Guislaine Refrégier

Speciation in fungi.

In this review on fungal speciation, we first contrast the issues of species definition and species criteria and show that by distinguishing the two concepts the approaches to studying the speciation can be clarified. We then review recent developments in the understanding of modes of speciation in fungi. Allopatric speciation raises no theoretical problem and numerous fungal examples exist from nature. We explain the theoretical difficulties raised by sympatric speciation, review the most recent models, and provide some natural examples consistent with speciation in sympatry. We describe the nature of prezygotic and postzygotic reproductive isolation in fungi and examine their evolution as functions of temporal and of the geographical distributions. We then review the theory and evidence for roles of cospeciation, host shifts, hybridization, karyotypic rearrangement, and epigenetic mechanisms in fungal speciation. Finally, we review the available data on the genetics of speciation in fungi and address the issue of speciation in asexual species.

Cophylogeny of the anther smut fungi and their caryophyllaceous hosts: prevalence of host shifts and importance of delimiting parasite species for inferring cospeciation

BackgroundUsing phylogenetic approaches, the expectation that parallel cladogenesis should occur between parasites and hosts has been validated in some studies, but most others provided evidence for frequent host shifts. Here we examine the evolutionary history of the association between Microbotryum fungi that cause anther smut disease and their Caryophyllaceous hosts. We investigated the congruence between host and parasite phylogenies, inferred cospeciation events and host shifts, and assessed whether geography or plant ecology could have facilitated the putative host shifts identified.For cophylogeny analyses on microorganisms, parasite strains isolated from different host species are generally considered to represent independent evolutionary lineages, often without checking whether some strains actually belong to the same generalist species. Such an approach may mistake intraspecific nodes for speciation events and thus bias the results of cophylogeny analyses if generalist species are found on closely related hosts. A second aim of this study was therefore to evaluate the impact of species delimitation on the inferences of cospeciation.ResultsWe inferred a multiple gene phylogeny of anther smut strains from 21 host plants from several geographic origins, complementing a previous study on the delimitation of fungal species and their host specificities. We also inferred a multi-gene phylogeny of their host plants, and the two phylogenies were compared. A significant level of cospeciation was found when each host species was considered to harbour a specific parasite strain, i.e. when generalist parasite species were not recognized as such. This approach overestimated the frequency of cocladogenesis because individual parasite species capable of infecting multiple host species (i.e. generalists) were found on closely related hosts. When generalist parasite species were appropriately delimited and only a single representative of each species was retained, cospeciation events were not more frequent than expected under a random distribution, and many host shifts were inferred.Current geographic distributions of host species seemed to be of little relevance for understanding the putative historical host shifts, because most fungal species had overlapping geographic ranges. We did detect some ecological similarities, including shared pollinators and habitat types, between host species that were diseased by closely related anther smut species. Overall, genetic similarity underlying the host-parasite interactions appeared to have the most important influence on specialization and host-shifts: generalist multi-host parasite species were found on closely related plant species, and related species in the Microbotryum phylogeny were associated with members of the same host clade.ConclusionWe showed here that Microbotryum species have evolved through frequent host shifts to moderately distant hosts, and we show further that accurate delimitation of parasite species is essential for interpreting cophylogeny studies.

Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method

We developed a new multiplexed-PCR assay to accurately classify Mycobacterium tuberculosis complex (MTC) isolates at the sublineage level by single nucleotide polymorphisms (SNPs). This method relies on 7 SNPs located in different genes of the MTC strains (recC, rec0, recR, ligB, ligC, alkA, and mgtC). Most of these genes are involved in replication, repair and recombination (3R) functions of M. tuberculosis strains, four of the mutations are synonymous, and thus neutral. Genes were chosen as a first empirical approach to assess the congruence between spoligotyping-based phylogeographical classification and SNP typing. This scheme efficiently classifies most of MTC phylogeographical groups: (1) confirming and identifying new sublineage-specific SNPs, (2) unraveling phylogenetical relationships between spoligotyping-defined MTC sublineages, (3) appropriately assigning sublineages to some spoligotypes and reassigning sublineages to other mis-labeled spoligotype signatures. This study opens the way to a more meaningful taxonomic, evolutionary and epidemiological classification. It also allows evaluation of spoligotype-signature significance towards a more comprehensive understanding of the evolutionary mechanisms of the clustered regularly interspaced short palindromic repeat (CRISPR) locus in MTC.

Hybrid sterility and inviability in the parasitic fungal species complex Microbotryum

Microbotryum violaceum, the anther‐smut fungus, forms a complex of sibling species which specialize on different plants. Previous studies have shown the presence of partial ecological isolation and F1 inviability, but did not detect assortative mating apart from a high selfing rate. We investigated other post‐mating barriers and show that F1 hybrid sterility, the inability of gametes to mate, increased gradually with the increasing genetic distance between the parents. F2 hybrids showed a reduced ability to infect the plants that was also correlated with the genetic distance. The host on which the F2 hybrids were passaged caused a selection for alleles derived from the pathogen species originally isolated from that host, but this effect was not detectable for the most closely related species. The post‐mating barriers thus remain weak among the closest species pairs, suggesting that premating barriers are sufficient to initiate divergence in this system.

Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices

ABSTRACT As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.

A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria

Background Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates. Methodology/Principal Findings DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.

“Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System

ABSTRACT We developed “spoligoriftyping,” a 53-plex assay based on two preexisting methods, the spoligotyping and “rifoligotyping” assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Spoligotyping of Mycobacterium africanum, Burkina Faso

Using Ziehl-Neelsen–positive slides collected from tuberculosis diagnostic centers in Burkina Faso, we showed that 20% of 80 spoligotyping-positive DNA samples had a characteristic Mycobacterium africanum–specific genomic signature. This result suggests that M. africanum is still present in Burkina Faso at almost the same prevalence as 15–20 years ago.

Chapter 3 Genome Evolution in Plant Pathogenic and Symbiotic Fungi

Abstract Approximately 100,000 species of fungi have been described so far, of which, a high percentage obtain nutrients by living in close association with other organisms (mainly plants), being pathogens or symbionts (i.e. commensalists or mutualists). At the genomic level, an association between broad-scale genetic changes and the emergences of the parasitic and symbiotic life style in fungi has been proposed. Although comparative genomic studies in fungi are still in the early stages, they have enormous potential to improve our understanding of how such life styles evolve. In this chapter, we review the main characteristics of genome evolution in fungi, particularly in species that are pathogenic or symbiotic with plants, by focusing on the mechanisms involved in host interactions. We address the following topics in relation to the pathogenic and symbiotic lifestyles in fungi: the evolution of genome organization, chromosomal rearrangements, evolution of gene families and clusters, suppression of recombination around mating type loci, rapidly evolving genes, horizontal transfer, hybridization, transposable elements, telomeres, introns, mitochondrial genomes, and we finally compare genome evolution between pathogenic bacteria and fungi.

PERMANENT GENETIC RESOURCES: Isolation of 60 polymorphic microsatellite loci in EST libraries of four sibling species of the phytopathogenic fungal complex Microbotryum.

We report the development of 60 microsatellite markers on four species of the fungal complex Microbotryum, causing anther smut of the Caryophyllaceae. Microsatellites were found in four expressed sequence tag (EST) libraries, built from isolates of M. lychnis‐dioicae, M. violaceum sensus stricto, M. lagerheimii and M. dianthorum, collected, respectively, from the plants Silene latifolia, S. nutans, S. vulgaris and Dianthus carthusianorum. Intrapopulation polymorphism was investigated using 24 isolates, and cross‐amplification was explored using 23 isolates belonging to at least 10 different Microbotryum species. This study provides numerous microsatellite markers for population genetics and mapping studies.