of the ectomycorrhiza at the cinder sites were formed by members of other fungal groups, probably the subphylum Ascomycotina. Fourth, a preliminary survey of 14-45 ectomycorrhizal root tips from each of 20 trees at one cinder site indicated that trees were dominated by one or a few ectomycorrhizal RFLP types. However, the same RFLP types did not dominate on all trees, and dominant types showed considerable spatial variation. Fifth, the RFLP patterns of some fungal sporocarps matched those of ectomycorrhizal root tips, but many did not, indicating that many of the ectomycorrhizal fungi at these sites fruit infrequently, whereas other fungi with more abundant sporocarps may not form ectomycorrhiza. This emphasizes the need to characterize the ectomycorrhizal communities formed on the plant roots themselves, rather than characterization based on sporocarps alone, particularly in arid environments. Finally, the differences in ectomycorrhizal fungal communities we observed between soil types supported the concept that conserving fungal diversity requires conservation of host plant species over their entire range, not just typical sites. If future studies corroborate these patterns, our results suggest that abiotically stressful environments are important to include in these conservation efforts. Key words: community composition; conservation; diversity; ectomycorrhizal fungi; environmental stress; RFLP analysis: species richness; sporocarp surveys., INTRODUCTION One of the major types of mycorrhiza, the ectomycorrhiza, involves an estimated 5000 species of fungi in the subphyla Ascomycotina and Basidiomycotina (Kendrick 1992, Molina et al. 1992) and [...]

We compared 1,047 base pairs (bp) of the mitochondrial cytochrome b gene from the three species of extant peccaries - the collared peccary (Pecari tajacu), the white-lipped peccary (Tayassu pecari), and the Chacoan peccary (Catagonus wagneri); three suids - domestic pig (Sus scrofa), warthog (Phacochoerus aethiopicus), and babirusa (Babyrousa babyrussa) - were used as outgroups. Phylogenetic analyses using parsimony and maximum likelihood resulted in trees that placed the Chacoan peccary and the white-lipped peccary in a clade separate from the collared peccary. This relationship remains tentative, however, as tree topologies supporting alternate hypotheses were not significantly worse than the maximum likelihood tree. Estimated times since divergence based on cytochrome b suggest that Tayassu and Catagonus diverged in the late Pliocene, perhaps concommitant with the invasion of South America by tayassuids. The divergence of the Pecari lineage from that of the other two species was estimated to be at 3.4-7.4 x [10.sup.6] years ago, supporting the hypothesis that these two clades diverged in North America before either clade colonized South America. Our results support the classification of extant species of peccaries into three genera. Key words: Catagonus, Dicotyles, Pecari, Tayassu, Tayassuidae, Suidae, cytochrome b, mitochondrial DNA

A bacterial biosensor for benzene, toluene and similar compounds appears to be capable of accurately measuring environmental contaminants, making it a potentially inexpensive tool for field-ready assays. A reporter plasmid was constructed by placing the luc gene for firefyl luciferase directly under the control of the P(sub u) promoter and the xylR transcriptional activator of Pseudomonas putida mt-2. The biosensor cells were shown to detect benzene, toluene and xylene contamination in soil and water samples with high sensitivity and quantitative accuracy.

An outbreak of human anthrax occurred in Sverdlovsk, Union of Soviet Socialists Republic (now Ekaterinburg, Russia) in April 1979. Officials attributed this to consumption of contaminated meat, but Western governments believed it resulted from inhalation of spores accidentally released from a nearby military research facility. Tissue samples from 11 victims were obtained and methods of efficiently extracting high-quality total DNA from these samples were developed. Extracted DNA was analyzed by using PCR to determine whether it contained Bacillus anthracis-specific sequences. Double PCR using 'nested primers' increased sensitivity of the assay significantly. Tissue samples from 11 persons who died during the epidemic were examined. Results demonstrated that the entire complement of B. anthracis toxin and capsular antigen genes required for pathogenicity were present in tissues from each of these victims. Tissue from a vaccination site contained primarily nucleic acids from a live vaccine, although traces of genes from the infecting organisms were also present. PCR analysis using primers that detect the vrrA gene variable region on the B. anthracis chromosome demonstrated that at least four of the five known strain categories defined by this region were present in the tissue samples. Only one category is found in a single B. anthracis strain.

We report the first detailed genetic linkage map of rainbow trout (Oncorhynchus mykiss). The segregation analysis was performed using 76 doubled haploid rainbow trout produced by androgenesis from a hybrid between the 'OSU' and 'Arlee' androgenetically derived homozygous lines. Four hundred and seventy-six markers segregated into 31 major linkage groups and 11 small groups ([less than]5 markers/group). The minimum genome size is estimated to be 2627.5 cM in length. The sex-determining locus segregated to a distal position on one of the linkage groups. We analyzed the chromosomal distribution of three classes of markers: (1) amplified fragment length polymorphisms, (2) variable number of tandem repeats, and (3) markers obtained using probes homologous to the 5[prime] or 3[prime] end of salmonid-specific small interspersed nuclear elements. Many of the first class of markers were clustered in regions that appear to correspond to centromeres. The second class of markers were more telomeric in distribution, and the third class were intermediate. Tetrasomic inheritance, apparently related to the tetraploid ancestry of salmonid fishes, was detected at one simple sequence repeat locus and suggested by the presence of one extremely large linkage group that appeared to consist of two smaller groups linked at their tips. The double haploid rainbow trout lines and linkage map present a foundation for further genomic studies.

The B chromosomes of maize typically undergo nondisjunction during the second microspore division (generative cell division). When the microspore nucleus contains only one B chromosome, two kinds of sperm result, one with two B chromosomes and one with no B chromosomes. The sperm with the B chromosomes preferentially fertilizes the egg cell. Previous studies of these phenomena have been limited to genetic analysis and chromosome spreads. In this study we show that a B chromosome-specific probe can be used with fluorescence in situ hybridization (FISH) analysis to detect the presence, location, and frequency of B chromosomes in intact interphase nuclei within mature pollen of maize. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe.

The use of molecular techniques for the assessment of familial relationships among social species of mammals has become relatively commonplace. However, some species represent poor candidates for such studies due to naturally low levels of genetic diversity, leading to unacceptably large standard errors associated with estimates of relatedness. Here, we report on a preliminary study of genetic diversity within two populations of a social species of ground squirrel, Gunnison's prairie dog (Cynomys gunnisoni) using DNA fingerprinting. We observed low levels of diversity in the form of large mean coefficients of genetic similarity among individuals occupying the same population. Overall similarity, determined from the combined data, yielded by three minisatellite probes, ranged from 55 to 61%. These values place Gunnison's prairie dog at the extreme upper end of the range of similarity values reported for outbred species of mammals (ca. 0.20-0.50). As a partial means of explaining these results, and as a means of comparing our results to those of similar studies using allozymes, we determined the level of differentiation between our two study colonies in the form of an F-statistic analog. A value of 0.11 ([+ or -] 2.26 x [10.sup.-3]) was obtained and is similar to values reported from allozyme studies (0.07-0.12). A significance test of this value yielded a positive result (D = 5.63, d.f. = 1, P < 0.025), demonstrating that gene flow between populations is limited, a factor that may help to maintain low levels of diversity. Key words: Cynomys gunnisoni, genetic diversity, population differentiation, DNA fingerprinting

PCR analysis of 198 Bacillus anthracis isolates indicated a variable region of DNA sequence differing in length among the isolates. Five polymorphisms were characterized by the presence of two to six copies of the 12-bp tandem repeat 5'-CAATATCAACAA-3'. This variable-number tandem repeat region is found within a larger sequence containing one complete open reading frame that encodes a putative 30-kDa protein. Length variation did not alter the reading frame of the encoded protein and only modified the copy number of a 4-amino-acid sequence (QYQQ) from 2 to 6.

Genetic marker systems have improved dramatically in the past 10 yr. Each new system needs to be evaluated for its distribution of markers on genetic linkage maps to validate its use for genetic analysis. The resulting maps an also useful for establishing the genetic positions of genes affecting important phenotypes. We have constructed a high density map in soybean [Glycine max (L.) Merr.] using a 300 RIL (recombinant inbred line) population from BSR101 x PI437.654 by first constructing an RFLP (restriction fragment length polymorphism) 'scaffold' map based on the entire population. The RFLP anchored map was then further populated with AFLP (amplified fragment length polymorphism) markers based on only a 42 RIL subset. We report here an 840 marker map consisting of 165 RFLP, 25 RAPD (random amplified polymorphic DNA), and 650 AFLP markers spread over 29 linkage groups representing 3441 cM distance. Although clustering of AFLP markers occurred, markers were mapped to every linkage group and were well distributed relative to other marker systems. The AFLP marker system appears to be a useful approach for generating high density genetic maps in soybean., GENETIC MAPPING has been greatly enhanced in the last decade with the introduction of novel genetic marker systems. These systems have resulted in increased capabilities for detecting DNA polymorphism and [...]

Amplified fragment length polymorphism (AFLP) markers were utilized to determine the phylogenetic relationship between Bacillus anthracis and related Bacillus species. Polymerase chain reaction analysis of the Bacillus genome with AFLP markers indicated the presence of low levels of molecular variation in Bacillus anthracis due to low rates of evolution that restricted the generation of molecular diversity. Furthermore, genomic analysis indicated the close phylogenetic relationship between Bacillus anthracis and Bacillus cereus.