Multilocus phylogenetic analysis of true morels (Morchella) reveals high levels of endemics in Turkey relative to other regions of Europe

The present study was conducted to better understand how the phylogenetic diversity of true morels (Morchella) in Turkey compares with species found in other regions of the world. The current research builds on our recently published surveys of 10 Turkish provinces and the northern hemisphere in which DNA sequence data from 247 and 562 collections respectively were analyzed phylogenetically. Herein we report on phylogenetic analyses of 243 additional collections made in spring 2009 and 2010 from eight additional provinces in the Aegean, Black Sea, central Anatolia, eastern Anatolia and Marmara regions of Turkey. Our analysis revealed that five species within the Esculenta clade (yellow morels) and 15 species within the Elata clade (black morels) were present in Turkey. Our preliminary results also indicate that M. anatolica, recently described from a collection in Mu?la province in the Aegean region of Turkey, is a closely related sister of M. rufobrunnea; these two species comprise a separate evolutionary lineage from the Esculenta and Elata clades. Nine species of Morchella currently are known only from Turkey, four species were present in Turkey and other European countries and seven species might have been introduced to Turkey anthropogenically. Three of the putatively exotic species in Turkey appear to be endemic to western North America; they are nested within a clade of fire-adapted morels that dates to the late Oligocene, 25 000 000 y ago. Our results indicate that there are roughly twice as many Morchella species in Turkey compared with the other regions of Europe sampled. Knowledge of Morchella species diversity and their biogeographic distribution are crucial for formulating informed conservation policies directed at preventing species loss and ensuring that annual morel harvests are sustainable and ecologically sound.     

DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella conica (black morel).

The internal transcribed spacer (ITS) of the gene coding for rRNA was sequenced in both directions with the gene walking technique in a black morel (Morchella conica) and a yellow morel (M. esculenta) to elucidate the ITS length discrepancy between the two species groups (750-bp ITS in black morels and 1,150-bp ITS in yellow morels.     

Phylogenetic diversity of true morels (Morchella), the main edible non-timber product from native Patagonian forests of Argentina

Morchella species are edible fungi in high demand and therefore command high prices in world markets. Phenotypic-based identification at the species-level remains inadequate because of their complex life cycles, minor differences and plasticity of morphological characteristics between species, and the lack of agreement between scientific and common names. In Patagonia–Argentina, morels are associated with native forests of Austrocedrus chilensis (Cordilleran or Chilean cypress) and Nothofagus antarctica (ñire) and several exotic conifers that were introduced from western North America. Little is known about their taxonomy and phylogenetic relationships with other species in the genus. This work focused on the identification of collections of Morchella from Patagonia and their phylogenetic relationships with other species from the Northern Hemisphere. The comparison was made by analysis of DNA sequences obtained from four loci: the nuclear ribosomal internal transcribed spacer region (ITS) and the partial RNA polymerase I gene (RPB1) for the complete collection; and ITS, RPB1, RNA polymerase II gene (RPB2), and translation elongation factor (EF1-α) for the species-rich Elata Subclade. Analyses of individual and combined data sets revealed that Patagonian morels belong to the Elata Clade and comprised three strongly supported species-level lineages from both Patagonian native forest, and exotic trees introduced from western North America. One lineage was identified as Morchella frustrata phylogenetic species Mel-2, which is known from the USA and Canada. The second lineage, which appeared to be ‘fire-adapted’, was identified as Morchella septimelata phylogenetic species (Mel-7), which is also known from the USA. This species was collected from burned native forests mainly composed of A. chilensis and N. antarctica but also Pseudotsuga menziesii (Mirb.) Blanco, which is native to western North America. The phylogenetic analyses suggested that the third species from Patagonia was nested within the species-rich Elata Subclade and represents a new species-level lineage (informally designated Mel-37) within Elata Clade. The present collections from Patagonia constitute the southernmost latitude from which Morchella has been reported to date. The identification of two Argentine morels as North American taxa is therefore a remarkable biogeographic pattern. In view of the hypothesis that the Elata Clade originated in western North America, we speculate that at least two of the lineages colonized South America from North America via long distance dispersal, migration or, more likely, they were introduced with the exotic tree species that they were collected near.     

Multigene molecular phylogenetics reveals true morels (Morchella) are especially species-rich in China

The phylogenetic diversity of true morels (Morchella) in China was estimated by initially analyzing nuclear ribosomal internal transcribed spacer (ITS) rDNA sequences from 361 specimens collected in 21 provinces during the 2003-2011 growing seasons, together with six collections obtained on loan from three Chinese herbaria. Based on the results of this preliminary screen, 40 Esculenta Clade (yellow morels) and 30 Elata Clade (black morels) were chosen to represent the full range of phylogenetic diversity sampled. To investigate their species limits, we generated DNA sequences from portions of three protein-coding genes (RPB1, RPB2 and EF-1α) and domains D1 and D2 of the nuclear large subunit (LSU) rDNA for all 70 collections. To fully assess evolutionary relationships, previously published multilocus DNA sequence data representing all known Morchella species was included in this study. Phylogenetic analyses employing maximum parsimony and maximum likelihood frameworks resolved 30 species in China compared with 22 in Europe and 19 within North America. Eleven novel phylogenetically distinct species were discovered in China, including two species within the Elata Clade and nine within the Esculenta Clade. Of the 30 species in China, 20 appear to be endemic, nine were also represented in Europe, and four putatively fire-adapted species have disjunct distributions in China, Europe and western North America. Although the diversification time estimates place the Esculenta Clade in China as early as the late Cretaceous and the Elata Clade by the early Oligocene, 27 of the 30 species evolved between the middle Miocene 12Mya and present.     

Characterization of mating-type idiomorphs suggests that <Emphasis Type="Italic">Morchella importuna,Mel-20</Emphasis> and <Emphasis Type="Italic">M. sextelata</Emphasis> are heterothallic

Morels (Morchella spp.) are highly prized for their culinary qualities and intensively collected worldwide by mycophiles. Morels are divided into three clades by phylogenetic analyses: black morels, yellow morels and the rufobrunnea clade. Morchella importuna, Mel-20 and M. sextelata are included in the black morel clade and are widely distributed in Yunnan province, China. M. importuna and M. sextelata have been artificially cultured in recent years, but their life cycles and reproductive systems are still poorly understood, which delays the progress of morel cultivation. In this study, the genomes of two ascospore isolates of M. importuna with opposite mating-type were sequenced and two idiomorphs, MAT1–1 and MAT1–2, were identified. The MAT1–2 idiomorph was 6.7 kb in length containing a single MAT1–2-1 gene, and the MAT1–1 idiomorph was 10.5 kb containing a MAT1–1-1 gene and two other open reading frames (ORFs), GME3123 and GME3124. These ORFs differed greatly from the homologues of previously published mating-type genes; therefore, we speculate that they are novel mating genes found only in morels. Single-ascospore populations of M. importuna, M. sextelata and Mel-20 were analysed, and the result indicated that the ratios of MAT1–1- and MAT1–2-harbouring idiomorphs were not significantly different from a 1:1 ratio. The results suggest that these three black morels are heterothallic.     

四川秋季发生的两种羊肚菌生境调查与鉴定

为了寻找四川秋季发生的羊肚菌资源,了解其产地环境及种属关系,探寻羊肚菌在秋季发生的适生条件,对四川省凉山州盐源县、会东县和冕宁县的羊肚菌发生地开展了生境调查,共采集到11份羊肚菌标本。采用形态学和分子鉴定相结合的方法,对标本进行鉴定,结果表明所采集的标本被鉴定为2种羊肚菌,即七妹羊肚菌Morchella septimelata（Mel-7）和Morchella sp.（Mes-16）,其中,七妹羊肚菌为四川新记录种。通过生境调查发现,秋季发生的羊肚菌适生环境为山地林下陡坡,可见,四川秋季发生的羊肚菌有一定的特殊性。     

A multigene molecular phylogenetic assessment of true morels (Morchella) in Turkey

A collection of 247 true morels (Morchella spp.) primarily from the Mediterranean and Aegean Regions of Southern Turkey, were analyzed for species diversity using partial RNA polymerase I (RPB1) and nuclear ribosomal large subunit (LSU) rDNA gene sequences. Based on the result of this initial screen, 62 collections representing the full range of genetic diversity sampled were subjected to multigene phylogenetic species recognition based on genealogical concordance (GCPSR). The 62-taxon dataset consisted of partial sequences from three nuclear protein-coding genes, RNA polymerase I (RPB1), RNA polymerase II (RPB2), translation elongation factor (EF1-α), and partial LSU rDNA gene sequences. Phylogenetic analyses of the individual and combined datasets, using maximum parsimony (MP) and maximum likelihood (ML), yielded nearly fully resolved phylogenies that were highly concordant topologically. GCPSR analysis of the 62-taxon dataset resolved 15 putative phylogenetically distinct species. The early diverging Elata (black morels) and Esculenta Clades (yellow morels) were represented, respectively, by 13 and two species. Because a Latin binomial can be applied with confidence to only one of the 15 species (Morchella semilibera), species were identified by clade (Mel for Elata and Mes for Esculenta) followed by a unique Arabic number for each species within these two clades. Eight of the species within the Elata Clade appear to be novel, including all seven species within the Mel-20-to-31 subclade and its sister designated Mel-25. Results of the present study provide essential data for ensuring the sustainability of morel harvests through the formulation of sound conservation policies.     

Phylogeny and historical biogeography of true morels (Morchella) reveals an early Cretaceous origin and high continental endemism and provincialism in the Holarctic

True morels (Morchella, Ascomycota) are arguably the most highly-prized of the estimated 1.5 million fungi that inhabit our planet. Field guides treat these epicurean macrofungi as belonging to a few species with cosmopolitan distributions, but this hypothesis has not been tested. Prompted by the results of a growing number of molecular studies, which have shown many microbes exhibit strong biogeographic structure and cryptic speciation, we constructed a 4-gene dataset for 177 members of the Morchellaceae to elucidate their origin, evolutionary diversification and historical biogeography. Diversification time estimates place the origin of the Morchellaceae in the middle Triassic 243.63 (95% highest posterior density [HPD] interval: 169.35-319.89) million years ago (Mya) and the divergence of Morchella from its closest relatives in the early Cretaceous 129.61 (95% HPD interval: 90.26-173.16) Mya, both within western North America. Phylogenetic analyses identified three lineages within Morchella: a basal monotypic lineage represented by Morchella rufobrunnea, and two sister clades comprising the black morels (Elata Clade, 26 species) and yellow morels (Esculenta Clade, 16 species). Morchella possesses a Laurasian distribution with 37/41 species restricted to the Holarctic. All 33 Holarctic species represented by multiple collections exhibited continental endemism. Moreover, 16/18 North American and 13/15 Eurasian species appeared to exhibit provincialism. Although morel fruit bodies produce thousands of explosively discharged spores that are well suited to aerial dispersal, our results suggest that they are poorly adapted at invading novel niches. Morels also appear to have retained the ancestral fruit body plan, which has remained remarkably static since the Cretaceous.     

Phylogeny of the lichen genus Placopsis and its allies based on Bayesian analyses of nuclear and mitochondrial sequences

The phylogenetic relationships of the lichen genus Placopsis and related genera in the Agyriales were analyzed using molecular data. We obtained a total of 66 new sequences from the nuclear ITS, LSU and the mitochondrial SSU rDNA. Phylogenetic analyses were conducted in a Bayesian and a maximum-parsimony framework. Our analyses show that Placopsis is paraphyletic with members of Orceolina nesting within the genus. A morphological character supporting the Placopsis-Orceolina clade is the non-amyloid ascus. The section Aspiciliopsis as defined by sunken fruiting bodies is not supported, but the type species of Aspiciliopsis is more closely related to Orceolina. This clade shares apothecia with reduced amphithecia as apomorphic character. We suggest resurrecting the generic name Aspiciliopsis. Trapelia is the sister genus to Placopsis and Aspiciliopsis/Orceolina.     

Phylogeny and taxonomy of the North American clade of the Ceratocystis fimbriata complex

Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C. caryae and is described as C. smalleyi.     

Arbitrary Primer Based RAPD — A Useful Genetic Marker for Species Identification in Morels

Internal transcribed spacer (ITS) regions of 5.8S ribosomal DNA gene of 46 monospore cultures representing different species of family Morchellaceae were sequenced. Eight putative species namely Morchella esculenta, M. crassipes, M. angusticeps, M. conice, Mitrophora semilibera, Morchella spongiola, M. vulgaris and Verpa conica were designated to the sequenced cultures analyzed based on similarity with the best aligned sequence of the BLAST search. We recorded interspecific polymorphism in ITS regions of the 5.8S r-DNA gene amongst putative species of morels. However no intraspecific ITS polymorphism could be visualized amongst monospores from single and or different ascocarps collected from various geographical regions. PCR RAPDamplified profiles of different monospores were identical at intraspecific levels and could not be distinguished by eight arbitrary primers tested. RAPD profiles exhibited significant interspecific polymorphism distinguishing all the eight putative species from each other by primer OPP-6 (5’- GTG GGT TGA C- 3’) and a custom primer (5’-CGC ACC GCA G-3’). RAPDprofiles generated using aforesaid arbitrary primers can serve as useful genetic markers for species identification and to improve morel systematics. Present study is the first report on exploitation of random primers in species differentiation in morels.     

Phylogenetic analyses reveal deeply divergent species lineages in the genus Sphaerobolus (Phallales: Basidiomycota)

Phylogenetic analyses of 27 artillery fungus (Sphaerobolus sp.) isolates were conducted to identify species boundaries in the genus Sphaerobolus. Multiple gene genealogies inferred from maximum likelihood, Bayesian, and maximum-parsimony analyses of sequence data from individual loci (mtSSU, ITS, EF 1-alpha, and LSU) and a combined dataset (mtSSU, ITS, and EF 1-alpha) concordantly indicate the existence of three deeply divergent lineages in the genus Sphaerobolus, each representing a phylogenetic species. These three phylogenetic species correspond to two known species: Sphaerobolus iowensis and Sphaerobolus stellatus, and a newly discovered species. Suprageneric phylogenetic analyses of the mtSSU and LSU datasets containing representatives of related genera of the gomphoid-phalloid clade of Homobasidiomycetes suggested that the undescribed taxon likely is more closely related to S. stellatus than to S. iowensis.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

Tubeufia asiana, the teleomorph of Aquaphila albicans in the tubeufiaceae, pleosporales, based on cultural and molecular data

The teleomorph of Aquaphila albicans was discovered on submerged wood collected in Thailand. Its black, soft-textured, setose ascomata, bitunicate asci and hyaline to pale brown, multiseptate ascospores indicated an affinity to Tubeufiaceae (Dothideomycetes). After morphological or molecular comparisons with related species in Tubeufia, Acanthostigma and Taphrophila, it is described and illustrated as a new species, T. asiana Sivichai & K.M. Tsui, sp. nov. Finding this Tubeufia teleomorph was surprising, given the falcate conidia of its A. albicans anamorph, which superficially resemble the conidia of Fusarium and not the coiled, helicosporous conidia of other species in Tubeufiaceae. We assessed the phylogenetic relationships of A. albicans-T. asiana with ribosomal sequences from SSU and ITS and partial LSU regions by parsimony and Bayesian analysis. An initial set of 40 taxa representing a wide range of ascomycete families and their SSU sequences from GenBank showed A. albicans-T. asiana to be nested within the Tubeufiaceae with 100% bootstrap support. Their placement was inferred with ITS and partial LSU ribosomal sequences. The nearly identical ITS sequences of two isolates of A. albicans and one isolate of Tubeufia asiana united these fungi as a monophyletic group with 100% bootstrap support and further nested them, with 88% bootstrap support, in a clade containing Helicoon gigantisporum and Helicoma chlamydosporum. This is the first molecular phylogenetic study to place a nonhelicosporous species within the Tubeufiaceae and to show that helical conidia were lost at least once within the family.     

冬季赤水河流域刚毛藻多样性调查及系统发育分析

研究于2019年冬季对赤水河流域开展刚毛藻多样性调查,共设计采样点38个,覆盖赤水河上、中、下游。调查发现:21个采样位点分布有刚毛藻目藻类,其中19个位点有刚毛藻分布。基于核糖体小亚基(SSU rDNA)、核糖体大亚基(LSU rDNA)和内转录间隔区(ITS)对采集样品进行系统发育分析,结果显示:(1)赤水河流域刚毛藻多样性较高,且该流域上、中、下游均有刚毛藻分布;(2)目前淡水刚毛藻类群包含至少10个支系,赤水河流域采集到的刚毛藻覆盖其中6个支系(分别是clade 1、clade 2、clade 4、clade 7、clade 9和clade 10);(3)相比基于SSU+LSU双分子标记构建的系统进化树,基于SSU+ITS+LSU三分子标记构建的进化树各支系支持度更高,可以较有效地将淡水刚毛藻不同支系区分开来。研究较好地展示了冬季赤水河流域刚毛藻的广泛分布及其分子多样性,丰富了中国淡水丝状绿藻的分类研究,也为赤水河段的水生态环境保护提供了基础数据支持。     

Morel Phylogeny and Diagnostics Based on Restriction Fragment Length Polymorphism Analysis of ITS Region of 5.8S Ribosomal RNA Gene

Thirty-six cultures representing eight Morchella and related genera, namely, Morchella esculenta, M. crassipes, M. spongiola, M. vulgaris, M. angusticeps, M. conica, Mitrophora semilibera and Verpa conica were subjected to restriction analysis of ITS1-5.8SITS2 region of rDNA. Six restriction endonuclease enzymes viz TaqI, EcoRl, Mspl, Rsal, Hinfl and BsuRl were used to generate restriction fragments and analysis of phylogenetic relationships among morels. The Amplified Ribosomal DNA Restriction Analysis (ARDRA) not only distinguished yellow morels from black morels but also separated related genera Mitrophora semilibera and Verpa conica from true morels. Simultaneously, each morel species could be separated from each other exhibiting considerable phylogenetic distances. The unique restriction fragment profiles generated by the restriction endonucleases enabled us to identify marker fragments to distinguish each species within and amongst the morel group. Since no intra-specific variation in restriction profiles by the six restriction endonucleases could be visualized among monospores, the technique could be used for rapid identification of wild morel specimens as a cheap alternative to direct sequencing for germplasm cataloguing.     

The phylogeny of the genus Indoplanorbis (Gastropoda,Planorbidae) from Africa and the French West Indies

Indoplanorbis exustus is a freshwater snail known as the intermediate host of various trematode parasites, including different species of the genus Schistosoma. Although its genetic diversity is well described in Asia, the phylogenetic diversity of strains from Africa and Guadeloupe (French West Indies) and their relationship to Asian and South‐East Asian strains remain unknown. To tackle this issue, we sampled individuals from Africa and Guadeloupe, and we computed phylogenetic reconstructions using five molecular markers: partial sequences of two mitochondrial genes, cox1 and 16S, and three nuclear markers, ITS1, ITS2 (Internal Transcribed Spacer 1 and 2) and 5.8S. Our results suggest that strains in Africa and Guadeloupe come from Asia and that they all belong to a single clade that is widespread around the globe.     

A first assessment of genetic variation among Morchella esculenta (morel) populations

Habitat loss and fragmentation have serious consequences for species diversity as well as genetic diversity within a species. As the most sought-after culinary fungus in the Midwest United States, morels (Morchella esculenta and related species) demand the attention of conservationists interested in preserving biological and genetic diversity. Little is known about the natural history of M. esculenta, which is critical information for understanding population dynamics as well as the impacts of habitat fragmentation and harvesting. We report initial results from our long-term studies of genetic variability among fruiting bodies at the Conard Environmental Research Area at Grinnell College, Grinnell, Iowa. Using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), a technique that has been successfully used to examine intrapopulation structure and detect clonal populations in numerous fungi, we found substantially higher levels of genetic polymorphism among 57 fruiting bodies than has been previously reported. Though laboratory studies indicate that the inbreeding potential for this fungus is high, we found little evidence for inbreeding, with only two pairs of the randomly chosen isolates having identical genotypes at the 34 loci examined. This work highlights the importance of further attempts to resolve important aspects of the morel life cycle regarding heterokaryosis and inbreeding potential.     

AFLP and single-strand conformation polymorphism studies of recombination in the entomopathogenic fungus Nomuraea rileyi

In most putative asexual fungi analysed through population genetic studies, recombination has been detected. However, the mechanism by which it is achieved is still not known. A parasexual cycle is known to occur in asexual fungi but there is no evidence, as yet, of its prevalence in natural populations. This study was undertaken to investigate the possibility of a parasexual cycle mediating recombination in the mitosporic fungus Nomuraea rileyi. The genotypic diversity in isolates sampled from an epizootic population from South India was studied through AFLP. The AFLP data were subjected to analysis of molecular variance (AMOVA) and cluster analysis. Great genetic variation was observed in the population including the isolates from a single insect. To assess the occurrence of recombination in the population, single-strand conformation polymorphism (SSCP) of partial regions of two mitochondrial (mt) genes (rRNA genes of LSU and SSU) and a nuclear gene (β tubulin) was performed. The SSCP data were analysed using MP, the tree length permutation test, and multilocus analysis. Recombination was inferred from the SSCP analysis. The occurrence of isolates with diverse genotypes in a single insect; the fact that fungi multiply as hyphal bodies (cell wall-less) in the insect haemolymph; and the inference of recombination in mitochondrial genes (suggesting cytomixis), all indicate that recombination is accomplished by fusion of hyphal bodies of different isolates infecting the insect.     

Presumptive horizontal symbiont transmission in the fungus-growing termite Macrotermes natalensis

All colonies of the fungus-growing termite Macrotermes natalensis studied so far are associated with a single genetically variable lineage of Termitomyces symbionts. Such limited genetic variation of symbionts and the absence of sexual fruiting bodies (mushrooms) on M. natalensis mounds would be compatible with clonal vertical transmission, as is known to occur in Macrotermes bellicosus. We investigated this hypothesis by analysing DNA sequence polymorphisms as codominant SNP markers of four single-copy gene fragments of Termitomyces isolates from 31 colonies of M. natalensis. A signature of free recombination was found, indicative of frequent sexual horizontal transmission. First, all 31 strains had unique multilocus genotypes. Second, SNP markers (n = 55) were largely in Hardy-Weinberg equilibrium (90.9%) and almost all possible pairs of SNPs between genetically unlinked loci were in linkage equilibrium (96.7%). Finally, extensive intragenic recombination was found, especially in the EF1alpha fragment. Substantial genetic variation and a freely recombining population structure can only be explained by frequent horizontal and sexual transmission of Termitomyces. The apparent variation in symbiont transmission mode among Macrotermes species implies that vertical symbiont transmission can evolve rapidly. The unexpected finding of horizontal transmission makes the apparent absence of Termitomyces mushrooms on M. natalensis mounds puzzling. To our knowledge, this is the first detailed study of the genetic population structure of a single lineage of Termitomyces.