A revised phylogeny of Boletaceae using whole genome sequences.

The porcini mushroom family Boletaceae is a diverse, widespread group of ectomycorrhizal (ECM) mushroom-forming fungi that so far has eluded intrafamilial phylogenetic resolution based on morphology and multilocus data sets. In this study, we present a genome-wide molecular data set of 1764 single-copy gene families from a global sampling of 418 Boletaceae specimens. The resulting phylogenetic analysis has strong statistical support for most branches of the tree, including the first statistically robust backbone. The enigmatic Phylloboletellus chloephorus from non-ECM Argentinian subtropical forests was recovered as a new subfamily sister to the core Boletaceae. Time-calibrated branch lengths estimate that the family first arose in the early to mid-Cretaceous and underwent a rapid radiation in the Eocene, possibly when the ECM nutritional mode arose with the emergence and diversification of ECM angiosperms. Biogeographic reconstructions reveal a complex history of vicariance and episodic long-distance dispersal correlated with historical geologic events, including Gondwanan origins and inferred vicariance associated with its disarticulation. Together, this study represents the most comprehensively sampled, data-rich molecular phylogeny of the Boletaceae to date, establishing a foundation for future robust inferences of biogeography in the group.

Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster.

Psychoactive mushrooms in the genus Psilocybe have immense cultural value and have been used for centuries in Mesoamerica. Despite the recent surge of interest in these mushrooms due to the psychotherapeutic potential of their natural alkaloid psilocybin, their phylogeny and taxonomy remain substantially incomplete. Moreover, the recent elucidation of the psilocybin biosynthetic gene cluster is known for only five of ~165 species of Psilocybe, four of which belong to only one of two major clades. We set out to improve the phylogeny of Psilocybe using shotgun sequencing of fungarium specimens, from which we obtained 71 metagenomes including from 23 types, and conducting phylogenomic analysis of 2,983 single-copy gene families to generate a fully supported phylogeny. Molecular clock analysis suggests the stem lineage of Psilocybe arose ~67 mya and diversified ~56 mya. We also show that psilocybin biosynthesis first arose in Psilocybe, with 4 to 5 possible horizontal transfers to other mushrooms between 40 and 9 mya. Moreover, predicted orthologs of the psilocybin biosynthetic genes revealed two distinct gene orders within the biosynthetic gene cluster that corresponds to a deep split within the genus, possibly a signature of two independent acquisitions of the cluster within Psilocybe.

Contrasting continental patterns of adaptive population divergence in the holarctic ectomycorrhizal fungus Boletus edulis.

In the hyperdiverse fungi, the process of speciation is virtually unknown, including for the > 20 000 species of ectomycorrhizal mutualists. To understand this process, we investigated patterns of genome-wide differentiation in the ectomycorrhizal porcini mushroom, Boletus edulis, a globally distributed species complex with broad ecological amplitude. By whole-genome sequencing 160 individuals from across the Northern Hemisphere, we genotyped 792 923 single nucleotide polymorphisms to characterize patterns of genome-wide differentiation and to identify the adaptive processes shaping global population structure. We show that B. edulis exhibits contrasting patterns of genomic divergence between continents, with multiple lineages present across North America, while a single lineage dominates Europe. These geographical lineages are inferred to have diverged 1.62-2.66 million years ago, during a period of climatic upheaval and the onset of glaciation in the Pliocene-Pleistocene boundary. High levels of genomic differentiation were observed among lineages despite evidence of substantial and ongoing introgression. Genome scans, demographic inference, and ecological niche models suggest that genomic differentiation is maintained by environmental adaptation, not physical isolation. Our study uncovers striking patterns of genome-wide differentiation on a global scale and emphasizes the importance of local adaptation and ecologically mediated divergence, rather than prezygotic barriers such as allopatry or genomic incompatibility, in fungal population differentiation.

Whose Ear?: Proposal to conserve the name Auricularia auricula (L.) Underw. for Auricularia auricula-judae (Bull.) Quél.

Auricularia auricula-judae is a saprobic European jelly fungus with traditional culinary and medicinal significance, often said to resemble a human ear. It was originally named Tremella auricula by Linnaeus and has been moved to different genera since, but its specific epithet was also changed from auricula to auricula-judae by Bulliard in 1789, which is not normally a valid nomenclatural alteration. However, due to the practice of "name sanctioning" in the mycological nomenclatural code, this change has been accepted. This article outlines the nomenclatural and cultural history of the controversial name Auricularia auricula-judae and suggests its return to the original specific epithet auricula, as well as the designation of an epitype specimen.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

The melanized layer of Armillaria ostoyae rhizomorphs: Its protective role and functions.

Armillaria ostoyae (Romagn.) Herink is a highly pathogenic fungus that uses exploratory, cordlike structures called rhizomorphs to seek out new sources of nutrition, posing a parasitic threat to natural stands of trees, orchards, and vineyards. Rhizomorphs are notoriously difficult to destroy, and this resilience is due in large part to a melanized layer that protects the rhizomorph. While this structure has been previously observed, its structural and chemical defenses are yet to be discerned. Research was conducted on both lab-cultured and wild-harvested rhizomorph samples. While both environments produce rhizomorphs, only the wild-harvested rhizomorphs produced the melanized layer, allowing for direct investigation of its structure and properties. Imaging, chemical analysis, mechanical testing, and finite element modeling were used to understand the defense mechanisms provided by the melanized layer. Imaging showed a porous outer layer in both types of rhizomorphs, though the pores were smaller in the harvested melanized layer. This melanized layer contained calcium, which provides chemical defense against both human and natural control methods, but was absent from cultured samples. Nanoindentation resulted in a larger variance of hardness values for cultured rhizomorphs than for wild-harvested. Finite element analysis proved that the smaller pore structure of the melanized porous layer had the best balance between maximum deformation and resulting permanent deformation. These results allow for a better understanding of the defenses of this pathogenic fungus, which may lead to better control methods.

What's for dinner this time?: DNA authentication of "wild mushrooms" in food products sold in the USA.

Mushrooms have been consumed by humans for thousands of years, and while some have gastronomic and nutritional value, it has long been recognized that only select species of mushrooms are suitable for consumption. Adverse health effects of consuming poisonous mushrooms range from mild illness to death. Many valuable edible mushrooms are either impractical or unable to be grown commercially, requiring them to be harvested from the wild. In the U.S., products containing these wild-collected mushrooms are often sold with the nonspecific and undefined label "wild mushrooms," although in some cases particular species are listed in the ingredients. However, the ambiguity of the definition of "wild mushrooms" in foods makes it impossible to know which species are involved or whether they are truly wild-collected or cultivated varieties. As a consequence, any individual adverse reactions to consuming the mushrooms in these products cannot be traced to the source due to the minimal regulations around the harvest and sale of wild mushrooms. For this study, we set out to shed light on what species of fungi are being sold as "wild mushrooms" using DNA metabarcoding to identify fungal contents of various food products acquired from locally sourced grocers and a large online retail site. Twenty-eight species of mushroom were identified across 16 food products, ranging from commonly cultivated species to wild species not represented in global DNA databases. Our results demonstrate that "wild mushroom" ingredients often consist entirely or in part of cultivated species such as the ubiquitous white and brown "button" mushrooms and portabella (Agaricus bisporus), oyster (Pleurotus spp.) and shiitake (Lentinula edodes). In other cases truly wild mushrooms were detected but they were not always consistent with the species on the label. More alarmingly, a few products with large distribution potential contained species whose edibility is at best dubious, and at worst potentially toxic.

Emerimicins V-X, 15-Residue Peptaibols Discovered from an Acremonium sp. through Integrated Genomic and Chemical Approaches.

Fermentation of Acremonium tubakii W. Gams isolated from a soil sample collected from the University of Utah led to the isolation and characterization of six new linear pentadecapeptides, emerimicins V-X (1-6). Peptaibols containing 15-residues are quite rare, with only 22 reported. Genome mining and bioinformatic analysis were used to identify the emerimicin 60 kbp eme biosynthetic cluster harboring a single 16-module hybrid polyketide-nonribosomal peptide synthetase. A detailed bioinformatic investigation of the corresponding 15 adenylation domains, combined with 1D and 2D NMR experiments, LC-MS/MS data, and advanced Marfey's method, allowed for the elucidation and absolute configuration of all proteinogenic and nonproteinogenic amino acid residues in 1-6. As some peptaibols possess cytotoxic activity, a zebrafish embryotoxicity assay was used to evaluate the toxicity of the six emerimicins and showed that emerimicin V (1) and VI (2) exhibit the most potent activity. Additionally, out of the six emerimicins, 1 displayed modest activity against Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with MIC values of 64, 32, and 64 µg/mL, respectively.

Reclassification of Pterulaceae Corner (Basidiomycota: Agaricales) introducing the ant-associated genus Myrmecopterula gen. nov., Phaeopterula Henn. and the corticioid Radulomycetaceae fam. nov.

Pterulaceae was formally proposed to group six coralloid and dimitic genera: Actiniceps (=Dimorphocystis), Allantula, Deflexula, Parapterulicium, Pterula, and Pterulicium. Recent molecular studies have shown that some of the characters currently used in Pterulaceae do not distinguish the genera. Actiniceps and Parapterulicium have been removed, and a few other resupinate genera were added to the family. However, none of these studies intended to investigate the relationship between Pterulaceae genera. In this study, we generated 278 sequences from both newly collected and fungarium samples. Phylogenetic analyses supported with morphological data allowed a reclassification of Pterulaceae where we propose the introduction of Myrmecopterula gen. nov. and Radulomycetaceae fam. nov., the reintroduction of Phaeopterula, the synonymisation of Deflexula in Pterulicium, and 53 new combinations. Pterula is rendered polyphyletic requiring a reclassification; thus, it is split into Pterula, Myrmecopterula gen. nov., Pterulicium and Phaeopterula. Deflexula is recovered as paraphyletic alongside several Pterula species and Pterulicium, and is sunk into the latter genus. Phaeopterula is reintroduced to accommodate species with darker basidiomes. The neotropical Myrmecopterula gen. nov. forms a distinct clade adjacent to Pterula, and most members of this clade are associated with active or inactive attine ant nests. The resupinate genera Coronicium and Merulicium are recovered in a strongly supported clade close to Pterulicium. The other resupinate genera previously included in Pterulaceae, and which form basidiomes lacking cystidia and with monomitic hyphal structure (Radulomyces, Radulotubus and Aphanobasidium), are reclassified into Radulomycetaceae fam. nov. Allantula is still an enigmatic piece in this puzzle known only from the type specimen that requires molecular investigation. A key for the genera of Pterulaceae and Radulomycetaceae fam. nov. is also provided here.

Lost in translation: Population genomics and long-read sequencing reveals relaxation of concerted evolution of the ribosomal DNA cistron.

Concerted evolution of the ribosomal DNA array has been studied in numerous eukaryotic taxa, yet is still poorly understood. rDNA genes are repeated dozens to hundreds of times in the eukaryotic genome (Eickbush and Eickbush, 2007) and it is believed that these arrays are homogenized through concerted evolution (Zimmer et al., 1980; Dover, 1993) preventing the accumulation of intragenomic, and intraspecific, variation. However, numerous studies have reported rampant intragenomic and intraspecific variation in the rDNA array (Ganley and Kobayashi, 2011; Naidoo et al., 2013; Hughes and Petersen, 2001; Lindner and Banik, 2011; Li et al., 2013; Lindner et al., 2013; Hughes et al., 2018), contradicting our current understanding of concerted evolution. The internal transcribed spacers (ITS) of the rDNA cistron are the most commonly used DNA barcoding region in Fungi (Schoch et al., 2012), and rely on concerted evolution to homogenize the rDNA array leading to a "barcode gap" (Puillandre et al., 2012). Here we show that in Boletus edulis Bull., ITS intragenomic variation persists at low allele frequencies throughout the rDNA array, this variation does not correlate with genomic relatedness between populations, and rDNA genes may not evolve in a strictly concerted fashion despite the presence of unequal recombination and gene conversion. Under normal assumptions, heterozygous positions found in ITS sequences represent hybridization between populations, yet through allelic mapping of the rDNA array we found numerous heterozygous alleles to be stochastically introgressed throughout, presenting a dishonest signal of gene flow. Moreover, despite the signal of gene flow in ITS, our organisms were highly inbred, indicating a disconnect between true gene flow and barcoding signals. In addition, we show that while the mechanisms of concerted evolution are ongoing in pseudo-heterozygous individuals, they are not fully homogenizing the ITS array. Concerted evolution of the rDNA array may insufficiently homogenize the ITS gene, allowing for misleading signals of gene flow to persist, vastly complicating the use of the ITS locus for DNA barcoding in Fungi.

Cryptic Diversity in Colombian Edible Leaf-Cutting Ants (Hymenoptera: Formicidae).

Leaf-cutting ants are often considered agricultural pests, but they can also benefit local people and serve important roles in ecosystems. Throughout their distribution, winged reproductive queens of leaf-cutting ants in the genus Atta Fabricius, 1804 are consumed as a protein-rich food source and sometimes used for medical purposes. Little is known, however, about the species identity of collected ants and the accuracy of identification when ants are sold, ambiguities that may impact the conservation status of Atta species as well as the nutritional value that they provide to consumers. Here, 21 samples of fried ants bought in San Gil, Colombia, were identified to species level using Cytochrome Oxidase I (COI) barcoding sequences. DNA was extracted from these fried samples using standard Chelex extraction methods, followed by phylogenetic analyses with an additional 52 new sequences from wild ant colonies collected in Panama and 251 publicly available sequences. Most analysed samples corresponded to Atta laevigata (Smith, 1858), even though one sample was identified as Atta colombica Guérin-Méneville, 1844 and another one formed a distinct branch on its own, more closely related to Atta texana (Buckley, 1860) and Atta mexicana (Smith, 1858). Analyses further confirm paraphyly within Atta sexdens (Linnaeus, 1758) and A. laevigata clades. Further research is needed to assess the nutritional value of the different species.

Reclassification of Parapterulicium Corner (Pterulaceae, Agaricales), contributions to Lachnocladiaceae and Peniophoraceae (Russulales) and introduction of Baltazaria gen. nov.

The genus Parapterulicium was first introduced to accommodate two Brazilian species of coralloid fungi with affinities to Pterulaceae (Agaricales). Despite the coralloid habit and the presence of skeletal hyphae, other features, notably the presence of gloeocystidia, dichophyses and papillate hyphal ends, differentiate this genus from Pterulaceaesensu stricto. Fieldwork in Brazil resulted in the rediscovery of two coralloid fungi identifiable as Parapterulicium, the first verified collections of this genus since Corner's original work in the 1950s. Molecular phylogenetic analyses of nrITS and nrLSU sequences from these modern specimens revealed affinities with the /peniophorales clade in the Russulales, rather than Pterulaceae. The presence of distinctive hyphal elements, homologous to the defining features of /peniophorales, is consistent with the phylogenetic evidence and thus clearly distinguished Parapterulicium and its type species P.subarbusculum from Pterulaceae, placing this genus within /peniophorales. Parapterulicium was also found to be polyphyletic so Baltazaria gen. nov. is proposed to accommodate P.octopodites, Scytinostromagalactinum, S.neogalactinum and S.eurasiaticogalactinum also within /peniophorales.

New species of Auritella (Inocybaceae) from Cameroon, with a worldwide key to the known species.

Two new species in the genus Auritella (Inocybaceae) are described as new from tropical rainforest in Cameroon. Descriptions, photographs, line drawings, and a worldwide taxonomic key to the described species of Auritella are presented. Phylogenetic analysis of 28S rDNA and rpb2 nucleotide sequence data suggests at least five phylogenetic species that can be ascribed to Auritella occur in the region comprising Cameroon and Gabon and constitute a strongly supported monophyletic subgroup within the genus. Phylogenetic analysis of ITS data supports the conspecificity of numerous collections attributed to the two new species as well as the monophyly of Australian species of Auritella. This work raises the known number of described species of Auritella to thirteen worldwide, four of which occur in tropical Africa, one in tropical India, and eight in temperate and tropical regions of Australia. This is the first study to confirm an ectomycorrhizal status of Auritella using molecular data.

Kombocles bakaiana gen. sp. nov. (Boletaceae), a new sequestrate fungus from Cameroon.

Kombocles bakaiana gen. sp. nov. is described as new to science. This sequestrate, partially hypogeous fungus was collected around and within the stilt root system of an ectomycorrhizal (ECM) tree of the genus Uapaca (Phyllanthaceae) in a Guineo-Congolian mixed tropical rainforest in Cameroon. Molecular data place this fungus in Boletaceae (Boletales, Agaricomycetes, Basidiomycota) with no clear relationship to previously described taxa within the family. Macro- and micromorphological characters, habitat, and DNA sequence data are provided. Unique morphological features and a molecular phylogenetic analysis of 304 sequences across the Boletales justify the recognition of the new taxa. Kombocles bakaiana is the fourth sequestrate Boletaceae described from the greater African tropics, and the first to be described from Cameroon.

Scaling up discovery of hidden diversity in fungi: impacts of barcoding approaches.

The fungal kingdom is a hyperdiverse group of multicellular eukaryotes with profound impacts on human society and ecosystem function. The challenge of documenting and describing fungal diversity is exacerbated by their typically cryptic nature, their ability to produce seemingly unrelated morphologies from a single individual and their similarity in appearance to distantly related taxa. This multiplicity of hurdles resulted in the early adoption of DNA-based comparisons to study fungal diversity, including linking curated DNA sequence data to expertly identified voucher specimens. DNA-barcoding approaches in fungi were first applied in specimen-based studies for identification and discovery of taxonomic diversity, but are now widely deployed for community characterization based on sequencing of environmental samples. Collectively, fungal barcoding approaches have yielded important advances across biological scales and research applications, from taxonomic, ecological, industrial and health perspectives. A major outstanding issue is the growing problem of 'sequences without names' that are somewhat uncoupled from the traditional framework of fungal classification based on morphology and preserved specimens. This review summarizes some of the most significant impacts of fungal barcoding, its limitations, and progress towards the challenge of effective utilization of the exponentially growing volume of data gathered from high-throughput sequencing technologies.This article is part of the themed issue 'From DNA barcodes to biomes'.

Cortinarius subgenus Callistei in North America and Europe-type studies, diversity, and distribution of species.

Five species of Cortinarius subgenus Callistei, are recognized in Europe and North America. Cortinarius callisteus, C. infucatus, and C. neocallisteus sp. nov. have a broad distribution, extending from western North America to Europe. Cortinarius tofaceus is known from eastern North America and Europe, while C. callistei sp. is known only from one locality in Sweden. All five species are primarily associated with coniferous trees. Previously the species were included either in subgenus Leprocybe or subgenus Cortinarius, but recently they have been separated into subgenus Callistei based on molecular data. Type specimens of the names associated with this subgenus were studied and a neotype proposed for C. tofaceus and an epitype for C. infucatus Barcodes for the species are deposited in RefSeq and UNITE.

New species of Elaphomyces (Elaphomycetaceae, Eurotiales, Ascomycota) from tropical rainforests of Cameroon and Guyana.

The sequestrate false truffles Elaphomyces favosus, E. iuppitercellus, and E. labyrinthinus spp. nov. are described as new to science from the Dja Biosphere Reserve, Cameroon. Elaphomyces adamizans sp. nov. is described as new from the Pakaraima Mountains of Guyana. The Cameroonian species are the first Elaphomyces taxa to be formally described from Africa, occurring in lowland Guineo-Congolian tropical rainforests dominated by the ectomycorrhizal (ECM) canopy tree Gilbertiodendron dewevrei (Fabaceae subfam. Caesalpinioideae). The Guyanese species is the third to be discovered in lowland tropical South America, occurring in forests dominated by the ECM trees Pakaraimaea dipterocarpacea (Dipterocarpaceae) and Dicymbe jenmanii (Fabaceae subfam. Caesalpinioideae). Macromorphological, micromorphological, habitat, and DNA sequence data are provided for each new species. Molecular and morphological data place these fungi in Elaphomycetaceae (Eurotiales, Ascomycota). Unique morphological features are congruent with molecular delimitation of each of the new species based on a phylogenetic analysis of the rDNA ITS and 28S loci across the Elaphomycetaceae. The phylogenetic analysis also suggests that a common ancestor is shared between some Elaphomyces species from Africa and South America, and that species of the stalked, volvate genus Pseudotulostoma may be nested in Elaphomyces.

New species of xerocomoid boletes (Boletaceae) from Himalayan India based on morphological and molecular evidence.

Xerocomus doodhcha and Hortiboletus indorubellus (Boletaceae) from broadleaf montane forest in Sikkim, India, are proposed as new. They are described in detail with supporting morphological illustrations and compared with related taxa using molecular phylogenetic analysis of ITS and 28S rDNA sequences. Xerocomus doodhcha is characterized by a pale brown pileus, basidiospores with a finely bacillate surface under SEM, and phylogenetic proximity to the type species of Xerocomus, X. subtomentosus Hortiboletus indorubellus is characterized by a dark brown to reddish brown pileus, context that turns brownish to brownish orange on bruising, and phylogenetic proximity to Hortiboletus rubellus.

Decoupled genomic elements and the evolution of partner quality in nitrogen-fixing rhizobia.

Understanding how mutualisms evolve in response to a changing environment will be critical for predicting the long-term impacts of global changes, such as increased N (nitrogen) deposition. Bacterial mutualists in particular might evolve quickly, thanks to short generation times and the potential for independent evolution of plasmids through recombination and/or HGT (horizontal gene transfer). In a previous work using the legume/rhizobia mutualism, we demonstrated that long-term nitrogen fertilization caused the evolution of less-mutualistic rhizobia. Here, we use our 63 previously isolated rhizobium strains in comparative phylogenetic and quantitative genetic analyses to determine the degree to which variation in partner quality is attributable to phylogenetic relationships among strains versus recent genetic changes in response to N fertilization. We find evidence of distinct evolutionary relationships between chromosomal and pSym genes, and broad similarity between pSym genes. We also find that nifD has a unique evolutionary history that explains much of the variation in partner quality, and suggest MoFe subunit interaction sites in the evolution of less-mutualistic rhizobia. These results provide insight into the mechanisms behind the evolutionary response of rhizobia to long-term N fertilization, and we discuss the implications of our results for the evolution of the mutualism.

Disentangling visual and olfactory signals in mushroom-mimicking Dracula orchids using realistic three-dimensional printed flowers.

Flowers use olfactory and visual signals to communicate with pollinators. Disentangling the relative contributions and potential synergies between signals remains a challenge. Understanding the perceptual biases exploited by floral mimicry illuminates the evolution of these signals. Here, we disentangle the olfactory and visual components of Dracula lafleurii, which mimics mushrooms in size, shape, color and scent, and is pollinated by mushroom-associated flies. To decouple signals, we used three-dimensional printing to produce realistic artificial flower molds that were color matched and cast using scent-free surgical silicone, to which we could add scent. We used GC-MS to measure scents in co-occurring mushrooms, and related orchids, and used these scents in field experiments. By combining silicone flower parts with real floral organs, we created chimeras that identified the mushroom-like labellum as a source of volatile attraction. In addition, we showed remarkable overlap in the volatile chemistry between D. lafleurii and co-occurring mushrooms. The characters defining the genus Dracula - a mushroom-like, 'gilled' labellum and a showy, patterned calyx - enhance pollinator attraction by exploiting the visual and chemosensory perceptual biases of drosophilid flies. Our techniques for the manipulation of complex traits in a nonmodel system not conducive to gene silencing or selective breeding are useful for other systems.