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Restoration of lost species ranges to their native distribution is key for the survival of endangered species. However, reintroductions often fail and long-term genetic consequences are poorly understood. Alpine ibex ( Capra ibex ) are wild goats that recovered from <100 individuals to ~50,000 within a century by population reintroductions. We analyzed the population genomic consequences of the Alpine ibex reintroduction strategy. We genotyped 101,822 genomewide single nucleotide polymorphism loci in 173 Alpine ibex, the closely related Iberian ibex ( Capra pyrenaica ) and domestic goat ( Capra hircus ). The source population of all Alpine ibex maintained genetic diversity comparable to Iberian ibex, which experienced less severe bottlenecks. All reintroduced Alpine ibex populations had individually and combined lower levels of genetic diversity than the source population. The reintroduction strategy consisted of primary reintroductions from captive breeding and secondary reintroductions from established populations. This stepwise reintroduction strategy left a strong genomic footprint of population differentiation, which increased with subsequent rounds of reintroductions. Furthermore, analyses of genomewide runs of homozygosity showed recent inbreeding primarily in individuals of reintroduced populations. We showed that despite the rapid census recovery, Alpine ibex carry a persistent genomic signature of their reintroduction history. We discuss how genomic monitoring can serve as an early indicator of inbreeding.

Fungal pathogens can rapidly evolve virulence towards resistant crops in agricultural ecosystems. Gains in virulence are often mediated by the mutation or deletion of a gene encoding a protein recognized by the plant immune system. However, the loci and the mechanisms of genome evolution enabling rapid virulence evolution are poorly understood. We performed genome-wide association mapping on a global collection of 106 strains of Zymoseptoria tritici, the most damaging pathogen of wheat in Europe, to identify polymorphisms linked to virulence on two wheat varieties. We found 25 distinct genomic loci associated with reproductive success of the pathogen. However, no locus was shared between the host genotypes, suggesting host specialization. The main locus associated with virulence encoded a highly expressed, small secreted protein. Population genomic analyses showed that the gain in virulence was explained by a segregating gene deletion polymorphism. The deletion was likely adaptive by preventing detection of the encoded protein. Comparative genomics of closely related species showed that the locus emerged de novo since speciation. A large cluster of transposable elements in direct proximity to the locus generated extensive rearrangements leading to multiple independent gene losses. Our study demonstrates that rapid turnover in the chromosomal structure of a pathogen can drive host specialization.

Local adaptation plays a key role in the evolutionary trajectory of host-pathogen interactions. However, the genetic architecture of local adaptation in host-pathogen systems is poorly understood. Fungal plant pathogens in agricultural ecosystems provide highly tractable models to quantify phenotypes and map traits to corresponding genomic loci. The outcome of crop-pathogen interactions is thought to be governed largely by gene-for-gene interactions. However, recent studies showed that virulence can be governed by quantitative trait loci and that many abiotic factors contribute to the outcome of the interaction. After introducing concepts of local adaptation and presenting examples from wild plant pathosystems, we focus this review on a major pathogen of wheat, Zymoseptoria tritici, to show how a multitude of traits can affect local adaptation. Zymoseptoria tritici adapted to different thermal environments across its distribution range, indicating that thermal adaptation may limit effective dispersal to different climates. The application of fungicides led to the rapid evolution of multiple, independent resistant populations. The degree of colony melanization showed strong pleiotropic effects with other traits, including trade-offs with colony growth rates and fungicide sensitivity. The success of the pathogen on its host can be assessed quantitatively by counting pathogen reproductive structures and measuring host damage based on necrotic lesions. Interestingly, these two traits can be weakly correlated and depend both on host and pathogen genotypes. Quantitative trait mapping studies showed that the genetic architecture of locally adapted traits varies from single loci with large effects to many loci with small individual effects. We discuss how local adaptation could hinder or accelerate the development of epidemics in agricultural ecosystems., (© 2016 John Wiley & Sons Ltd.)

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, a major pathogen of wheat globally and the most damaging pathogen of wheat in Europe. A gene-for-gene (GFG) interaction between Z. tritici and wheat cultivars carrying the Stb6 resistance gene has been postulated for many years, but the genes have not been identified. We identified AvrStb6 by combining quantitative trait locus mapping in a cross between two Swiss strains with a genome-wide association study using a natural population of c. 100 strains from France. We functionally validated AvrStb6 using ectopic transformations. AvrStb6 encodes a small, cysteine-rich, secreted protein that produces an avirulence phenotype on wheat cultivars carrying the Stb6 resistance gene. We found 16 nonsynonymous single nucleotide polymorphisms among the tested strains, indicating that AvrStb6 is evolving very rapidly. AvrStb6 is located in a highly polymorphic subtelomeric region and is surrounded by transposable elements, which may facilitate its rapid evolution to overcome Stb6 resistance. AvrStb6 is the first avirulence gene to be functionally validated in Z. tritici, contributing to our understanding of avirulence in apoplastic pathogens and the mechanisms underlying GFG interactions between Z. tritici and wheat., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

A simplistic view of the adaptive process pictures a hillside along which a population can climb: when ecological 'demands' change, evolution 'supplies' the variation needed for the population to climb to a new peak. Evolutionary ecologists point out that this simplistic view can be incomplete because the fitness landscape changes dynamically as the population evolves. Geneticists meanwhile have identified complexities relating to the nature of genetic variation and its architecture, and the importance of epigenetic variation is under debate. In this review, we highlight how complexity in both ecological 'demands' and the evolutionary 'supply' influences organisms' ability to climb fitness landscapes that themselves change dynamically as evolution proceeds, and encourage new synthetic effort across research disciplines towards ecologically realistic studies of adaptation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Zymoseptoria tritici is an ascomycete fungus that causes Septoria tritici blotch, a globally distributed foliar disease on wheat. Z. tritici populations are highly polymorphic and exhibit significant quantitative variation for virulence. Despite its importance, the genes responsible for quantitative virulence in this pathogen remain largely unknown. We investigated the expression profiles of four Z. tritici strains differing in virulence in an experiment conducted under uniform environmental conditions. Transcriptomes were compared at four different infection stages to characterize the regulation of gene families thought to be involved in virulence and to identify new virulence factors. The major components of the fungal infection transcriptome showed consistent expression profiles across strains. However, strain-specific regulation was observed for many genes, including some encoding putative virulence factors. We postulate that strain-specific regulation of virulence factors can determine the outcome of Z. tritici infections. We show that differences in gene expression may be major determinants of virulence variation among Z. tritici strains, adding to the already known contributions to virulence variation based on differences in gene sequence and gene presence/absence polymorphisms.

Epidemics caused by fungal plant pathogens pose a major threat to agro-ecosystems and impact global food security. High-throughput sequencing enabled major advances in understanding how pathogens cause disease on crops. Hundreds of fungal genomes are now available and analyzing these genomes highlighted the key role of effector genes in disease. Effectors are small secreted proteins that enhance infection by manipulating host metabolism. Fungal genomes carry 100s of putative effector genes, but the lack of homology among effector genes, even for closely related species, challenges evolutionary and functional analyses. Furthermore, effector genes are often found in rapidly evolving chromosome compartments which are difficult to assemble. We review how population and comparative genomics toolsets can be combined to address these challenges. We highlight studies that associated genome-scale polymorphisms with pathogen lifestyles and adaptation to different environments. We show how genome-wide association studies can be used to identify effectors and other pathogenicity-related genes underlying rapid adaptation. We also discuss how the compartmentalization of fungal genomes into core and accessory regions shapes the evolution of effector genes. We argue that an understanding of genome evolution provides important insight into the trajectory of host-pathogen co-evolution.

Ustilago maydis is an obligate biotrophic fungal pathogen which causes common smut disease of corn. To proliferate in host tissue, U. maydis must gain access to nutrients and overcome plant defence responses, such as the production of reactive oxygen species. The elucidation of the mechanisms by which U. maydis meets these challenges is critical for the development of strategies to combat smut disease. In this study, we focused on the contributions of phospholipases (PLs) to the pathogenesis of corn smut disease. We identified 11 genes encoding putative PLs and characterized the transcript levels for these genes in the fungus grown in culture and during infection of corn tissue. To assess the contributions of specific PLs, we focused on two genes, lip1 and lip2, which encode putative phospholipase A 2 (PLA 2 ) enzymes with similarity to platelet-activating factor acetylhydrolases. PLA 2 enzymes are known to counteract oxidative damage to lipids in other organisms. Consistent with a role in the mitigation of oxidative damage, lip2 mutants were sensitive to oxidative stress provoked by hydrogen peroxide and by increased production of reactive oxygen species caused by inhibitors of mitochondrial functions. Importantly, mutants defective in lip2, but not lip1, were attenuated for virulence in corn seedlings. Finally, a comparative analysis of fatty acid and cardiolipin profiles in the wild-type strain and a lip2 mutant revealed differences consistent with a protective role for Lip2 in maintaining lipid homeostasis and mitochondrial health during proliferation in the hostile host environment., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Evolution of fungicide resistance is a major threat to food production in agricultural ecosystems. Fungal pathogens rapidly evolved resistance to all classes of fungicides applied to the field. Resistance to the commonly used azole fungicides is thought to be driven mainly by mutations in a gene (CYP51) encoding a protein of the ergosterol biosynthesis pathway. However, some fungi gained azole resistance independently of CYP51 mutations and the mechanisms leading to CYP51-independent resistance are poorly understood. We used whole-genome sequencing and genome-wide association studies (GWAS) to perform an unbiased screen of azole resistance loci in Rhynchosporium commune, the causal agent of the barley scald disease. We assayed cyproconazole resistance in 120 isolates collected from nine populations worldwide. We found that mutations in highly conserved genes encoding the vacuolar cation channel YVC1, a transcription activator, and a saccharopine dehydrogenase made significant contributions to fungicide resistance. These three genes were not previously known to confer resistance in plant pathogens. However, YVC1 is involved in a conserved stress response pathway known to respond to azoles in human pathogenic fungi. We also performed GWAS to identify genetic polymorphism linked to fungal growth rates. We found that loci conferring increased fungicide resistance were negatively impacting growth rates, suggesting that fungicide resistance evolution imposed costs. Analyses of population structure showed that resistance mutations were likely introduced into local populations through gene flow. Multilocus resistance evolution to fungicides shows how pathogen populations can evolve a complex genetic architecture for an important phenotypic trait within a short time span., (© 2016 John Wiley & Sons Ltd.)

Fungal plant pathogens rapidly evolve virulence on resistant hosts through mutations in genes encoding proteins that modulate the host immune responses. The mutational spectrum likely includes chromosomal rearrangements responsible for gains or losses of entire genes. However, the mechanisms creating adaptive structural variation in fungal pathogen populations are poorly understood. We used complete genome assemblies to quantify structural variants segregating in the highly polymorphic fungal wheat pathogen Zymoseptoria tritici The genetic basis of virulence in Z. tritici is complex, and populations harbor significant genetic variation for virulence; hence, we aimed to identify whether structural variation led to functional differences. We combined single-molecule real-time sequencing, genetic maps, and transcriptomics data to generate a fully assembled and annotated genome of the highly virulent field isolate 3D7. Comparative genomics analyses against the complete reference genome IPO323 identified large chromosomal inversions and the complete gain or loss of transposable-element clusters, explaining the extensive chromosomal-length polymorphisms found in this species. Both the 3D7 and IPO323 genomes harbored long tracts of sequences exclusive to one of the two genomes. These orphan regions contained 296 genes unique to the 3D7 genome and not previously known for this species. These orphan genes tended to be organized in clusters and showed evidence of mutational decay. Moreover, the orphan genes were enriched in genes encoding putative effectors and included a gene that is one of the most upregulated putative effector genes during wheat infection. Our study showed that this pathogen species harbored extensive chromosomal structure polymorphism that may drive the evolution of virulence., Importance: Pathogen outbreak populations often harbor previously unknown genes conferring virulence. Hence, a key puzzle of rapid pathogen evolution is the origin of such evolutionary novelty in genomes. Chromosomal rearrangements and structural variation in pathogen populations likely play a key role. However, identifying such polymorphism is challenging, as most genome-sequencing approaches only yield information about point mutations. We combined long-read technology and genetic maps to assemble the complete genome of a strain of a highly polymorphic fungal pathogen of wheat. Comparisons against the reference genome of the species showed substantial variation in the chromosome structure and revealed large regions unique to each assembled genome. These regions were enriched in genes encoding likely effector proteins, which are important components of pathogenicity. Our study showed that pathogen populations harbor extensive polymorphism at the chromosome level and that this polymorphism can be a source of adaptive genetic variation in pathogen evolution., (Copyright © 2016 Plissonneau et al.)

Background: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields., Results: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae., Conclusion: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.

Hybrid zones allow the measurement of gene flow across the genome, producing insight into the genomic architecture of speciation. Such analysis is particularly powerful when applied to multiple pairs of hybridizing species, as patterns of genomic differentiation can then be related to age of the hybridizing species, providing a view into the build-up of differentiation over time. We examined 33 809 single nucleotide polymorphisms (SNPs) in three hybridizing woodpecker species: Red-breasted, Red-naped and Yellow-bellied sapsuckers (Sphyrapicus ruber, Sphyrapicus nuchalis and Sphyrapicus varius), two of which (ruber and nuchalis) are much more closely related than each is to the third (varius). To identify positions of SNPs on chromosomes, we developed a localization method based on comparative genomics. We found narrow clines, bimodal distributions of hybrid indices and genomic regions with decreased rates of introgression. These results suggest moderately strong reproductive isolation among species and selection against specific hybrid genotypes. We found 19 small regions of strong differentiation between species, partly shared among species pairs, but no large regions of differentiation. An association analysis revealed a single strong-effect candidate locus associated with plumage, possibly explaining mismatch among the three species in genomic relatedness and plumage similarity. Our comparative analysis of species pairs of different age and their hybrid zones showed that moderately strong reproductive isolation can occur with little genomic differentiation, but that reproductive isolation is incomplete even with much greater genomic differentiation, implying there are long periods of time when hybridization is possible if diverging populations are in geographic contact., (© 2016 John Wiley & Sons Ltd.)

Zymoseptoria tritici causes Septoria tritici blotch (STB) on wheat. The disease interaction is characterized by clearly defined temporal phases of infection, ultimately resulting in the death of host tissue. Zymoseptoria tritici is a highly polymorphic species with significant intraspecific variation in virulence profiles. We generated a deep transcriptomic sequencing dataset spanning the entire time course of an infection using a previously uncharacterized, highly virulent Z. tritici strain isolated from a Swiss wheat field. We found that seven clusters of gene transcription profiles explained the progression of the infection. The earliest highly up-regulated genes included chloroperoxidases, which may help the fungus cope with plant defences. The onset of necrotrophy was characterized by a concerted up-regulation of proteases, plant cell wall-degrading enzymes and lipases. Functions related to nutrition and growth characterized late necrotrophy and the transition to saprotrophic growth on dead plant tissue. We found that the peak up-regulation of genes essential for mating coincided with the necrotrophic phase. We performed an intraspecies comparative transcriptomics analysis using a comparable time course infection experiment of the genome reference isolate IPO323. Major components of the fungal infection transcriptome were conserved between the two strains. However, individual small, secreted proteins, proteases and cell wall-degrading enzymes showed strongly differentiated transcriptional profiles between isolates. Our analyses illustrate that successful STB infections involve complex transcriptomic remodelling to up-regulate distinct gene functions. Heterogeneity in transcriptomes among isolates may explain some of the considerable variation in virulence and host specialization found within the species., (© 2015 BSPP AND JOHN WILEY & SONS LTD.)

Background: Pathogenic intestinal protozoa infections are responsible for substantial mortality and morbidity, particularly in settings where people lack improved sanitation and safe drinking water. We assessed the relation between access to, and use of, sanitation facilities and water treatment and infection with intestinal protozoa., Methods: We did a systematic review and searched PubMed, ISI Web of Science, and Embase from inception to June 30, 2014, without restrictions on language. All publications were examined by two independent reviewers and were included if they presented data at the individual level about access or use of sanitation facilities or water treatment, in combination with individual-level data on human intestinal protozoa infections. Meta-analyses using random effects models were used to calculate overall estimates., Findings: 54 studies were included and odds ratios (ORs) extracted or calculated from 2 × 2 contingency tables. The availability or use of sanitation facilities was associated with significantly lower odds of infection with Entamoeba histolytica or Entamoeba dispar (OR 0·56, 95% CI 0·42-0·74) and Giardia intestinalis (0·64, 0·51-0·81), but not for Blastocystis hominis (1·03, 0·87-1·23), and Cryptosporidium spp (0·68, 0·17-2·68). Water treatment was associated with significantly lower odds of B hominis (0·52, 0·34-0·78), E histolytica or E dispar (0·61, 0·38-0·99), G intestinalis (0·63, 0·50-0·80), and Cryptosporidium spp infections (0·83, 0·70-0·98)., Interpretation: Availability and use of sanitation facilities and water treatment is associated with lower odds of intestinal protozoa infections. Interventions that focus on water and sanitation, coupled with hygiene behaviour, should be emphasised to sustain the control of intestinal protozoa infections., Funding: Swiss National Science Foundation (project numbers PBBSP3-146869 and P300P3-154634), Medicor Foundation, European Research Council (614739-A&#95;HERO)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host., (Copyright © 2015 by the Genetics Society of America.)

Background: The pathogenic yeast Cryptococcus neoformans causes life-threatening meningoencephalitis in individuals suffering from HIV/AIDS. The cyclic-AMP/protein kinase A (PKA) signal transduction pathway regulates the production of extracellular virulence factors in C. neoformans, but the influence of the pathway on the secretome has not been investigated. In this study, we performed quantitative proteomics using galactose-inducible and glucose-repressible expression of the PKA1 gene encoding the catalytic subunit of PKA to identify regulated proteins in the secretome., Methods: The proteins in the supernatants of cultures of C. neoformans were precipitated and identified using liquid chromatography-coupled tandem mass spectrometry. We also employed multiple reaction monitoring in a targeted approach to identify fungal proteins in samples from macrophages after phagocytosis of C. neoformans cells, as well as from the blood and bronchoalveolar fluid of infected mice., Results: We identified 61 secreted proteins and found that changes in PKA1 expression influenced the extracellular abundance of five proteins, including the Cig1 and Aph1 proteins with known roles in virulence. We also observed a change in the secretome profile upon induction of Pka1 from proteins primarily involved in catabolic and metabolic processes to an expanded set that included proteins for translational regulation and the response to stress. We further characterized the secretome data using enrichment analysis and by predicting conventional versus non-conventional secretion. Targeted proteomics of the Pka1-regulated proteins allowed us to identify the secreted proteins in lysates of phagocytic cells containing C. neoformans, and in samples from infected mice. This analysis also revealed that modulation of PKA1 expression influences the intracellular survival of cryptococcal cells upon phagocytosis., Conclusions: Overall, we found that the cAMP/PKA pathway regulates specific components of the secretome including proteins that affect the virulence of C. neoformans. The detection of secreted cryptococcal proteins from infected phagocytic cells and tissue samples suggests their potential utility as biomarkers of infection. The proteomics data are available via ProteomeXchange with identifiers PXD002731 and PASS00736.

A major problem associated with the intensification of agriculture is the emergence of fungicide resistance. Azoles are ergosterol biosynthesis inhibitors that have been widely used in agriculture and medicine since the 1970s, leading to emergence of increasingly resistant fungal populations. The known genetic mechanisms underlying lower azole sensitivity include mutations affecting the CYP51 gene that encodes the target protein, but in many cases azole resistance is a more complex trait with an unknown genetic basis. We used quantitative trait locus (QTL) mapping to identify genes affecting azole sensitivity in two crosses of Zymoseptoria tritici, the most damaging wheat pathogen in Europe. Restriction site associated DNA sequencing (RADseq) was used to genotype 263 (cross 1) and 261 (cross 2) progeny at ∼ 8500 single nucleotide polymorphisms (SNP) and construct two dense linkage maps. Azole sensitivity was assessed using high-throughput digital image analysis of colonies growing on Petri dishes with or without the fungicide propiconazole. We identified three QTLs for azole sensitivity, including two that contained novel fungicide sensitivity genes. One of these two QTLs contained only 16 candidate genes, among which four most likely candidates were identified. The third QTL contained ERG6, encoding another protein involved in ergosterol biosynthesis. Known genes in QTLs affecting colony growth included CYP51 and PKS1, a gene affecting melanization in Z. tritici. PKS1 showed compelling evidence for pleiotropy, with a rare segregating allele that increased melanization while decreasing growth rate and propiconazole sensitivity. This study resolved the genetic architecture of an important agricultural trait and led to identification of novel genes that are likely to affect azole sensitivity in Z. tritici. It also provided insight into fitness costs associated with lowered azole sensitivity and suggests a novel fungicide mixture strategy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Schistosomiasis is a disease caused by infection with blood flukes of the genus Schistosoma. Transmission of, and exposure to, the parasite result from faecal or urinary contamination of freshwater containing intermediate host snails, and dermal contact with the same water. The World Health Assembly resolution 65.21 from May 2012 urges member states to eliminate schistosomiasis through preventive chemotherapy (i.e. periodic large-scale administration of the antischistosomal drug praziquantel to school-aged children and other high-risk groups), provision of water, sanitation and hygiene (WASH) and snail control. However, control measures focus almost exclusively on preventive chemotherapy, while only few studies made an attempt to determine the impact of upgraded access to safe water, adequate sanitation and good hygiene on schistosome transmission. We recently completed a systematic review and meta-analysis pertaining to WASH and schistosomiasis and found that people with safe water and adequate sanitation have significantly lower odds of a Schistosoma infection. Importantly though, the transmission of schistosomiasis is deeply entrenched in social-ecological systems, and hence is governed by setting-specific cultural and environmental factors that determine human behaviour and snail populations. Here, we provide a comprehensive review of the literature, which explores the transmission routes of schistosomes, particularly focussing on how these might be disrupted with WASH-related technologies and human behaviour. Additionally, future research directions in this area are highlighted.

Advances in culture-independent sequencing methods have been utilised in recent studies to understand the phylogenetic composition of the human microbiome of healthy and diseased skin. Allergic rhinitis (AR) is an inflammatory condition of the nasal cavity caused by environmental allergens. Although nasal microbial communities have been considered important contributors in human health, no studies to date have comprehensively compared fungal communities (mycobiome) of the nasal vestibule using the culture-independent pyrosequencing method. This study aimed to investigate how fungal communities of the nasal vestibule skin surface are influenced by AR. The phylogenetic composition of the nasal vestibule mycobiome of patients with AR was analysed by culture-independent pyrosequencing methods and compared with healthy individuals. A total of 69 fungal genera were identified from both AR samples and healthy controls, and the genus Malassezia predominated in the nasal vestibule. Species-level analysis classified eight different Malassezia species including M. pachydermatis and M. cuniculi, which were normally isolated from animals, and revealed M. restricta to be the most abundant species in the nasal vestibule. Although high interpersonal variation was observed, some of the AR samples displayed significantly higher diversities than healthy controls at both the genus and species level., (© 2015 Blackwell Verlag GmbH.)

Background: Access to "safe" water and "adequate" sanitation are emphasized as important measures for schistosomiasis control. Indeed, the schistosomes' lifecycles suggest that their transmission may be reduced through safe water and adequate sanitation. However, the evidence has not previously been compiled in a systematic review., Methodology: We carried out a systematic review and meta-analysis of studies reporting schistosome infection rates in people who do or do not have access to safe water and adequate sanitation. PubMed, Web of Science, Embase, and the Cochrane Library were searched from inception to 31 December 2013, without restrictions on year of publication or language. Studies' titles and abstracts were screened by two independent assessors. Papers deemed of interest were read in full and appropriate studies included in the meta-analysis. Publication bias was assessed through the visual inspection of funnel plots and through Egger's test. Heterogeneity of datasets within the meta-analysis was quantified using Higgins' I2., Principal Findings: Safe water supplies were associated with significantly lower odds of schistosomiasis (odds ratio (OR) = 0.53, 95% confidence interval (CI): 0.47-0.61). Adequate sanitation was associated with lower odds of Schistosoma mansoni, (OR = 0.59, 95% CI: 0.47-0.73) and Schistosoma haematobium (OR = 0.69, 95% CI: 0.57-0.84). Included studies were mainly cross-sectional and quality was largely poor., Conclusions/significance: Our systematic review and meta-analysis suggests that increasing access to safe water and adequate sanitation are important measures to reduce the odds of schistosome infection. However, most of the studies were observational and quality was poor. Hence, there is a pressing need for adequately powered cluster randomized trials comparing schistosome infection risk with access to safe water and adequate sanitation, more studies which rigorously define water and sanitation, and new research on the relationships between water, sanitation, hygiene, human behavior, and schistosome transmission.

Melanin plays an important role in virulence and antimicrobial resistance in several fungal pathogens. The wheat pathogen Zymoseptoria tritici is important worldwide, but little is known about the genetic architecture of pathogenicity, including the production of melanin. Because melanin production can exhibit complex inheritance, we used quantitative trait locus (QTL) mapping in two crosses to identify the underlying genes. Restriction site-associated DNA sequencing was used to genotype 263 (cross 1) and 261 (cross 2) progeny at ~8500 single-nucleotide polymorphisms and construct two dense linkage maps. We measured gray values, representing degrees of melanization, for single-spore colonies growing on Petri dishes by using a novel image-processing approach that enabled high-throughput phenotyping. Because melanin production can be affected by stress, each offspring was grown in two stressful environments and one control environment. We detected six significant QTL in cross 1 and nine in cross 2, with three QTL shared between the crosses. Different QTL were identified in different environments and at different colony ages. By obtaining complete genome sequences for the four parents and analyzing sequence variation in the QTL confidence intervals, we identified 16 candidate genes likely to affect melanization. One of these candidates was PKS1, a polyketide synthase gene known to play a role in the synthesis of dihydroxynaphthalene melanin. Three candidate quantitative trait nucleotides were identified in PKS1. Many of the other candidate genes were not previously associated with melanization., (Copyright © 2014 Lendenmann et al.)

An outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C. gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV(+) patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors. Importance: Cryptococcus gattii is the causative agent responsible for ongoing infections in the Pacific Northwest of the United States and western Canada. The incidence of these infections increased dramatically in the 1990s and remains elevated. These infections are attributable to three clonal lineages of C. gattii, VGIIa, VGIIb, and VGIIc, with only VGIIa identified once previously in the Pacific Northwest prior to the start of the outbreak, albeit in a less virulent form. This study addresses the origin and emergence of this outbreak, using whole-genome sequencing and comparison of both outbreak and global isolates. We show that VGIIa arose mitotically from a less virulent clonal group, possibly via the action of a mutator phenotype, while VGIIb was likely introduced from Australia, and VGIIc appears to have emerged in the United States or in an undersampled locale via sexual reproduction. This work shows that multiple processes can contribute to the emergence of an outbreak., (Copyright © 2014 Billmyre et al.)

The variability of intron density among eukaryotes is puzzling and still debated. Most previous studies have been limited because of the near absence of intron presence-absence polymorphism (IPAP) within species or because comparisons could be made only between distantly related species. We conducted population genetic analyses on eight loci showing IPAP to investigate the effect of natural selection on intron dynamics in a global collection of the panmictic fungal plant pathogen Zymoseptoria tritici and its very close relatives. Five of these loci likely represent recent intron gains because their absence is fixed among the closest relatives of Z. tritici, and three likely represent recent intron losses because their presence is fixed among the close relatives. We analyzed signatures of selection by comparing allele frequencies, nucleotide diversities, and rates of recombination and found compelling evidence that at least two out of the five intron-gain loci, a SWIM zinc-finger gene and a sugar transporter, are under directional selection favoring alleles that gained the intron. Our results suggest that the intron-present alleles of these loci are sweeping to fixation, providing a genetic hitchhiking mechanism to explain rapid intron gain in Z. tritici. The overall findings are consistent with the hypothesis that intron gains are more likely to be driven by selection while intron losses are more likely to be due to neutral processes such as genetic drift., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Native plant species that have lost their mutualist partners may require non-native pollinators or seed dispersers to maintain reproduction. When natives are highly specialized, however, it appears doubtful that introduced generalists will partner effectively with them. We used visitation observations and pollination treatments (experimental manipulations of pollen transfer) to examine relationships between the introduced, generalist Japanese White-eye (Zosterops japonicus) and 3 endemic Hawaiian plant species (Clermontia parviflora, C. montis-loa, and C. hawaiiensis). These plants are characterized by curved, tubular flowers, apparently adapted for pollination by curve-billed Hawaiian honeycreepers. Z. japonicus were responsible for over 80% of visits to flowers of the small-flowered C. parviflora and the midsize-flowered C. montis-loa. Z. japonicus-visited flowers set significantly more seed than did bagged flowers. Z. japonicus also demonstrated the potential to act as an occasional Clermontia seed disperser, although ground-based frugivory by non-native mammals likely dominates seed dispersal. The large-flowered C. hawaiiensis received no visitation by any birds during observations. Unmanipulated and bagged C. hawaiiensis flowers set similar numbers of seeds. Direct examination of Z. japonicus and Clermontia morphologies suggests a mismatch between Z. japonicus bill morphology and C. hawaiiensis flower morphology. In combination, our results suggest that Z. japonicus has established an effective pollination relationship with C. parviflora and C. montis-loa and that the large flowers of C. hawaiiensis preclude effective visitation by Z. japonicus., (© 2013 Society for Conservation Biology.)

Energy expenditure in wild animals can be limited (i) intrinsically by physiological processes that constrain an animal's capacity to use energy, (ii) extrinsically by energy availability in the environment and/or (iii) strategically based on trade-offs between elevated metabolism and survival. Although these factors apply to all individuals within a population, some individuals expend more or less energy than other individuals. To examine the role of an energy ceiling in a species with a high and individually repeatable metabolic rate, we compared energy expenditure of thick-billed murres (Uria lomvia) with and without handicaps during a period of peak energy demand (chick-rearing, N = 16). We also compared energy expenditure of unencumbered birds (N = 260) across 8 years exhibiting contrasting environmental conditions and correlated energy expenditure with fitness (reproductive success and survival). Murres experienced an energy ceiling mediated through behavioural adjustments. Handicapped birds decreased time spent flying/diving and chick-provisioning rates such that overall daily energy expenditure remained unchanged across the two treatments. The energy ceiling did not reflect energy availability or trade-offs with fitness, as energy expenditure was similar across contrasting foraging conditions and was not associated with reduced survival or increased reproductive success. We found partial support for the trade-off hypothesis as older murres, where prospects for future reproduction would be relatively limited, did overcome an energy ceiling to invest more in offspring following handicapping by reducing their own energy reserves. The ceiling therefore appeared to operate at the level of intake (i.e. digestion) rather than expenditure (i.e. thermal constraint, oxidative stress). A meta-analysis comparing responses of breeding animals to handicapping suggests that our results are typical: animals either reduced investment in themselves or in their offspring to remain below an energy ceiling. Across species, whether a handicapped individual invested in its own energy stores or its offspring's growth was not explained by life history (future vs. current reproductive potential). Many breeding animals apparently experience an intrinsic energy ceiling, and increased energy costs lead to a decline in self-maintenance and/or offspring provisioning., (© 2013 The Authors. Journal of Animal Ecology © 2013 British Ecological Society.)

Background: As global environmental change accelerates, biodiversity losses can disrupt interspecific interactions. Extinctions of mutualist partners can create "widow" species, which may face reduced ecological fitness. Hypothetically, such mutualism disruptions could have cascading effects on biodiversity by causing additional species coextinctions. However, the scope of this problem - the magnitude of biodiversity that may lose mutualist partners and the consequences of these losses - remains unknown., Methodology/principal Findings: We conducted a systematic review and synthesis of data from a broad range of sources to estimate the threat posed by vertebrate extinctions to the global biodiversity of vertebrate-dispersed and -pollinated plants. Though enormous research gaps persist, our analysis identified Africa, Asia, the Caribbean, and global oceanic islands as geographic regions at particular risk of disruption of these mutualisms; within these regions, percentages of plant species likely affected range from 2.1-4.5%. Widowed plants are likely to experience reproductive declines of 40-58%, potentially threatening their persistence in the context of other global change stresses., Conclusions: Our systematic approach demonstrates that thousands of species may be impacted by disruption in one class of mutualisms, but extinctions will likely disrupt other mutualisms, as well. Although uncertainty is high, there is evidence that mutualism disruption directly threatens significant biodiversity in some geographic regions. Conservation measures with explicit focus on mutualistic functions could be necessary to bolster populations of widowed species and maintain ecosystem functions.

Zymoseptoria tritici is an important fungal pathogen on wheat that originated in the Fertile Crescent. Its closely related sister species Z. pseudotritici and Z. ardabiliae infect wild grasses in the same region. This recently emerged host-pathogen system provides a rare opportunity to investigate the evolutionary processes shaping the genome of an emerging pathogen. Here, we investigate genetic signatures in plant cell wall degrading enzymes (PCWDEs) that are likely affected by or driving coevolution in plant-pathogen systems. We hypothesize four main evolutionary scenarios and combine comparative genomics, transcriptomics, and selection analyses to assign the majority of PCWDEs in Z. tritici to one of these scenarios. We found widespread differential transcription among different members of the same gene family, challenging the idea of functional redundancy and suggesting instead that specialized enzymatic activity occurs during different stages of the pathogen life cycle. We also find that natural selection has significantly affected at least 19 of the 48 identified PCWDEs. The majority of genes showed signatures of purifying selection, typical for the scenario of conserved substrate optimization. However, six genes showed diversifying selection that could be attributed to either host adaptation or host evasion. This study provides a powerful framework to better understand the roles played by different members of multigene families and to determine which genes are the most appropriate targets for wet laboratory experimentation, for example, to elucidate enzymatic function during relevant phases of a pathogen's life cycle.

Chromosomal rearrangements are a major driver of eukaryotic genome evolution, affecting speciation, pathogenicity and cancer progression. Changes in chromosome structure are often initiated by mis-repair of double-strand breaks in the DNA. Mis-repair is particularly likely when telomeres are lost or when dispersed repeats misalign during crossing-over. Fungi carry highly polymorphic chromosomal complements showing substantial variation in chromosome length and number. The mechanisms driving chromosome polymorphism in fungi are poorly understood. We aimed to identify mechanisms of chromosomal rearrangements in the fungal wheat pathogen Zymoseptoria tritici. We combined population genomic resequencing and chromosomal segment PCR assays with electrophoretic karyotyping and resequencing of parents and offspring from experimental crosses to show that this pathogen harbors a highly diverse complement of accessory chromosomes that exhibits strong global geographic differentiation in numbers and lengths of chromosomes. Homologous chromosomes carried highly differentiated gene contents due to numerous insertions and deletions. The largest accessory chromosome recently doubled in length through insertions totaling 380 kb. Based on comparative genomics, we identified the precise breakpoint locations of these insertions. Nondisjunction during meiosis led to chromosome losses in progeny of three different crosses. We showed that a new accessory chromosome emerged in two viable offspring through a fusion between sister chromatids. Such chromosome fusion is likely to initiate a breakage-fusion-bridge (BFB) cycle that can rapidly degenerate chromosomal structure. We suggest that the accessory chromosomes of Z. tritici originated mainly from ancient core chromosomes through a degeneration process that included BFB cycles, nondisjunction and mutational decay of duplicated sequences. The rapidly evolving accessory chromosome complement may serve as a cradle for adaptive evolution in this and other fungal pathogens., Competing Interests: The authors have declared that no competing interests exist.

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Extinctions can leave species without mutualist partners and thus potentially reduce their fitness. In cases where non-native species function as mutualists, mutualism disruption associated with species' extinction may be mitigated. To assess the effectiveness of mutualist species with different origins, we conducted a meta-analysis in which we compared the effectiveness of pollination and seed-dispersal functions of native and non-native vertebrates. We used data from 40 studies in which a total of 34 non-native vertebrate mutualists in 20 geographic locations were examined. For each plant species, opportunistic non-native vertebrate pollinators were generally less effective mutualists than native pollinators. When native mutualists had been extirpated, however, plant seed set and seedling performance appeared elevated in the presence of non-native mutualists, although non-native mutualists had a negative overall effect on seed germination. These results suggest native mutualists may not be easily replaced. In some systems researchers propose taxon substitution or the deliberate introduction of non-native vertebrate mutualists to reestablish mutualist functions such as pollination and seed dispersal and to rescue native species from extinction. Our results also suggest that in places where all native mutualists are extinct, careful taxon substitution may benefit native plants at some life stages., (©2012 Society for Conservation Biology.)

The presence of spliceosomal introns in eukaryotic genes poses a major puzzle for the study of genome evolution. Intron densities vary enormously among distant lineages. However, the mechanisms driving intron gains are poorly understood and very few intron gains and losses have been documented over short evolutionary time spans. Fungi emerged recently as excellent models to study intron evolution and "reverse splicing" was found to be a major driver of recent intron gains in a clade of ascomycete fungi. We screened a total of 38 genomes from two fungal clades important in medicine and agriculture to identify intron gains and losses both within and between species. We detected 86 and 198 variable intron positions in the Cryptococcus and Fusarium clades, respectively. Some genes underwent extensive changes in their exon-intron structure, with up to six variable intron positions per gene. We identified a very recently gained intron in a group of tomato-infecting strains belonging to the F. oxysporum species complex. In the human pathogen C. gattii, we found recent intron losses in subtypes of the species. The two studied fungal clades provided evidence for extensive changes in their exon-intron structure within and among closely related species. We show that both intronization of previously coding DNA and insertion of exogenous DNA are the major drivers of intron gains.

Though spliceosomal introns are a major structural component of most eukaryotic genes and intron density varies by more than three orders of magnitude among eukaryotes [1-3], the origins of introns are poorly understood, and only a few cases of unambiguous intron gain are known [4-8]. We utilized population genomic comparisons of three closely related fungi to identify crucial transitory phases of intron gain and loss. We found 74 intron positions showing intraspecific presence-absence polymorphisms (PAPs) for the entire intron. Population genetic analyses identified intron PAPs at different stages of fixation and showed that intron gain or loss was very recent. We found direct support for extensive intron transposition among unrelated genes. A substantial proportion of highly similar introns in the genome either were recently gained or showed a transient phase of intron PAP. We also identified an intron transfer among paralogous genes that created a new intron. Intron loss was due mainly to homologous recombination involving reverse-transcribed mRNA. The large number of intron positions in transient phases of either intron gain or loss shows that intron evolution is much faster than previously thought and provides an excellent model to study molecular mechanisms of intron gain., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants, improving plant nutrition and diversity. Evidence exists suggesting that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. This potentially has two important consequences for their genetics. First, by random distribution of nuclei at spore formation, new offspring of an AMF could receive different complements of nucleotypes compared to the parent or siblings-we consider this as segregation. Second, genetic exchange between AMF would allow the mixing of nuclei, altering nucleotype diversity in new spores. Because segregation was assumed not to occur and genetic exchange has only recently been demonstrated, no attempts have been made to test whether this affects the symbiosis with plants. Here, we show that segregation occurs in the AMF Glomus intraradices and can enhance the growth of rice up to five times, even though neither parental nor crossed AMF lines induced a positive growth response. This process also resulted in an alteration of symbiosis-specific gene transcription in rice. Our results demonstrate that manipulation of AMF genetics has important consequences for the symbiotic effects on plants and could be used to enhance the growth of globally important crops., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Arbuscular mycorrhizal symbioses occur between fungi and the majority of plant species. They are important for plant nutrition, plant growth, protection from pathogens, plant diversity, nutrient cycling, and ecosystem processes. A key goal in research is to understand the molecular basis of the establishment, regulation, and functioning of the symbiosis. However, lack of knowledge on the genetics of the fungal side of this association has hindered progress. Here, we show how several key, recently discovered processes concerning the genetics of arbuscular mycorrhizal fungi could be essential for ultimately understanding the molecular genetics of this important symbiosis with plants.

In the ecologically important arbuscular mycorrhizal fungi (AMF), Sod1 encodes a functional polypeptide that confers increased tolerance to oxidative stress and that is upregulated inside the roots during early steps of the symbiosis with host plants. It is still unclear whether its expression is directed at scavenging reactive oxygen species (ROS) produced by the host, if it plays a role in the fungus-host dialogue, or if it is a consequence of oxidative stress from the surrounding environment. All these possibilities are equally likely, and molecular variation at the Sod1 locus can possibly have adaptive implications for one or all of the three mentioned functions. In this paper, we analyzed the diversity of the Sod1 gene in six AMF species, as well as 14 Glomus intraradices isolates from a single natural population. By sequencing this locus, we identified a large amount of nucleotide and amino acid molecular diversity both among AMF species and individuals, suggesting a rapid divergence of its codons. The Sod1 gene was monomorphic within each isolate we analyzed, and quantitative PCR strongly suggest this locus is present as a single copy in G. intraradices. Maximum-likelihood analyses performed using a variety of models for codon evolution indicated that a number of amino acid sites most likely evolved under the regime of positive selection among AMF species. In addition, we found that some isolates of G. intraradices from a natural population harbor very divergent orthologous Sod1 sequences, and our analysis suggested that diversifying selection, rather than recombination, was responsible for the persistence of this molecular diversity within the AMF population.

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants and form extensive underground hyphal networks simultaneously connecting the roots of different plant species. No empirical evidence exists for either anastomosis between genetically different AMF or genetic exchange.Five isolates of one population of Glomus intraradices were used to study anastomosis between hyphae of germinating spores. We show that genetically distinct AMF, from the same field, anastomose, resulting in viable cytoplasmic connections through which genetic exchange could potentially occur.Pairs of genetically different isolates were then co-cultured in an in vitro system.Freshly produced spores were individually germinated to establish new cultures.Using several molecular tools, we show that genetic exchange occurred between genetically different AMF. Specific genetic markers from each parent were transmitted to the progeny. The progeny were viable, forming symbioses with plant roots. The phenotypes of some of the progeny were significantly different from either parent.Our results indicate that considerable promiscuity could occur in these fungi because nine out of 10 combinations of different isolates anastomosed. The ability to perform genetic crosses between AMF experimentally lays a foundation for understanding the genetics and evolutionary biology of these important plants symbionts.

Background: Arbuscular mycorrhizal fungi (AMF) are important symbionts of most plant species, promoting plant diversity and productivity. This symbiosis is thought to have contributed to the early colonisation of land by plants. Morphological stasis over 400 million years and the lack of an observed sexual stage in any member of the phylum Glomeromycota led to the controversial suggestion of AMF being ancients asexuals. Evidence for recombination in AMF is contradictory., Results: We addressed the question of recombination in the AMF Glomus intraradices by sequencing 11 polymorphic nuclear loci in 40 morphologically identical isolates from one field. Phylogenetic relationships among genotypes showed a reticulate network pattern providing a rationale to test for recombination. Five statistical tests predicted multiple recombinant regions in the genome of a core set of isolates. In contrast, five clonal lineages had fixed a large number of differences., Conclusion: Our data show that AMF from one field have undergone recombination but that clonal lineages coexist. This finding has important consequences for understanding AMF evolution, co-evolution of AMF and plants and highlights the potential for commercially introduced AMF inoculum recombining with existing local populations. Finally, our results reconcile seemingly contradictory studies on whether AMF are clonal or form recombining populations.

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.

Gene copy number polymorphism was studied in a population of the arbuscular mycorrhizal fungus Glomus intraradices by using a quantitative PCR approach on four different genomic regions. Variation in gene copy number was found for a pseudogene and for three ribosomal genes, providing conclusive evidence for a widespread occurrence of macromutational events in the population.

Novel indolylindazolylmaleimides were synthesized and examined for kinase inhibition. We identified low-nanomolar inhibitors of PKC-beta with good to excellent selectivity vs other PKC isozymes and GSK-3beta. In a cell-based functional assay, 8f and 8i effectively blocked IL-8 release induced by PKC-betaII (IC(50) = 20-25 nM). In cardiovascular safety assessment, representative lead compounds bound to the hERG channel with high affinity, potently inhibited ion current in a patch-clamp experiment, and caused a dose-dependent increase of QT(c) in guinea pigs.

For fast flapping flight of birds in air, the maximum power and efficiency of the muscles occur over a limited range of contraction speeds and loads. Thus, contraction frequency and work per stroke tend to stay constant for a given species. In birds such as auks (Alcidae) that fly both in air and under water, wingbeat frequencies in water are far lower than in air, and it is unclear to what extent contraction frequency and work per stroke are conserved. During descent, compression of air spaces dramatically lowers buoyant resistance, so that maintaining a constant contraction frequency and work per stroke should result in an increased swimming speed. However, increasing speed causes exponential increases in drag, thereby reducing mechanical versus muscle efficiency. To investigate these competing factors, we have developed a biomechanical model of diving by guillemots (Uria spp.). The model predicted swimming speeds if stroke rate and work per stroke stay constant despite changing buoyancy. We compared predicted speeds with those of a free-ranging Brünnich's guillemot (U. lomvia) fitted with a time/depth recorder. For descent, the model predicted that speed should gradually increase to an asymptote of 1.5-1.6 m s-1 at approximately 40 m depth. In contrast, the instrumented guillemot typically reached 1.5 m s-1 within 10 m of the water surface and maintained that speed throughout descent to 80 m. During ascent, the model predicted that guillemots should stroke steadily at 1.8 m s-1 below their depth of neutral buoyancy (62 m), should alternate stroking and gliding at low buoyancies from 62 to 15 m, and should ascend passively by buoyancy alone above 15 m depth. However, the instrumented guillemot typically ascended at 1.25 m s-1 when negatively buoyant, at approximately 1.5 m s-1 from 62 m to 25 m, and supplemented buoyancy with stroking above 25 m. Throughout direct descent, and during ascent at negative and low positive buoyancies (82-25 m), the guillemot maintained its speed within a narrow range that minimized the drag coefficient. In films, guillemots descending against high buoyancy at shallow depths increased their stroke frequency over that of horizontal swimming, which had a substantial glide phase. Model simulations also indicated that stroke duration, relative thrust on the downstroke versus the upstroke, and the duration of gliding can be varied to regulate swimming speed with little change in contraction speed or work per stroke. These results, and the potential use of heat from inefficient muscles for thermoregulation, suggest that diving guillemots can optimize their mechanical efficiency (drag) with little change in net physiological efficiency.

1. Cholesterol metabolism has been characterized in three species of New World primates, the cotton-top tamarin, the saddle-back tamarin, and the squirrel monkey. 2. When fed a diet containing cholesterol, the three species exhibited differing responses of plasma cholesterol levels. 3. Dietary cholesterol absorption was determined and plasma cholesterol die-away kinetics were analyzed in terms of a two-pool model. 4. The results of the analyses of cholesterol turnover are consistent with the observed species-specific differences in plasma cholesterol values and cholesterol absorption. 5. Cholesterol metabolism differs between the two tamarin species, as well as between the tamarins and the squirrel monkey. 6. Implications of species-specific differences between tamarin species are discussed in terms of the use of tamarin species as animal models for comparative studies of cholesterol metabolism and the etiology of cancer and cardiovascular disease.

The diving and thermoregulatory metabolic rates of two species of diving seabird, common (Uria aalge) and thick-billed murres (U. lomvia), were studied in the laboratory. Post-absorptive resting metabolic rates were similar in both species, averaging 7.8 W.kg-1, and were not different in air or water (15-20 degrees C). These values were 1.5-2 times higher than values predicted from published allometric equations. Feeding led to increases of 36 and 49%, diving caused increases of 82 and 140%, and preening led to increases of 107 and 196% above measured resting metabolic rates in common and thick-billed murres, respectively. Metabolic rates of both species increased linearly with decreasing water temperature; lower critical temperature was 15 degrees C in common murres and 16 degrees C in thick-billed murres. Conductance (assuming a constant body temperature) did not change with decreasing temperature, and was calculated at 3.59 W.m-2 x degrees C-1 and 4.68 W.m-2 x degrees C-1 in common and thick-billed murres, respectively. Murres spend a considerable amount of time in cold water which poses a significant thermal challenge to these relatively small seabirds. If thermal conductance does not change with decreasing water temperature, murres most likely rely upon increasing metabolism to maintain body temperature. The birds probably employ activities such as preening, diving, or food-induced thermogenesis to meet this challenge.