Rosanne A. Healy

Historical biogeography and diversification of truffles in the Tuberaceae and their newly identified southern hemisphere sister lineage.

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.

High diversity and widespread occurrence of mitotic spore mats in ectomycorrhizal Pezizales

Fungal mitospores may function as dispersal units and/ or spermatia and thus play a role in distribution and/or mating of species that produce them. Mitospore production in ectomycorrhizal (EcM) Pezizales is rarely reported, but here we document mitospore production by a high diversity of EcM Pezizales on three continents, in both hemispheres. We sequenced the internal transcribed spacer (ITS) and partial large subunit (LSU) nuclear rDNA from 292 spore mats (visible mitospore clumps) collected in Argentina, Chile, China, Mexico and the USA between 2009 and 2012. We collated spore mat ITS sequences with 105 fruit body and 47 EcM root sequences to generate operational taxonomic units (OTUs). Phylogenetic inferences were made through analyses of both molecular data sets. A total of 48 OTUs from spore mats represented six independent EcM Pezizales lineages and included truffles and cup fungi. Three clades of seven OTUs have no known meiospore stage. Mitospores failed to germinate on sterile media, or form ectomycorrhizas on Quercus, Pinus and Populus seedlings, consistent with a hypothesized role of spermatia. The broad geographic range, high frequency and phylogenetic diversity of spore mats produced by EcM Pezizales suggests that a mitospore stage is important for many species in this group in terms of mating, reproduction and/or dispersal.

Orbilia ultrastructure, character evolution and phylogeny of Pezizomycotina

Molecular phylogenetic analyses indicate that the monophyletic classes Orbiliomycetes and Pezizomycetes are among the earliest diverging branches of Pezizomycotina, the largest subphylum of the Ascomycota. Although Orbiliomycetes is resolved as the most basal lineage in some analyses, molecular support for the node resolving the relationships between the two classes is low and topologies are unstable. We provide ultrastructural evidence to inform the placement of Orbiliomycetes by studying an Orbilia, a member of the only order (Orbiliales) of the class. The truncate ascus apex in the Orbilia is thin-walled except at the margin, and an irregular wall rupture of the apex permits ascospore discharge. Ascus, ascogenous and non-ascogenous hyphae were simple septate, with septal pores plugged by unelaborated electron-dense, non-membranous occlusions. Globose Woronin bodies were located on both sides of the septum. Nuclear division was characterized by the retention of an intact nuclear envelope, and a two-layered disk-shaped spindle pole body. The less differentiated nature of the spore discharge apparatus and septal pore organization supports an earliest diverging position of Orbiliomycetes within the subphylum, while the closed nuclear division and disk-shaped spindle pole body are interpreted as ancestral state characters for Ascomycota.

How to know the fungi: combining field inventories and DNA‐barcoding to document fungal diversity

The fungi kingdom is among the most diverse eukaryotic lineages on Earth with estimates of several million extant species (O’Brien et al., 2005; Blackwell, 2011; Taylor et al., 2014). Fungi play critical roles in carbon andnutrient cycling of terrestrial and aquatic ecosystems, and they are important pathogens and mutualists (Read & Perez-Moreno, 2003; Taylor et al., 2012; Grossart et al., 2016). More than 80% of plant species form symbioses with fungi and these symbioses have been crucial to the colonization of terrestrial ecosystems (Field et al., 2015a; Selosse et al., 2015). Despite their impacts on primary ecosystem functions, assessments of fungal biodiversity estimate that only c. 10% of fungal species have been described (Bass & Richards, 2011; Hibbett et al., 2011). Traditionally, specimen-based taxonomic studies have been the only way to discover new species. Because most fungi have microscopic life-stages and convergent morphological features (Rivas-Plata & Lumbsch, 2011; Wynns, 2015), many fungal groups remain severely undersampled. DNA-barcoding and highthroughput sequencing methods have provided a new framework for studying fungal biodiversity (Fierer et al., 2012; Schoch et al., 2012; Myrold et al., 2014), and diversity estimates based on environmental sequences have increased exponentially. Although these ‘sequence-based classification and identification’ methods are a powerful means to rapidly detect hidden diversity, careful interpretation of these data is needed to make accurate inferences (K~oljalg et al., 2013; Lindahl et al., 2013; Nguyen et al., 2015; Hibbett et al., 2016). In particular, many environmental sequences cannot be associated with a known fungal species or lineage. This remains a major challenge to decipher fungal community composition and understand ecological roles of fungi in leaf litter, soil, or inside plants (Yahr et al., 2016). In some cases, these fungi are truly undescribed and their ecological roles are unknown but in other cases they represent described taxa for which no sequence is available (Nagy et al., 2011; Nilsson et al., 2016). DNA barcoding of herbarium specimens and culture collections is extremely valuable to link unidentified sequences to known taxa (e.g. Brock et al., 2009; Nagy et al., 2011; Osmundson et al., 2013; Garnica et al., 2016).DNA sequences have been generated from fungal type specimens > 200 years old (Larsson & Jacobsson, 2004), but in many cases obtaining sequences from historical material is challenging (Dentinger et al., 2010). Today’s threats to biodiversity from habitat loss and climate change are occurring at an unprecedented scale, and it is possible that many species may become extinct before they have been discovered (Costello et al., 2013; Monastersky, 2014). In the need to describe and protect as many species as possible we addressed the following questions: what are the best methods to rapidly document fungal biodiversity? Are traditional, specimen-based approaches still useful?

Terfezia disappears from the American truffle mycota as two new genera and Mattirolomyces species emerge

Reexamination and molecular phylogenetic analyses of American Terfezia species and Mattirolomyces tiffanyae revealed that their generic assignments were wrong. Therefore we here propose these combinations: Mattirolomyces spinosus comb. nov. (≡ Terfezia spinosa), Stouffera longii gen. & comb. nov. (≡ Terfezia longii) and Temperantia tiffanyae gen. & comb. nov. (≡ Mattirolomyces tiffanyae). In addition we describe a new species, Mattirolomyces mexicanus spec. nov. All species belong to the Pezizaceae. Based on these results Terfezia is not known from North America, Mattirolomyces is represented by two species and two new monotypic genera are present.

Functional and phylogenetic implications of septal pore ultrastructure in the ascoma of Neolecta vitellina.

Neolecta represents the earliest derived extant ascomycete lineage (Taphrinomycotina) to produce ascomata. For this reason the genus has been of interest with regard to ascoma evolution in ascomycetes. However, the evidence is equivocal regarding whether the Neolecta ascoma is homologous or analogous to ascomata produced in the later derived ascomycete lineages (Pezizomycotina). We investigated phylogenetically informative septal pore ultrastructure of Neolecta vitellina to compare with Pezizomycotina. We found that crystalline bodies that block nonascogenous septal pores in Neolecta differ from Woronin bodies, a synapomorphy for the Pezizomycotina, in three ways: (i) vacuolar origin, (ii) associated material and (iii) being loosely membrane bound. We also observed a unique type of membranous material within the septal pore, as well as distant from the septal pore, that appears to be associated with the endoplasmic reticulum. The vacuolar crystals and membranous material might have a function analagous to septal pore structures (e.g. Woronin bodies, lamellate structures) in the Pezizomycotina. Morphological evidence from our study supports an independently derived septal pore-occluding structure in the Neolecta lineage.

Mycorrhizal detection of native and non-native truffles in a historic arboretum and the discovery of a new North American species, Tuber arnoldianum sp. nov.

During a study comparing the ectomycorrhizal root communities in a native forest with those at the Arnold Arboretum in Massachusetts (USA), the European species Tuber borchii was detected on the roots of a native red oak in the arboretum over two successive years. Since T. borchii is an economically important edible truffle native to Europe, we conducted a search of other roots in the arboretum to determine the extent of colonization. We also wanted to determine whether other non-native Tuber species had been inadvertently introduced into this 140-year-old Arboretum because many trees were imported into the site with intact soil and roots prior to the 1921 USDA ban on these horticultural practices in the USA. While T. borchii was not found on other trees, seven other native and exotic Tuber species were detected. Among the North American Tuber species detected from ectomycorrhizae, we also collected ascomata of a previously unknown species described here as Tuber arnoldianum. This new species was found colonizing both native and non-native tree roots. Other ectomycorrhizal taxa that were detected included basidiomycetes in the genera Amanita, Russula, Tomentella, and ascomycetes belonging to Pachyphlodes, Helvella, Genea, and Trichophaea. We clarify the phylogenetic relationships of each of the Tuber species detected in this study, and we discuss their distribution on both native and non-native host trees.

Atractiella rhizophila, sp. nov., an endorrhizal fungus isolated from the Populus root microbiome

ABSTRACT Among fungi isolated from healthy root mycobiomes of Populus, we discovered a new endorrhizal fungal species belonging to the rust lineage Pucciniomycotina, described here as Atractiella rhizophila. We characterized this species by transmission electron microscopy (TEM), phylogenetic analysis, and plant bioassay experiments. Phylogenetic sequence analysis of isolates and available environmental and reference sequences indicates that this new species, A. rhizophila, has a broad geographic and host range. Atractiella rhizophila appears to be present in North America, Australia, Asia, and Africa and is associated with trees, orchids, and other agriculturally important species, including soybean, corn, and rice. Despite the large geographic and host range of this species sampling, A. rhizophila appears to have exceptionally low sequence variation within nuclear rDNA markers examined. With inoculation studies, we demonstrate that A. rhizophila is nonpathogenic, asymptomatically colonizes plant roots, and appears to foster plant growth and elevated photosynthesis rates.

Evolution of zygomycetous spindle pole bodies: Evidence from Coemansia reversa mitosis

PREMISE OF THE STUDY The earliest eukaryotes were likely flagellates with a centriole that nucleates the centrosome, the microtubule-organizing center (MTOC) for nuclear division. The MTOC in higher fungi, which lack flagella, is the spindle pole body (SPB). Can we detect stages in centrosome evolution leading to the diversity of SPB forms observed in terrestrial fungi? Zygomycetous fungi, which consist of saprobes, symbionts, and parasites of animals and plants, are critical in answering the question, but nuclear division has been studied in only two of six clades. METHODS Ultrastructure of mitosis was studied in Coemansia reversa (Kickxellomycotina) germlings using cryofixation or chemical fixation. Character evolution was assessed by parsimony analysis, using a phylogenetic tree assembled from multigene analyses. KEY RESULTS At interphase the SPB consisted of two components: a cytoplasmic, electron-dense sphere containing a cylindrical structure with microtubules oriented nearly perpendicular to the nucleus and an intranuclear component appressed to the nuclear envelope. Markhams rotation was used to reinforce the image of the cylindrical structure and determine the probable number of microtubules as nine. The SPB duplicated early in mitosis and separated on the intact nuclear envelope. Nuclear division appears to be intranuclear with spindle and kinetochore microtubules interspersed with condensed chromatin. CONCLUSIONS This is the sixth type of zygomycetous SPB, and the third type that suggests a modified centriolar component. Coemansia reversa retains SPB character states from an ancestral centriole intermediate between those of fungi with motile cells and other zygomycetous fungi and Dikarya.

Luteoamylascus aculeatus (Pezizomycetes, Pezizaceae): a new genus and species near the Pachyphlodes–Amylascus lineage

The new taxon Luteoamylascus aculeatus described in this article is proposed to accommodate two collections of a hypogeous ascomycete from central Spain, characterized by a tomentose yellowish peridium, labyrinth-like gleba filled with whitish hyphae, and intensely reacting amyloid asci. ITS, 28S, and RPB2 data suggest that this new taxon is an independent lineage proposed here as the new genus Luteoamylascus. Until now, this lineage was only known from ectomycorrhizal root tips and mitotic spore mats. In phylogenetic analyses, the Luteoamylascus lineage is placed close to the genera Amylascus, Pachyphlodes, and Scabropezia. Morphological data suggest an affinity with Amylascus.