Significant diversity and potential problems associated with inferring population structure within the Cenococcum geophilum species complex

Cenococcum geophilum is perhaps the most widely distributed and most recognized ectomycorrhizal fungus with a host range of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. We conducted a phylogenetic analysis on a large collection of isolates (n=74) from North America and Europe based on glyceraldehyde 3-phosphate dehydrogenase (gpd). A subset of isolates (n=22) also was analyzed with the more conservative LSU-rDNA locus. Significant nucleotide diversity was detected (approximately 20%) in the gpd region and the LSU-rDNA analysis supported that the C. geophilum isolates studied were monophyletic but distinct from two isolates, Am5-1 and N2-10, which previously were used in population genetic studies of this species. These results suggest that Am5-1 and N2-10 are likely two undescribed species or even genera. Our results suggest that C. geophilum sensu lato is a species complex and support previous molecular, physiological and morphological studies that have shown significant diversity in C. geophilum. This study also revealed that caution is advised when conducting population genetic studies in C. geophilum due to the possibility of pooling unrelated isolates. This potential problem also has implications for other fungal taxa because cryptic species routinely have been found in recent years based on molecular data.     

土生空团菌的遗传多样性分析

为了研究土生空团菌的遗传多样性,对来自中国、美国、瑞士和法国等菌株的rDNA ITS区进行序列分析,利用Popgene32和phylip软件进行数据计算和聚类分析。序列分析表明土生空团菌rDNA ITS区的序列长度为422–447bp,遗传距离在0.000–0.051之间。居群结构和聚类分析结果表明:(1)土生空团菌有一定的遗传多样性,且遗传差异主要来自于居群内;(2)基因流Nm>1,遗传漂变不是导致土生空团菌居群遗传分化的主要因素;(3)土生空团菌的遗传分化受到地理环境的影响,而与宿主来源没有明显相关性。     

Genetic diversity of Fusarium oxysporum populations isolated from different soils in France

The genetic diversity of soil-borne populations of Fusarium oxysporum was assessed using 350 isolates collected from six different French soils. All isolates were characterised by restriction fragment analysis of the PCR-amplified ribosomal intergenic spacer (IGS). Twenty-six IGS types were identified among the 350 isolates analysed. Five to nine different IGS types were detected in each soil. None of the IGS types was common to all of the soils. An analysis of the molecular variance based on IGS type relationships and frequency revealed that the genetic structure of the populations of F. oxysporum varied widely among the soils. Some populations were both highly diverse within the soils and differentiated between the soils. A possible relationship between the intrapopulation or interpopulation level of diversity and some external factors such as the soil type or the crop history was evaluated. A subsample representative of the diversity of the six populations was further characterised by analysing the genomic distribution of two transposable elements, impala and Fot1. One to 10 copies of the impala element were present in most of the isolates, irrespective of their soil of origin. The Fot1 element was only detected in 40% of the isolates originating from the three populations less diverse in terms of IGS types, but in 82.6% of the isolates originating from the three more diverse populations.     

Genetic diversity and differential in vitro responses to Ni in <Emphasis Type="Italic">Cenococcum geophilum</Emphasis> isolates from serpentine soils in Portugal

Amplified fragment length polymorphism (AFLP) analysis was used to investigate the genetic diversity in isolates of the ectomycorrhizal fungus Cenococcum geophilum from serpentine and non-serpentine soils in Portugal. A high degree of genetic diversity was found among C. geophilum isolates; AFLP fingerprints showed that all the isolates were genetically distinct. We also assessed the in vitro Ni sensitivity in three serpentine isolates and one non-serpentine isolate. Only the non-serpentine isolate was significantly affected by the addition of Ni to the growth medium. At 30 microg g(-1) Ni, radial growth rate and biomass accumulation decreased to 73.3 and 71.6% of control, respectively, a highly significant inhibitory effect. Nickel at this concentration had no significant inhibitory effect on serpentine isolates, and so the fitness of serpentine isolates, as evaluated by radial growth rate and biomass yield, is likely unaffected by Ni in the field. In all isolates, the Ni concentration in the mycelia increased with increasing Ni concentration in the growth medium, but two profiles of Ni accumulation were identified. One serpentine isolate showed a linear trend of Ni accumulation. At the highest Ni exposure, the concentration of Ni in the mycelium of this isolate was in the hyperaccumulation range for Ni as defined for higher plants. In the remaining isolates, Ni accumulation was less pronounced and seems to approach a plateau at 30 microg g(-1) Ni. Because two profiles of Ni accumulation emerged among our Ni-insensitive serpentine isolates, this result suggests that different Ni detoxification pathways may be operating. The non-serpentine isolate whose growth was significantly affected by Ni was separated from the other isolates in the genetic analysis, suggesting a genetic basis for the Ni-sensitivity trait. This hypothesis is further supported by the fact that all isolates were maintained on medium without added Ni to avoid carry-over effects. However, because AFLP analysis failed to distinguish between serpentine and non-serpentine isolates, we cannot conclude that Ni insensitivity among our serpentine isolates is due to evolutionary adaptation. Screening a larger number of isolates, from different geographical origins and environments, should clarify the relationships between genetic diversity, morphology, and physiology in this important species.     

Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species

The genetic structure of the root-endophyte Phialocephala fortinii was analyzed in three study sites using 11 single-copy RFLP probes. A total of 541 strains isolated from surface-sterilized, fine roots (diameter 0.5-3 mm) of Norway spruce (Picea abies) were examined. The average gene diversity (H) was high in all three study sites. Cluster analysis showed that up to four well-separated clusters of multi-locus haplotypes were present within the sites. Significant population subdivision was detected among these clusters, suggesting that groups of multi-locus haplotypes were reproductively isolated and that P. fortinii is a species complex composed of several cryptic species. This hypothesis was supported by ISSR-PCR which showed clusters consistent with those of the multi-locus haplotypes identified by RFLP analysis. In contrast, ITS sequence analysis did not allow to separate the species as clearly. The index of association (IA) did not deviate significantly from zero within any cryptic species, suggesting that recombination occurs within these species. Cryptic species occurred sympatrically. Thalli of two cryptic species were detected in the same 5-mm-long root segment in one instance. No significant differentiation was observed among populations of the same cryptic species in forest stands located approximately 5 km from each other. This finding is consistent with significant gene flow over this spatial scale. In addition, several isolates with both identical multi-locus haplotype and identical ISSR fingerprint were found at each study site indicating genotype flow or a recent common history between study sites.     

Comparing the hyper-variable V4 and V9 regions of the small subunit rDNA for assessment of ciliate environmental diversity

The hyper-variable V4 and V9 regions of the small subunit (SSU) rDNA have been targeted for assessing environmental diversity of microbial eukaryotes using next generation sequencing technologies. Here, we explore how the genetic distances among these short fragments compare with the distances obtained from near full-length SSU-rDNA sequences by comparing all pairwise estimates, as well as within and among species of ciliates. Results show that pairwise distances from V4 more closely match the near full-length SSU-rDNA and are more comparable with previous studies based on much longer SSU-rDNA fragments, then pairwise distances from V9. Thus, studies that use the V4 will estimate similar values of phylotype richness and community structure as would have been estimated using the full-length SSU-rDNA.     

Oscheius tipulae, a widespread hermaphroditic soil nematode, displays a higher genetic diversity and geographical structure than Caenorhabditis elegans

The nematode Oscheius tipulae belongs to the same family (Rhabditidae) as the model species Caenorhabditis elegans . Both species reproduce through self-fertilizing hermaphrodites and facultative males. Recent studies have shown that the self-fertile C. elegans and C. briggsae displayed a 20-fold lower genetic diversity than the male–female species C. remanei . Several explanations have been put forward to account for this difference, including their mode of reproduction and dynamic population structure. Here, we present the results of extensive worldwide sampling of O. tipulae , which we previously used as a laboratory organism for developmental genetics. We found that O. tipulae is much more widespread and common in soil throughout the world than Caenorhabditis species. We analysed 63 O. tipulae isolates from several continents using amplified fragment length polymorphism (AFLP). We found that O. tipulae harbours a 5-fold higher genetic diversity than C. elegans and C. briggsae . As in C. elegans , a high proportion of this diversity was found locally. Yet, we detected significant geographical differentiation, both at the worldwide scale with a latitudinal structure and between three localities in France. In summary, O. tipulae exhibited significantly higher levels of genetic diversity and large-scale geographical structure than C. elegans , despite their shared mode of reproduction. This species difference in genetic diversity may be explained by a number of other differences, such as population size, distribution, migration and dynamics. Due to its widespread occurrence and relatively high genetic diversity, O. tipulae may be a promising study species for evolutionary studies.     

Genetic and phenotypic microdiversity of Ochrobactrum spp

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.     

Assessing whether alpha-tubulin sequences are suitable for phylogenetic reconstruction of Ciliophora with insights into its evolution in euplotids

The current understanding of ciliate phylogeny is mainly based on analyses of a single gene, the small subunit ribosomal RNA (SSU-rDNA). However, phylogenetic trees based on single gene sequence are not reliable estimators of species trees, and SSU-rDNA genealogies are not useful for resolution of some branches within Ciliophora. Since congruence between multiple loci is the best tool to determine evolutionary history, we assessed the usefulness of alpha-tubulin gene, a protein-coding gene that is frequently sequenced, for ciliate phylogeny. Here, we generate alpha-tubulin gene sequences of 12 genera and 30 species within the order Euplotida, one of the most frequently encountered ciliate clades with numerous apparently cosmopolitan species, as well as four genera within its putative sister order Discocephalida. Analyses of the resulting data reveal that: 1) the alpha-tubulin gene is suitable phylogenetic marker for euplotids at the family level, since both nucleotide and amino acid phylogenies recover all monophyletic euplotid families as defined by both morphological criteria and SSU-rDNA trees; however, alpha-tubulin gene is not a good marker for defining species, order and subclass; 2) for seven out of nine euplotid species for which paralogs are detected, gene duplication appears recent as paralogs are monophyletic; 3) the order Euplotida is non-monophyletic, and the family Uronychiidae with sequences from four genera, is non-monophyletic; and 4) there is more genetic diversity within the family Euplotidae than is evident from dargyrome (geometrical pattern of dorsal "silverline system" in ciliates) patterns, habit and SSU-rDNA phylogeny, which indicates the urgent need for taxonomic revision in this area.     

Genetic diversity of rhizobia nodulating Trifolium ambiguum in North America

Kura clover (Trifolium ambiguum M.B.) is a persistent rhizomatous forage legume, whose use in the U.S.A. is limited by establishment difficulties in part attributable to nodulation problems. In this study, soil was collected from established stands of Kura clover growing in 9 diverse North American environments. Rhizobia were plant-trapped using Kura clover cv. Endura as host, then rhizobia from nodules fingerprinted using BOX-PCR. The diversity of isolates from North America was then contrasted to that of rhizobia from a single Caucasian environment (Russia), the center of origin for this species. Populations were characterized using clustering methods, and genetic diversity estimated using the Shannon-Weaver diversity index. The genetic diversity of the North American populations was extremely limited, all isolates being closely related to two of the strains found in a locally available commercial inoculant. In contrast, Russian isolates formed a distinct cluster with significant internal genetic diversity. Genetic diversity indices for the North American and Russian populations were 3.5 and 10.76, respectively. The implication of this and other studies is that Kura clover is highly specific in Rhizobium requirement. If the performance of this legume in the U.S.A. is to be improved, either by modifying current establishment practices or plant breeding, it is essential that these studies be paralleled by more collections and evaluation of rhizobia from its center of origin, given the extremely limited diversity of rhizobia found in North America.     

Cryptic species in Stachybotrys chartarum

Stachybotrys chartarum has received much attention as a possible cause of sick-building syndrome. Because morphological species recognition in fungi can hide diversity, we applied a phylogenetic approach to search for cryptic species. We examined 23 isolates from the San Francisco Bay Area, and another seven from around the US. Using markers we developed for three polymorphic protein coding loci (chitin synthase 1, beta-tubulin 2, and trichodiene synthase 5), we infer that two distinct phylogenetic species exist within the single described morphological species. We have found no correlation between genetic isolation and geographic distance.     

Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample

The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a (60)Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.     

A single European aspen (Populus tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS genotypes and hundreds of species of ectomycorrhizal fungi

Ectomycorrhizal fungi (EcMF) form diverse communities and link different host plants into mycorrhizal networks, yet little is known about the magnitude of mycobiont diversity of a single tree individual. This study addresses species richness and spatial structure of EcMF in the root system of a single European aspen (Populus tremula) individual in an old-growth boreal mixed forest ecosystem in Estonia. Combining morphological and molecular identification methods for both plant and fungi, 122 species of EcMF were recovered from 103 root samples of the single tree. Richness estimators predicted the total EcMF richness to range from 182 to 207 species, reflecting the observation of 62.3% singletons and doubletons within the community. Fine-scale genetic diversity in Cenococcum geophilum indicates the presence of 23 internal transcribed spacer genotypes. EcMF community was significantly spatially autocorrelated only at the lineage level up to 3 m distance, but not at the species level. Proximity of other hosts had a significant effect on the spatial distribution of EcMF lineages. This study demonstrates that a single tree may host as many EcMF species and individuals as recovered on multiple hosts in diverse communities over larger areas.     

Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Ciliophora: Peritrichia) inferred from mitochondrial, nuclear, and morphological markers

Protist diversity is currently a much debated issue in eukaryotic microbiology. Recent evidence suggests that morphological and genetic diversity might be decoupled in some groups of protists, including ciliates, and that these organisms might be much more diverse than their morphology implies. We sought to assess the genetic and morphological diversity of Carchesium polypinum, a widely distributed peritrich ciliate. The mitochondrial marker cytochrome c oxidase subunit I and the nuclear small subunit ribosomal RNA were used to examine genetic diversity. For the morphological assessment, live microscopy and Protargol staining were used. The mitochondrial marker revealed six robust, deeply diverging, and strongly supported clades, while the nuclear gene was congruent for three of these clades. There were no major differences among individuals from the different clades in any of the morphological features examined. Thus, the underlying genetic diversity in C. polypinum is greater than what its morphology suggests, indicating that morphology and genetics are not congruent in this organism. Furthermore, because the clades identified by the mitochondrial marker are so genetically diverse and are confirmed by a conserved nuclear marker in at least three cases, we propose that C. polypinum be designated as a "cryptic species complex." Our results provide another example where species diversity can be underestimated in microbial eukaryotes when using only morphological criteria to estimate species richness.     

Hidden Biodiversity in an Ecologically Important Freshwater Amphipod: Differences in Genetic Structure between Two Cryptic Species

Cryptic species, i.e. species that are morphologically hard to distinguish, have been detected repeatedly in various taxa and ecosystems. In order to evaluate the importance of this finding, we have to know in how far cryptic species differ in various aspects of their biology. The amphipod Gammarus fossarum is a key invertebrate in freshwater streams and contains several cryptic species. We examined the population genetic structure, genetic diversity and demographic history of two of them (type A and type B) using microsatellite markers and asked whether they show significant differences. We present results of population genetic analyses based on a total of 37 populations from the headwaters of two major European drainages, Rhine and Rhone. We found that, in both species, genetic diversity was geographically structured among and within drainages. For type A in the Rhine and type B in the Rhone, we detected significant patterns of isolation by distance. The increase of genetic differentiation with geographical distance, however, was much higher in type A than in type B. This result indicates substantial interspecific differences in population history and/or the extent of current gene flow between populations. In the Rhine, type B does not show evidence of isolation by distance, and population differentiation is relatively low across hundreds of kilometres. The majority of these populations also show signatures of recent bottlenecks. These patterns are consistent with a recent expansion of type B into the Rhine drainage. In summary, our results suggest considerable and previously unrecognized interspecific differences in the genetic structure of these cryptic keystone species.     

Discordance Between Spatial Distributions of Y-Chromosomal and Mitochondrial Haplotypes in African Green Monkeys (Chlorocebus spp.): A Result of Introgressive Hybridization or Cryptic Diversity?

Introgressive hybridization may cause substantial discordances among phylogenies based on different genetic markers. Such discordances have been found in diverse mammal species including primates. A recent study of mitochondrial DNA (mtDNA) revealed several poly- and paraphyletic relationships in African green monkeys (Chlorocebus), suggesting contemporary and/or ancient introgressive hybridization among almost all parapatric species of the genus. However, mtDNA analyses alone do not allow us to draw conclusions concerning introgression events. In this study we analyzed two Y chromosomal (Y-chr) markers for 30 African green monkey samples and compared the resulting genetic relationships to those based on published mtDNA data. In line with the results for mtDNA, we found no Y-chr evidence of hypothesized hybridization among Chlorocebus sabaeus and C. tantalus in the northern part of the contact zone in West Africa, and we found two distinct and distantly related Y-chr haplotypes within the range of C. tantalus, suggesting possible cryptic genetic diversity rather than ancient introgressive hybridization in this species. In contrast, Y-chr data revealed monophyletic relationships within Chlorocebus pygerythrus from East Africa, suggesting that mtDNA paraphylies found in this species are most likely to be the result of ancient introgressive hybridization and subsequent cytonuclear extinction of an earlier taxon. Our results accentuate the importance of analyzing sex chromosomal data in addition to mtDNA to obtain more information on the potential outcomes of hybridization with respect to genetic and species diversity. Analysis of more diverse nuclear marker sets is needed to obtain a more complete picture of the African green monkey evolution.     

The molecular systematics of Leiopotherapon unicolor (Günther, 1859): testing for cryptic speciation in Australia's most widespread freshwater fish

Leiopotherapon unicolor is the most widespread freshwater fish species in Australia. A comprehensive allozyme and mitochondrial DNA 16S rRNA data set was assembled from 141 specimens of L. unicolor collected Australia-wide in order to test for cryptic speciation in this far-ranging species. Surprisingly, little genetic diversity was observed within L. unicolor and provided no evidence for the existence of cryptic species within this lineage. In contrast, a small sample set of L. aheneus used as the outgroup showed two highly divergent haplotypes strongly suggestive of cryptic speciation. L. unicolor has a number of ecological and life history attributes that may explain the lack of significant genetic divergence over substantial geographical distances. The occurrence of other widespread fish and crustacean species that also display only limited genetic diversity indicate that climate conditions more favourable to dispersal across central and northern Australia than is suggested by the extent of present-day aridity have occurred in the relatively recent geological past.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 537–552.     

Comparative studies of the population genetic structure of the Brachionus calyciflorus species complex from four inland lakes in Wuhu,China

To study how the population genetic structure in zooplankton respond to environmental conditions, using comparative limnology, the genetic diversity and genetic differentiation of the B. calyciflorus complex collected from four inland lakes in Wuhu City, China were investigated based on the 16S rRNA gene and nuDNA ITS sequences. The results displayed a high genetic diversity, and the nucleotide diversity of the 16S rRNA gene was higher than that of the ITS sequence. The phylogenetic analyses grouped the four populations into two cryptic species (Bc-JT and Bc-FL) with strong support. The two cryptic species were found in lakes with different trophic levels, demonstrating significant ecological specialization. The origins of clone TW12 were not consistent in the phylogenetic trees between two genetic markers, which might be attributed to the effects of male-mediated gene flow on the phylogenetic relationships of rotifers. The nucleotide diversity of the cryptic species Bc-JT was higher than that of Bc-FL, indicating that eutrophication might decrease the genetic diversity of cryptic species. The total phosphorus concentration in water bodies might be the most important factor affecting the genetic diversity of species.     

Contrasting root associated fungi of three common oak-woodland plant species based on molecular identification: host specificity or non-specific amplification?

An increasingly popular approach used to identify arbuscular mycorrhizal (AM) fungi in planta is to amplify a portion of AM fungal small subunit ribosomal DNA (SSU-rDNA) from whole root DNA extractions using the primer pair AM1-NS31, followed by cloning and sequencing. We used this approach to study the AM fungal community composition of three common oak-woodland plant species: a grass (Cynosurus echinatus), blue oak (Quercus douglasii), and a forb (Torilis arvensis). Significant diversity of AM fungi were found in the roots of C. echinatus, which is consistent with previous studies demonstrating a high degree of AM fungal diversity from the roots of various hosts. In contrast, clones from Q. douglasii and T. arvensis were primarily from non-AM fungi of diverse origins within the Ascomycota and Basidiomycota. This work demonstrates that caution must be taken when using this molecular approach to determine in planta AM fungal diversity if non-sequence based methods such as terminal restriction fragment length polymorphisms, denaturing gradient gel electrophoresis, or temperature gradient gel electrophoresis are used.     

Genetic diversity, reproductive biology, and speciation in the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin.

Beauveria bassiana, a mitosporic fungus used for the biological control of many insect species, is recognized as a "species complex" comprising genetically diverse lineages. Being predominantly asexual, mating tests cannot be applied to delimit species in this species complex. Genetic tests offer an indirect means of identifying species among isolates. To this end, molecular genetic analysis of a sample of B. bassiana isolates with 2 subsamples, 1 representing a worldwide collection and another from a localized epizootic population was carried out. DNA markers generated through AFLPs (amplified fragment length polymorphisms) and SSCPs (single-strand conformation poly morphisms) and nucleotide sequence data of different allelic forms of 3 genes (large and small subunits of rRNA and beta-tubulin) were evaluated. The B. bassiana isolates from the worldwide sample showed 11% overall similarity and no closely clustered groups. Phylogenetic trees generated from the AFLP and SSCP data of this sample resolved the different isolates into distinct phylogenetic lineages. In the epizootic B. bassiana population, prevalence of recombination was evident from random association of alleles in multilocus tests and lack of phylogenetic concordance among 3 gene genealogies. Thus, the worldwide sample of B. bassiana exhibits a predominantly clonal structure, hinting at species divergence leading to cryptic speciation with recombination being customary among isolates sharing a close ecological niche.