RESUMEN
Dinoflagellates of the genus Alexandrium are responsible for harmful algal blooms and produce paralytic shellfish toxins (PSTs). Their very large and complex genomes make it challenging to identify the genes responsible for toxin synthesis. A family-based genomic association study was developed to determine the inheritance of toxin production in Alexandrium minutum and identify genomic regions linked to this production. We show that the ability to produce toxins is inheritable in a Mendelian way, while the heritability of the toxin profile is more complex. We developed the first dinoflagellate genetic linkage map. Using this map, several major results were obtained: 1. A genomic region related to the ability to produce toxins was identified. 2. This region does not contain any polymorphic sxt genes, known to be involved in toxin production in cyanobacteria. 3. The sxt genes, known to be present in a single cluster in cyanobacteria, are scattered on different linkage groups in A. minutum. 4. The expression of two sxt genes not assigned to any linkage group, sxtI and sxtG, may be regulated by the genomic region related to the ability to produce toxins. Our results provide new insights into the organization of toxicity-related genes in A. minutum, suggesting a dissociated genetic mechanism for the production of the different analogues and the ability to produce toxins. However, most of the newly identified genes remain unannotated. This study therefore proposes new candidate genes to be further explored to understand how dinoflagellates synthesize their toxins.
Asunto(s)
Dinoflagelados , Dinoflagelados/genética , Dinoflagelados/metabolismo , Toxinas Marinas/genética , Toxinas Marinas/metabolismoRESUMEN
Despite theoretical expectations, marine microeukaryote population are often highly structured and the mechanisms behind such patterns remain to be elucidated. These organisms display huge census population sizes, yet genotyping usually requires clonal strains originating from single cells, hindering proper population sampling. Estimating allelic frequency directly from population wide samples, without any isolation step, offers an interesting alternative. Here, we validate the use of meta-transcriptome environmental samples to determine the population genetic structure of the dinoflagellate Alexandrium minutum. Strain and meta-transcriptome based results both indicated a strong genetic structure for A. minutum in Western Europe, to the level expected between cryptic species. The presence of numerous private alleles, and even fixed polymorphism, would indicate ancient divergence and absence of gene flow between populations. Single nucleotide polymorphisms (SNPs) displaying strong allele frequency differences were distributed throughout the genome, which might indicate pervasive selection from standing genetic variation (soft selective sweeps). However, a few genomic regions displayed extremely low diversity that could result from the fixation of adaptive de novo mutations (hard selective sweeps) within the populations.
Asunto(s)
Dinoflagelados , Dinoflagelados/genética , Transcriptoma , Metagenómica , Flujo Génico , Densidad de PoblaciónRESUMEN
Paralytic shellfish poisoning (PSP) is a human foodborne syndrome caused by the consumption of shellfish that accumulate paralytic shellfish toxins (PSTs, saxitoxin group). In PST-producing dinoflagellates such as Alexandrium spp., toxin synthesis is encoded in the nuclear genome via a gene cluster (sxt). Toxin production is supposedly associated with the presence of a 4th domain in the sxtA gene (sxtA4), one of the core genes of the PST gene cluster. It is postulated that gene expression in dinoflagellates is partially constitutive, with both transcriptional and post-transcriptional processes potentially co-occurring. Therefore, gene structure and expression mode are two important features to explore in order to fully understand toxin production processes in dinoflagellates. In this study, we determined the intracellular toxin contents of twenty European Alexandrium minutum and Alexandrium pacificum strains that we compared with their genome size and sxtA4 gene copy numbers. We observed a significant correlation between the sxtA4 gene copy number and toxin content, as well as a moderate positive correlation between the sxtA4 gene copy number and genome size. The 18 toxic strains had several sxtA4 gene copies (9-187), whereas only one copy was found in the two observed non-toxin producing strains. Exploration of allelic frequencies and expression of sxtA4 mRNA in 11 A. minutum strains showed both a differential expression and specific allelic forms in the non-toxic strains compared with the toxic ones. Also, the toxic strains exhibited a polymorphic sxtA4 mRNA sequence between strains and between gene copies within strains. Finally, our study supported the hypothesis of a genetic determinism of toxin synthesis (i.e., the existence of several genetic isoforms of the sxtA4 gene and their copy numbers), and was also consistent with the hypothesis that constitutive gene expression and moderation by transcriptional and post-transcriptional regulation mechanisms are the cause of the observed variability in the production of toxins by A. minutum.
RESUMEN
Parasites in the genus Amoebophrya sp. infest dinoflagellate hosts in marine ecosystems and can be determining factors in the demise of blooms, including toxic red tides. These parasitic protists, however, rarely cause the total collapse of Dinophyceae blooms. Experimental addition of parasite-resistant Dinophyceae (Alexandrium minutum or Scrippsiella donghaienis) or exudates into a well-established host-parasite coculture (Scrippsiella acuminata-Amoebophrya sp.) mitigated parasite success and increased the survival of the sensitive host. This effect was mediated by waterborne molecules without the need for a physical contact. The strength of the parasite defenses varied between dinoflagellate species, and strains of A. minutum and was enhanced with increasing resistant host cell concentrations. The addition of resistant strains or exudates never prevented the parasite transmission entirely. Survival time of Amoebophrya sp. free-living stages (dinospores) decreased in presence of A. minutum but not of S. donghaienis. Parasite progeny drastically decreased with both species. Integrity of the dinospore membrane was altered by A. minutum, providing a first indication on the mode of action of anti-parasitic molecules. These results demonstrate that extracellular defenses can be an effective strategy against parasites that protects not only the resistant cells producing them, but also the surrounding community.
RESUMEN
Sexual reproduction remains poorly characterized in dinoflagellates. This is especially the case at the molecular level. Here crossing experiments were performed among strains of the toxic dinoflagellate Alexandrium minutum belonging to two genetically divergent groups. Gene expression was compared between sexually compatible and incompatible crosses at the time of gamete fusion and resting cyst (~zygote) formation. Not a single transcript was identified as differentially expressed between compatible and incompatible crosses at these two crucial time points of the dinoflagellate life cycle. However, several thousands of transcripts displayed constitutive expression differences between strains. This was especially the case between the strains belonging to the genetically divergent groups. A few hundreds of transcripts were also identified as differentially expressed between strains belonging to opposite mating types. Some of these transcripts displayed homology with the SxtA protein, known to be involved in saxitoxin production in cyanobacteria, as well as with proteins potentially involved in mating in fungi.
Asunto(s)
Cianobacterias , Dinoflagelados , Dinoflagelados/genética , Expresión Génica , Reproducción , SaxitoxinaRESUMEN
Alexandrium minutum and Alexandrium pacificum are representatives of the dinoflagellate genus that regularly proliferate on the French coasts and other global coastlines. These harmful species may threaten shellfish harvest and human health due to their ability to synthesize neurotoxic alkaloids of the saxitoxin group. However, some dinoflagellates such as A. minutum, and as reported here A. pacificum as well, may also have a beneficial impact on the environment by producing dimethylsulfoniopropionate-DMSP, the precursor of dimethylsulfur-DMS and sulfate aerosols involved in climate balance. However, environmental conditions might influence Alexandrium physiology towards the production of harmful or environmentally friendly compounds. After assessing the influence of two salinity regimes (33 and 38) relative to each species origin (Atlantic French coast and Mediterranean Lagoon respectively), it appears that DMSP and toxin content was variable between the three experimented strains and that higher salinity disadvantages toxin production and tends to favor the production of the osmolytes DMSP and glycine betaine. Hence, this key metabolite production is strain and species-dependent and is influenced by environmental conditions of salinity which in turn, can diversely affect the environment. Widespread coastal blooms of A. minutum and A. pacificum, although being a risk for seafood contamination with toxins, are also a DMSP and DMS source that potentially contribute to the ecosystem structuration and climate. Regarding recent advances in DMSP biosynthesis pathway, 3 dsyB homologs were found in A. minutum but no homolog of the diatom sequence TpMMT.
Asunto(s)
Diatomeas , Dinoflagelados , Ecosistema , Floraciones de Algas Nocivas , Humanos , Dinámica Poblacional , Salinidad , MariscosRESUMEN
Harmful algal blooms are caused by specific members of microbial communities. Understanding the dynamics of these events requires comparing the strategies developed by the problematic species to cope with environmental fluctuations to the ones developed by the other members of the community. During three consecutive years, the meta-transcriptome of micro-eukaryote communities was sequenced during blooms of the toxic dinoflagellate Alexandrium minutum. The dataset was analyzed to investigate species specific gene expression dynamics. Major shifts in gene expression were explained by the succession of different species within the community. Although expression patterns were strongly correlated with fluctuation of the abiotic environment, and more specifically with nutrient concentration, transcripts specifically involved in nutrient uptake and metabolism did not display extensive changes in gene expression. Compared to the other members of the community, A. minutum displayed a very specific expression pattern, with lower expression of photosynthesis transcripts and central metabolism genes (TCA cycle, glucose metabolism, glycolysis ) and contrasting expression pattern of ion transporters across environmental conditions. These results suggest the importance of mixotrophy, cell motility and cell-to-cell interactions during A. minutum blooms.
Asunto(s)
Dinoflagelados/genética , Floraciones de Algas Nocivas/fisiología , Microbiota/genética , Océano Atlántico , Código de Barras del ADN Taxonómico , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Canales Iónicos/genética , Transporte Iónico/genética , Fotosíntesis/genética , Especificidad de la EspecieRESUMEN
We used a multistrain approach to study the intra- and interspecific variability of the growth rates of three Pseudo-nitzschia species - P. australis, P. fraudulenta, and P. pungens - and of their domoic acid (DA) production. We carried out mating and batch experiments to investigate the respective effects of strain age and cell size, and thus the influence of their life cycle on the physiology of these species. The cell size - life cycle relationship was characteristic of each species. The influence of age and cell size on the intraspecific variability of growth rates suggests that these characteristics should be considered cautiously for the strains used in physiological studies on Pseudo-nitzschia species. The results from all three species do not support the hypothesis of a decrease in DA production with time since isolation from natural populations. In P. australis, the cellular DA content was rather a function of cell size. More particularly, cells at the gametangia stage of their life cycle contained up to six times more DA than smaller or larger cells incapable of sexual reproduction. These findings reveal a link between P. australis life cycle and cell toxicity. This suggest that life cycle dynamics in Pseudo-nitzschia natural populations may influence bloom toxicity.
Asunto(s)
Diatomeas , Animales , Ácido Kaínico , Estadios del Ciclo de VidaRESUMEN
Untangling the functional basis of divergence between closely related species is a step toward understanding species dynamics within communities at both the evolutionary and ecological scales. We investigated cellular (i.e., growth, domoic acid production, and nutrient consumption) and molecular (transcriptomic analyses) responses to varying nutrient concentrations across several strains belonging to three species of the toxic diatom genus Pseudo-nitzschia. Three main results were obtained. First, strains from the same species displayed similar transcriptomic, but not necessarily cellular, responses to the experimental conditions. It showed the importance of considering intraspecific diversity to investigate functional divergence between species. Second, a major exception to the first finding was a strain recently isolated from the natural environment and displaying contrasting gene expression patterns related to cell motility and domoic acid production. This result illustrated the profound modifications that may occur when transferring a cell from the natural to the in vitro environment and asks for future studies to better understand the influence of culture duration and life cycle on expression patterns. Third, transcriptomic responses were more similar between the two species displaying similar ecology in situ, irrespective of the genetic distance. This was especially true for molecular responses related to TCA cycle, photosynthesis, and nitrogen metabolism. However, transcripts related to phosphate uptake were variable between species. It highlighted the importance of considering both overall genetic distance and ecological divergence to explain functional divergence between species.
Asunto(s)
Evolución Biológica , Diatomeas/fisiología , Ácido Kaínico/análogos & derivados , Ácido Kaínico/metabolismo , Familia de Multigenes , Nutrientes , FenotipoRESUMEN
The factors responsible for inducing the synthesis of toxins and responses from toxic phytoplankton blooms remain unclear. In this study we compare the influence of genotypic (at both the intra and interspecific levels) and environmental factors (nutrient concentration and ratio) on growth (in terms of cell densities) and domoic acid (DA) production in three Pseudo-nitzschia species: P. australis, P.pungens and P.fradulenta. A strong phosphate effect was detected. More precisely, a low initial concentration in phosphate, even at high initial nitrogen and silicate concentrations, induced the highest DA concentrations and the lowest cell densities in all strains/species studied. In contrast, a low initial concentration of nitrogen and silicate combined, with a higher phosphate concentration resulted in low cell densities, but without high DA production. Inter-species effects were also observed in DA production, where P. australis represented the most toxigenic species of all. Intra-specific variations were only moderate, except for a recently isolated P. australis strain, suggesting the influence of time since isolation on the physiology and DA production of Pseudo-nitzschia species. Overall, the lack of strong interaction between environmental and genotypic factors showed that the various genotypes investigated did not extensively diverge in their ability to respond (in terms of DA production and cell densities) to contrasting nutrient supply.
Asunto(s)
Diatomeas/crecimiento & desarrollo , Diatomeas/metabolismo , Floraciones de Algas Nocivas , Ácido Kaínico/análogos & derivados , Fosfatos/metabolismo , Diatomeas/genética , Genotipo , Ácido Kaínico/metabolismo , Nutrientes/metabolismoRESUMEN
Understanding divergence in the highly dispersive and seemingly homogeneous pelagic environment for organisms living as free drifters in the water column remains a challenge. Here, we analysed the transcriptome-wide mRNA sequences, as well as the morphology of 18 strains of Alexandrium minutum, a dinoflagellate responsible for harmful algal blooms worldwide, to investigate the functional bases of a divergence event. Analysis of the joint site frequency spectrum (JSFS) pointed towards an ancestral divergence in complete isolations followed by a secondary contact resulting in gene flow between the two diverging groups, but heterogeneous across sites. The sites displaying fixed SNPs were associated with a highly restricted gene flow and a strong overrepresentation of nonsynonymous polymorphism, suggesting the importance of selective pressures as drivers of the divergence. The most divergent transcripts were homologs to genes involved in calcium/potassium fluxes across the membrane, calcium transduction signal and saxitoxin production. The implication of these results in terms of ecological divergence and build-up of reproductive isolation is discussed. Dinoflagellates are especially difficult to study in the field at the ecological level due to their small size and the dynamic nature of their natural environment, but also at the genomic level due to their huge and complex genome and the absence of closely related model organism. This study illustrates the possibility to identify the traits of primary importance in ecology and evolution starting from high-throughput sequencing data, even for such organisms.
Asunto(s)
Dinoflagelados/genética , Evolución Molecular , Flujo Génico , Selección Genética , Variación Genética , Modelos Genéticos , Filogenia , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Aislamiento Reproductivo , TranscriptomaRESUMEN
The multiannual dynamic of the cyst-forming and toxic marine dinoflagellate Alexandrium minutum was studied over a time scale of about 150 years by a paleoecological approach based on ancient DNA (aDNA) quantification and cyst revivification data obtained from two dated sediment cores of the Bay of Brest (Brittany, France). The first genetic traces of the species presence in the study area dated back to 1873 ± 6. Specific aDNA could be quantified by a newly developed real-time PCR assay in the upper core layers, in which the germination of the species (in up to 17-19-year-old sediments) was also obtained. In both cores studied, our quantitative paleogenetic data showed a statistically significant increasing trend in the abundance of A. minutum ITS1 rDNA copies over time, corroborating three decades of local plankton data that have documented an increasing trend in the species cell abundance. By comparison, paleogenetic data of the dinoflagellate Scrippsiella donghaienis did not show a coherent trend between the cores studied, supporting the hypothesis of the existence of a species-specific dynamic of A. minutum in the study area. This work contributes to the development of paleoecological research, further showing its potential for biogeographical, ecological and evolutionary studies on marine microbes.
Asunto(s)
Dinoflagelados/aislamiento & purificación , Sedimentos Geológicos/parasitología , Bahías , ADN Ribosómico/genética , Dinoflagelados/clasificación , Dinoflagelados/genética , Dinoflagelados/metabolismo , Ecosistema , Francia , Sedimentos Geológicos/química , Historia del Siglo XX , Historia del Siglo XXI , Paleografía/historia , Reacción en Cadena en Tiempo Real de la Polimerasa , Especificidad de la EspecieRESUMEN
BACKGROUND: The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment. RESULTS: Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations. CONCLUSIONS: Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map.
Asunto(s)
Escherichia coli/genética , Adaptación Fisiológica , Ambiente , Evolución Molecular , Interacción Gen-Ambiente , Genoma Bacteriano , FenotipoRESUMEN
The evolutionary stability of haploid-diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long-standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year-round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid-diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.
Asunto(s)
Evolución Biológica , Phaeophyceae/crecimiento & desarrollo , Phaeophyceae/genética , Diploidia , Ecosistema , Francia , Haploidia , ItaliaRESUMEN
Ecological opportunities promote population divergence into coexisting lineages. However, the genetic mechanisms that enable new lineages to exploit these opportunities are poorly understood except in cases of single mutations. We examined how two Escherichia coli lineages diverged from their common ancestor at the outset of a long-term coexistence. By sequencing genomes and reconstructing the genetic history of one lineage, we showed that three mutations together were sufficient to produce the frequency-dependent fitness effects that allowed this lineage to invade and stably coexist with the other. These mutations all affected regulatory genes and collectively caused substantial metabolic changes. Moreover, the particular derived alleles were critical for the initial divergence and invasion, indicating that the establishment of this polymorphism depended on specific epistatic interactions.
Asunto(s)
Epistasis Genética , Escherichia coli/genética , Escherichia coli/fisiología , Mutación , Polimorfismo Genético , Alelos , Escherichia coli/metabolismo , Evolución Molecular , Genes Bacterianos , Genes Reguladores , Aptitud Genética , Genotipo , Glucosa/metabolismo , Interacciones MicrobianasRESUMEN
We investigated the relationship between genomic and phenotypic evolution among replicate populations of Escherichia coli evolved for 1000 generations in four different environments. By resequencing evolved genomes, we identified parallel changes in genes encoding transcription regulators within and between environments. Depending on both the environment and the altered gene, genetic parallelism at the gene level involved mutations that affected identical codons, protein domains or were widely distributed across the gene. Evolved clones were characterized by parallel phenotypic changes in their respective evolution environments but also in the three alternative environments. Phenotypic parallelism was high for clones that evolved in the same environment, even in the absence of genetic parallelism. By contrast, clones that evolved in different environments revealed a higher parallelism in correlated responses when they shared mutated genes. Altogether, this work shows that after an environmental change or the colonization of a new habitat, similar ecological performance might be expected from individuals that share mutated genes or that experienced similar past selective pressures.
Asunto(s)
Ambiente , Escherichia coli/genética , Evolución Molecular , Genes Bacterianos , ADN Bacteriano/genética , Escherichia coli/crecimiento & desarrollo , Aptitud Genética , Mutación , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Closely related organisms usually occupy similar ecological niches, leading to intense competition and even extinction. Such competition also can promote rapid phenotypic evolution and ecological divergence. This process may end with the stable occupation of distinct niches or, alternatively, may entail repeated bouts of evolution. Here we examine two Escherichia coli lineages, called L and S, that coexisted for more than 30,000 generations after diverging from a common ancestor. Both lineages underwent sustained phenotypic evolution based on global transcription and resource utilization profiles, with L seeming to encroach over time on the catabolic profile of S. Reciprocal invasion experiments with L and S clones from the same or different generations revealed evolutionary changes in their interaction, including an asymmetry that confirmed the encroachment by L on the niche of the S lineage. In general, L and S clones from the same generation showed negative frequency-dependent effects, consistent with stable coexistence. However, L clones could invade S clones from both earlier and later generations, whereas S clones could invade only L clones from earlier generations. In this system, the long-term coexistence of competing lineages evidently depended on successive rounds of evolution, rather than on initial divergence followed by a static equilibrium.
Asunto(s)
Ecología , Escherichia coli/genética , Evolución Molecular , Análisis por Conglomerados , Escherichia coli/crecimiento & desarrollo , Perfilación de la Expresión GénicaRESUMEN
The opportunity for a mutation to invade a population can dramatically vary depending on the context in which this mutation occurs. Such context dependence is difficult to document as it requires the ability to measure how a mutation affects phenotypes and fitness and to manipulate the context in which the mutation occurs. We identified a mutation in a gene encoding a global regulator in one of two ecotypes that diverged from a common ancestor during 1200 generations of experimental evolution. We replaced the ancestral allele by the mutant allele, and vice versa, in several clones isolated during the time course of the evolution experiment, and compared the phenotype and fitness of clones isogenic except for the focal mutation. We show that the fitness and phenotype of the mutation are strongly affected by epistatic interactions between genes in the same genome, as well as by frequency dependent selection resulting from biotic interactions between individuals in the same population. We conclude that amongst the replicate population in which it spread, the mutation we identified is only adaptive when occurring in specific genomes and competing with specific individuals. This study thus demonstrates that the opportunity for an adaptive mutation to spread in an evolutionary lineage can only be understood in the light of its genomic and competitive environments.
Asunto(s)
Epistasis Genética , Evolución Molecular , Aptitud Genética , Mutación , Alelos , ADN Bacteriano/genética , Escherichia coli/genética , Modelos Estadísticos , FenotipoRESUMEN
Cooperation should be favored under environmental conditions allowing the preferential interaction of cooperators among themselves and limiting interactions with defectors. Bacteria cooperating to kill competitors by secreting a toxin evolved during several hundred generations in two environments: a viscous environment that should promote cooperator assortment, and a nonviscous environment that should not allow such preferential interaction. A quantitative decrease in cooperation was observed in all populations, but as expected, cooperation was maintained at a higher level in the viscous environment. Mutants that are resistant against but not producing the toxin were identified at a low frequency in a few populations from the viscous environment and at a high frequency in all the populations from the nonviscous environment. The underlying mutations were identified. Relative fitness of the cooperator and mutant genotypes were obtained with bacteria that were isogenic, except for the identified mutations. Competition experiments indicated that cooperation is not favored by environmental viscosity as imposed in our system and suggested that when it comes to cooperation, environmental viscosity should be considered not only in terms of individual movement, but also in terms of the distribution of the public good.
Asunto(s)
Bacterias/genética , Evolución Biológica , Viscosidad , Bacterias/metabolismo , Secuencia de Bases , Colicinas/biosíntesis , Cartilla de ADN , Genes Bacterianos , MutaciónRESUMEN
BACKGROUND: Using 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. RESULTS: We 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. CONCLUSION: We 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.