Adaptations of Pseudoxylaria towards a comb-associated lifestyle in fungus-farming termite colonies.

Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria (Xylariaceae), named Pseudoxylaria, whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended ("sit and wait" strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces, and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity.

Resolution of eleven reported and five novel Podaxis species based on ITS phylogeny, phylogenomics, morphology, ecology, and geographic distribution.

The genus Podaxis was first described from India by Linnaeus in 1771, but several revisions of the genus have left the taxonomy unclear. Forty-four Podaxis species names and nine intraspecific varieties are currently accepted, but most fungarium specimens are labelled Podaxis pistillaris. Recent molecular analyses based on barcoding genes suggest that the genus comprises several species, but their status is largely unresolved. Here we obtained basidiospores and photographs from 166 fungarium specimens from around the world and generated a phylogeny based on rDNA internal transcribed spacer ITS1,5.8S and ITS2 (ITS), and a phylogenomic analysis of 3 839 BUSCO genes from low-coverage genomes for a subset of the specimens. Combining phylogenetics, phylogenomics, morphology, ecology, and geographical distribution, spanning 250 years of collections, we propose that the genus includes at least 16 unambiguous species. Based on 10 type specimens (holotype, paratype, and syntype), four recorded species were confirmed, P. carcinomalis, P. deflersii, P. emerici, and P. farlowii. Comparing phylogenetic analysis with described species, including morphology, ecology, and distribution, we resurrected P. termitophilus and designated neotypes, epitypes, or lectotypes for five previously described species, P. aegyptiacus, P. africana, P. beringamensis, P. calyptratus, and P. perraldieri. Lastly, based on phylogenies and morphology of type material, we synonymized three reported species, P. algericus, P. arabicus, and P. rugospora with P. pistillaris, and described five new species that we named P. desolatus, P. inyoensis, P. mareebaensis, P. namaquensis, and P. namibensis. Citation: Li GS, Leal-Dutra CA, Cuesta-Maté A, et al. 2023. Resolution of eleven reported and five novel Podaxis species based on ITS phylogeny, phylogenomics, morphology, ecology, and geographic distribution. Persoonia 51: 257-279. doi: 10.3767/persoonia.2023.51.07.

Generic boundaries in the Ophiostomatales reconsidered and revised.

The Ophiostomatales was erected in 1980. Since that time, several of the genera have been redefined and others have been described. There are currently 14 accepted genera in the Order. They include species that are the causal agents of plant and human diseases and common associates of insects such as bark beetles. Well known examples include the Dutch elm disease fungi and the causal agents of sporotrichosis in humans and animals. The taxonomy of the Ophiostomatales was confused for many years, mainly due to the convergent evolution of morphological characters used to delimit unrelated fungal taxa. The emergence of DNA-based methods has resolved much of this confusion. However, the delineation of some genera and the placement of various species and smaller lineages remains inconclusive. In this study we reconsidered the generic boundaries within the Ophiostomatales. A phylogenomic framework constructed from genome-wide sequence data for 31 species representing the major genera in the Order was used as a guide to delineate genera. This framework also informed our choice of the best markers from the currently most commonly used gene regions for taxonomic studies of these fungi. DNA was amplified and sequenced for more than 200 species, representing all lineages in the Order. We constructed phylogenetic trees based on the different gene regions and assembled a concatenated data set utilising a suite of phylogenetic analyses. The results supported and confirmed the delineation of nine of the 14 currently accepted genera, i.e. Aureovirgo, Ceratocystiopsis, Esteya, Fragosphaeria, Graphilbum, Hawksworthiomyces, Ophiostoma, Raffaelea and Sporothrix. The two most recently described genera, Chrysosphaeria and Intubia, were not included in the multi-locus analyses. This was due to their high sequence divergence, which was shown to result in ambiguous taxonomic placement, even though the results of phylogenomic analysis supported their inclusion in the Ophiostomatales. In addition to the currently accepted genera in the Ophiostomatales, well-supported lineages emerged that were distinct from those genera. These are described as novel genera. Two lineages included the type species of Grosmannia and Dryadomyces and these genera are thus reinstated and their circumscriptions redefined. The descriptions of all genera in the Ophiostomatales were standardised and refined where this was required and 39 new combinations have been provided for species in the newly emerging genera and one new combination has been provided for Sporothrix. The placement of Afroraffaelea could not be confirmed using the available data and the genus has been treated as incertae sedis in the Ophiostomatales. Paleoambrosia was not included in this study, due to the absence of living material available for this monotypic fossil genus. Overall, this study has provided the most comprehensive and robust phylogenies currently possible for the Ophiostomatales. It has also clarified several unresolved One Fungus-One Name nomenclatural issues relevant to the Order. Taxonomic novelties: New genera: Harringtonia Z.W. de Beer & M. Procter, Heinzbutinia Z.W. de Beer & M. Procter, Jamesreidia Z.W. de Beer & M. Procter, Masuyamyces Z.W. de Beer & M. Procter. New species: Masuyamyces massonianae M. Procter & Z.W. de Beer. New combinations: Dryadomyces montetyi (M. Morelet) M. Procter & Z.W. de Beer, Dryadomyces quercivorus (Kubono & Shin. Ito) M. Procter & Z.W. de Beer, Dryadomyces quercus-mongolicae (K.H. Kim et al.) M. Procter & Z.W. de Beer, Dryadomyces sulphureus (L.R. Batra) M. Procter & Z.W. de Beer, Graphilbum pusillum (Masuya) M. Procter & Z.W. de Beer, Grosmannia abieticolens (K. Jacobs & M.J. Wingf.) M. Procter & Z.W. de Beer, Grosmannia altior (Paciura et al.) M. Procter & Z.W. de Beer, Grosmannia betulae (Jankowiak et al.) M. Procter & Z.W. de Beer, Grosmannia curviconidia (Paciura et al.) M. Procter & Z.W. de Beer, Grosmannia euphyes (K. Jacobs & M.J. Wingf.) M. Procter & Z.W. de Beer, Grosmannia fenglinhensis (R. Chang et al.) M. Procter & Z.W. de Beer, Grosmannia gestamen (de Errasti & Z.W. de Beer) M. Procter & Z.W. de Beer, Grosmannia innermongolica (X.W. Liu et al.) M. Procter & Z.W. de Beer, Grosmannia pistaciae (Paciura et al.) M. Procter & Z.W. de Beer, Grosmannia pruni (Masuya & M.J. Wingf.) M. Procter & Z.W. de Beer, Grosmannia taigensis (Linnak. et al.) M. Procter & Z.W. de Beer, Grosmannia trypodendri (Jankowiak et al.) M. Procter & Z.W. de Beer, Harringtonia aguacate (D.R. Simmons et al.) M. Procter & Z.W. de Beer, Harringtonia brunnea (L.R. Batra) M. Procter & Z.W. de Beer, Harringtonia lauricola (T.C. Harr. et al.) Z.W. de Beer & M. Procter, Heinzbutinia grandicarpa (Kowalski & Butin) Z.W. de Beer & M. Procter, Heinzbutinia microspora (Arx) M. Procter & Z.W. de Beer, Heinzbutinia solheimii (B. Strzalka & Jankowiak) Z.W. de Beer & M. Procter, Jamesreidia coronata (Olchow. & J. Reid) M. Procter & Z.W. de Beer, Jamesreidia nigricarpa (R.W. Davidson) M. Procter & Z.W. de Beer, Jamesreidia rostrocoronata (R.W. Davidson & Eslyn) M. Procter & Z.W. de Beer, Jamesreidia tenella (R.W. Davidson) Z.W. de Beer & M. Procter, Leptographium cainii (Olchow. & J. Reid) M. Procter & Z.W. de Beer, Leptographium europioides (E.F. Wright & Cain) M. Procter & Z.W. de Beer, Leptographium galeiforme (B.K. Bakshi) M. Procter & Z.W. de Beer, Leptographium pseudoeurophioides (Olchow. & J. Reid) M. Procter & Z.W. de Beer, Leptographium radiaticola (J.J. Kim et al.) M. Procter & Z.W. de Beer, Masuyamyces acarorum (R. Chang & Z.W. de Beer) M. Procter & Z.W. de Beer, Masuyamyces ambrosius (B.K. Bakshi) M. Procter & Z.W. de Beer, Masuyamyces botuliformis (Masuya) Z.W. de Beer & M. Procter, Masuyamyces jilinensis (R. Chang et al.) M. Procter & Z.W. de Beer, Masuyamyces lotiformis (Z. Wang & Q. Lu) M. Procter & Z.W. de Beer, Masuyamyces pallidulus (Linnak. et al.) M. Procter & Z.W. de Beer, Masuyamyces saponiodorus (Linnak. et al.) M. Procter & Z.W. de Beer, Sporothrix longicollis (Massee & E.S. Salmon) M. Procter & Z.W. de Beer. Citation: de Beer W, Procter M, Wingfield MJ, Marincowitz S, Duong TA (2022). Generic boundaries in the Ophiostomatales reconsidered and revised. Studies in Mycology 101: 57-120. doi: 10.3114/sim.2022.101.02.

An Assessment of the Potential Economic Impacts of the Invasive Polyphagous Shot Hole Borer (Coleoptera: Curculionidae) in South Africa.

Studies addressing the economic impacts of invasive alien species are biased towards ex-post assessments of the costs and benefits of control options, but ex-ante assessments are also required to deal with potentially damaging invaders. The polyphagous shot hole borer Euwallacea fornicatus (Coleoptera: Curculionidae) is a recent and potentially damaging introduction to South Africa. We assessed the potential impact of this beetle by working across economic and biological disciplines and developing a simulation model that included dynamic mutualistic relations between the beetle and its symbiotic fungus. We modeled the potential growth in beetle populations and their effect on the net present cost of damage to natural forests, urban trees, commercial forestry, and the avocado industry over 10 yr. We modeled high, baseline, and low scenarios using discount rates of 8, 6, and 4%, and a plausible range of costs and mortality rates. Models predicted steady growth in the beetle and fungus populations, leading to average declines in tree populations of between 3.5 and 15.5% over 10 yr. The predicted net present cost was 18.45 billion international dollars (Int. $), or about 0.66% of the country's GDP for our baseline scenario ($2.7 billion to $164 billion for low and high scenarios). Most of the costs are for the removal of urban trees that die as a result of the beetle and its fungal symbiont, as has been found in other regions. We conclude that an ex-ante economic assessment system dynamics model can be useful for informing national strategies on invasive alien species management.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

Fire impacts bacterial composition in Protea repens (Proteaceae) infructescences.

The diverse bacterial communities in and around plants provide important benefits, such as protection against pathogens and cycling of essential minerals through decomposition of moribund plant biomass. Biodiverse fynbos landscapes generally have limited deadwood habitats due to the absence of large trees and frequent fire. In this study, we determined the effect of a fire disturbance on the bacterial communities in a fynbos landscape dominated by the shrub Protea repens using 16S ribosomal RNA amplicon sequencing. The bacterial community composition in newly formed fruiting structures (infructescences) and soil at a recently burnt site was different from that in an unburnt site. Bacteria inhabiting P. repens infructescences were similar to well-known taxa from decomposing wood and litter. This suggests a putative role for these aboveground plant structures as reservoirs for postfire decomposer bacteria. The results imply that inordinately frequent fires, which are commonplace in the Anthropocene, are a significant disturbance to bacterial communities and could affect the diversity of potentially important microbes from these landscapes.

Phylogenetic and phylogenomic analyses reveal two new genera and three new species of ophiostomatalean fungi from termite fungus combs.

The Ophiostomatales (Ascomycota) accommodates more than 300 species characterized by similar morphological adaptations to arthropod dispersal. Most species in this order are wood-inhabiting fungi associated with bark or ambrosia beetles. However, a smaller group of species occur in other niches such as in soil and Protea infructescences. Recent surveys of Termitomyces fungus gardens (fungus combs) of fungus-growing termites led to the discovery of characteristic ophiostomatalean-like fruiting structures. In this study, these ophiostomatalean-like fungi were identified using morphological characteristics, conventional molecular markers, and whole genome sequencing. In addition, the influence of the extracts derived from various parts of Termitomyces combs on the growth of these fungi in culture was considered. Based on phylogenomic analyses, two new genera (Intubia and Chrysosphaeria) were introduced to accommodate these ophiostomatalean species. Phylogenetic analyses revealed that the isolates resided in three well-supported lineages, and these were described as three new species (Intubia macrotermitinarum, I. oerlemansii, and Chrysosphaeria jan-nelii). Culture-based studies showed that these species do not depend on the Termitomyces comb material for growth.

Ancestral predisposition toward a domesticated lifestyle in the termite-cultivated fungus Termitomyces.

The ancestor of termites relied on gut symbionts for degradation of plant material, an association that persists in all termite families.1,2 However, the single-lineage Macrotermitinae has additionally acquired a fungal symbiont that complements digestion of food outside the termite gut.3 Phylogenetic analysis has shown that fungi grown by these termites form a clade-the genus Termitomyces-but the events leading toward domestication remain unclear.4 To address this, we reconstructed the lifestyle of the common ancestor of Termitomyces using a combination of ecological data with a phylogenomic analysis of 21 related non-domesticated species and 25 species of Termitomyces. We show that the closely related genera Blastosporella and Arthromyces also contain insect-associated species. Furthermore, the genus Arthromyces produces asexual spores on the mycelium, which may facilitate insect dispersal when growing on aggregated subterranean fecal pellets of a plant-feeding insect. The sister-group relationship between Arthromyces and Termitomyces implies that insect association and asexual sporulation, present in both genera, preceded the domestication of Termitomyces and did not follow domestication as has been proposed previously. Specialization of the common ancestor of these two genera on an insect-fecal substrate is further supported by similar carbohydrate-degrading profiles between Arthromyces and Termitomyces. We describe a set of traits that may have predisposed the ancestor of Termitomyces toward domestication, with each trait found scattered in related taxa outside of the termite-domesticated clade. This pattern indicates that the origin of the termite-fungus symbiosis may not have required large-scale changes of the fungal partner.

Comparative Genomic and Metabolic Analysis of Streptomyces sp. RB110 Morphotypes Illuminates Genomic Rearrangements and Formation of a New 46-Membered Antimicrobial Macrolide.

Morphotype switches frequently occur in Actinobacteria and are often associated with disparate natural product production. Here, we report on differences in the secondary metabolomes of two morphotypes of a Streptomyces species, including the discovery of a novel antimicrobial glycosylated macrolide, which we named termidomycin A. While exhibiting an unusual 46-member polyene backbone, termidomycin A (1) shares structural features with the clinically important antifungal agents amphotericin B and nystatin A1. Genomic analyses revealed a biosynthetic gene cluster encoding for a putative giant type I polyketide synthase (PKS), whose domain structure allowed us to propose the relative configuration of the 46-member macrolide. The architecture of the biosynthetic gene cluster was different in both morphotypes, thus leading to diversification of the product spectrum. Given the high frequency of genomic rearrangements in Streptomycetes, the metabolic analysis of distinct morphotypes as exemplified in this study is a promising approach for the discovery of bioactive natural products and pathways of diversification.

Genome reduction and relaxed selection is associated with the transition to symbiosis in the basidiomycete genus Podaxis.

Insights into the genomic consequences of symbiosis for basidiomycete fungi associated with social insects remain sparse. Capitalizing on viability of spores from centuries-old herbarium specimens of free-living, facultative, and specialist termite-associated Podaxis fungi, we obtained genomes of 10 specimens, including two type species described by Linnaeus >240 years ago. We document that the transition to termite association was accompanied by significant reductions in genome size and gene content, accelerated evolution in protein-coding genes, and reduced functional capacities for oxidative stress responses and lignin degradation. Functional testing confirmed that termite specialists perform worse under oxidative stress, while all lineages retained some capacity to cleave lignin. Mitochondrial genomes of termite associates were significantly larger; possibly driven by smaller population sizes or reduced competition, supported by apparent loss of certain biosynthetic gene clusters. Our findings point to relaxed selection that mirrors genome traits observed among obligate endosymbiotic bacteria of many insects.

New and Interesting Fungi. 4.

An order, family and genus are validated, seven new genera, 35 new species, two new combinations, two epitypes, two lectotypes, and 17 interesting new host and / or geographical records are introduced in this study. Validated order, family and genus: Superstratomycetales and Superstratomycetaceae (based on Superstratomyces ). New genera: Haudseptoria (based on Haudseptoria typhae); Hogelandia (based on Hogelandia lambearum); Neoscirrhia (based on Neoscirrhia osmundae); Nothoanungitopsis (based on Nothoanungitopsis urophyllae); Nothomicrosphaeropsis (based on Nothomicrosphaeropsis welwitschiae); Populomyces (based on Populomyces zwinianus); Pseudoacrospermum (based on Pseudoacrospermum goniomae). New species: Apiospora sasae on dead culms of Sasa veitchii (Netherlands); Apiospora stipae on dead culms of Stipa gigantea (Spain); Bagadiella eucalyptorum on leaves of Eucalyptus sp. (Australia); Calonectria singaporensis from submerged leaf litter (Singapore); Castanediella neomalaysiana on leaves of Eucalyptus sp. (Malaysia); Colletotrichum pleopeltidis on leaves of Pleopeltis sp. (South Africa); Coniochaeta deborreae from soil (Netherlands); Diaporthe durionigena on branches of Durio zibethinus (Vietnam); Floricola juncicola on dead culm of Juncus sp. (France); Haudseptoria typhae on leaf sheath of Typha sp. (Germany); Hogelandia lambearum from soil (Netherlands); Lomentospora valparaisensis from soil (Chile); Neofusicoccum mystacidii on dead stems of Mystacidium capense (South Africa); Neomycosphaerella guibourtiae on leaves of Guibourtia sp. (Angola); Niesslia neoexosporioides on dead leaves of Carex paniculata (Germany); Nothoanungitopsis urophyllae on seed capsules of Eucalyptus urophylla (South Africa); Nothomicrosphaeropsis welwitschiae on dead leaves of Welwitschia mirabilis (Namibia); Paracremonium bendijkiorum from soil (Netherlands); Paraphoma ledniceana on dead wood of Buxus sempervirens (Czech Republic); Paraphoma salicis on leaves of Salix cf. alba (Ukraine); Parasarocladium wereldwijsianum from soil (Netherlands); Peziza ligni on masonry and plastering (France); Phyllosticta phoenicis on leaves of Phoenix reclinata (South Africa); Plectosphaerella slobbergiarum from soil (Netherlands); Populomyces zwinianus from soil (Netherlands); Pseudoacrospermum goniomae on leaves of Gonioma kamassi (South Africa); Pseudopyricularia festucae on leaves of Festuca californica (USA); Sarocladium sasijaorum from soil (Netherlands); Sporothrix hypoxyli in sporocarp of Hypoxylon petriniae on Fraxinus wood (Netherlands); Superstratomyces albomucosus on Pycnanthus angolensis (Netherlands); Superstratomyces atroviridis on Pinus sylvestris (Netherlands); Superstratomyces flavomucosus on leaf of Hakea multilinearis (Australia); Superstratomyces tardicrescens from human eye specimen (USA); Taeniolella platani on twig of Platanus hispanica (Germany), and Tympanis pini on twigs of Pinus sylvestris (Spain). Citation: Crous PW, Hernández-Restrepo M, Schumacher RK, Cowan DA, Maggs-Kölling G, Marais E, Wingfield MJ, Yilmaz N, Adan OCG, Akulov A, Álvarez Duarte E, Berraf-Tebbal A, Bulgakov TS, Carnegie AJ, de Beer ZW, Decock C, Dijksterhuis J, Duong TA, Eichmeier A, Hien LT, Houbraken JAMP, Khanh TN, Liem NV, Lombard L, Lutzoni FM, Miadlikowska JM, Nel WJ, Pascoe IG, Roets F, Roux J, Samson RA, Shen M, Spetik M, Thangavel R, Thanh HM, Thao LD, van Nieuwenhuijzen EJ, Zhang JQ, Zhang Y, Zhao LL, Groenewald JZ (2021). New and Interesting Fungi. 4. Fungal Systematics and Evolution 7: 255-343. doi: 10.3114/fuse.2021.07.13.

The Termite Fungal Cultivar Termitomyces Combines Diverse Enzymes and Oxidative Reactions for Plant Biomass Conversion.

Macrotermitine termites have domesticated fungi in the genus Termitomyces as their primary food source using predigested plant biomass. To access the full nutritional value of lignin-enriched plant biomass, the termite-fungus symbiosis requires the depolymerization of this complex phenolic polymer. While most previous work suggests that lignocellulose degradation is accomplished predominantly by the fungal cultivar, our current understanding of the underlying biomolecular mechanisms remains rudimentary. Here, we provide conclusive omics and activity-based evidence that Termitomyces employs not only a broad array of carbohydrate-active enzymes (CAZymes) but also a restricted set of oxidizing enzymes (manganese peroxidase, dye decolorization peroxidase, an unspecific peroxygenase, laccases, and aryl-alcohol oxidases) and Fenton chemistry for biomass degradation. We propose for the first time that Termitomyces induces hydroquinone-mediated Fenton chemistry (Fe2+ + H2O2 + H+ → Fe3+ + •OH + H2O) using a herein newly described 2-methoxy-1,4-dihydroxybenzene (2-MH2Q, compound 19)-based electron shuttle system to complement the enzymatic degradation pathways. This study provides a comprehensive depiction of how efficient biomass degradation by means of this ancient insect's agricultural symbiosis is accomplished. IMPORTANCE Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems. To date, most research has focused on the enzymatic contributions of microbial partners to carbohydrate decomposition. Here, we provide genomic, transcriptomic, and enzymatic evidence that Termitomyces also employs redox mechanisms, including diverse ligninolytic enzymes and a Fenton chemistry-based hydroquinone-catalyzed lignin degradation mechanism, to break down lignin-rich plant material. Insights into these efficient decomposition mechanisms reveal new sources of efficient ligninolytic agents applicable for energy generation from renewable sources.

Female-biased sex allocation and lack of inbreeding avoidance in Cubitermes termites.

Sexually reproducing organisms face a strong selective pressure to find a mate and ensure reproduction. An important criterion during mate-selection is to avoid closely related individuals and subsequent potential fitness costs of resulting inbred offspring. Inbreeding avoidance can be active through kin recognition during mate choice, or passive through differential male and female-biased sex ratios, which effectively prevents sib-mating. In addition, sex allocation, or the resources allotted to male and female offspring, can impact mating and reproductive success. Here, we investigate mate choice, sex ratios, and sex allocation in dispersing reproductives (alates) from colonies of the termite Cubitermes tenuiceps. Termites have a short time to select a mate for life, which should intensify any fitness consequences of inbreeding. However, alates did not actively avoid inbreeding through mate choice via kin recognition based on genetic or environmental cues. Furthermore, the majority of colonies exhibited a female-biased sex ratio, and none exhibited a male-bias, indicating that differential bias does not reduce inbreeding. Sex allocation was generally female-biased, as females also were heavier, but the potential fitness effect of this costly strategy remains unclear. The bacterium Wolbachia, known in other insects to parasitically distort sex allocation toward females, was present within all alates. While Wolbachia is commonly associated with termites, parasitism has yet to be demonstrated, warranting further study of the nature of the symbiosis. Both the apparent lack of inbreeding avoidance and potential maladaptive sex allocation implies possible negative effects on mating and fitness.

Novel Fusarium mutualists of two Euwallacea species infesting Acacia crassicarpa in Indonesia.

Several species in the Euwallacea fornicatus complex have emerged as important pests of woody plants globally, particularly in habitats where they are invasive aliens. These beetles live in obligate symbioses with fungi in the genus Fusarium. In this study, we identified Euwallacea spp. and their fungal mutualists that have emerged as pests of planted Acacia crassicarpa in Riau, Indonesia. Morphological identification and phylogenetic analyses of the mitochondrial cytochrome oxidase c subunit I (COI) gene confirmed that E. similis and E. perbrevis are the most abundant beetles infesting these trees. Multilocus phylogenetic analyses of their fungal mutualists revealed their nonspecific association with six Fusarium species. These included F. rekanum and five novel Fusarium mutualists within the Fusarium solani species complex (FSSC), four of which reside in the Ambrosia Fusarium Clade (AFC). These new species are described here as F. akasia, F. awan, F. mekan, F. variasi, and F. warna.

Comparative Genomics Reveals Prophylactic and Catabolic Capabilities of Actinobacteria within the Fungus-Farming Termite Symbiosis.

Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth's ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis.IMPORTANCEActinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis.

Ophiostomatalean fungi associated with wood boring beetles in South Africa including two new species.

Ambrosia beetles are small wood inhabiting members of the Curculionidae that have evolved obligate symbioses with fungi. The fungal symbionts concentrate nutrients from within infested trees into a usable form for their beetle partners, which then utilize the fungi as their primary source of nutrition. Ambrosia beetle species associate with one or more primary symbiotic fungal species, but they also vector auxiliary symbionts, which may provide the beetle with developmental or ecological advantages. In this study we isolated and identified ophiostomatalean fungi associated with ambrosia beetles occurring in a native forest area in South Africa. Using a modified Bambara beetle trap, living ambrosia beetle specimens were collected and their fungal symbionts isolated. Four beetle species, three Scolytinae and one Bostrichidae, were collected. Five species of ophiostomatalean fungi were isolated from the beetles and were identified using both morphological characters and DNA sequence data. One of these species, Raffaelea sulphurea, was recorded from South Africa for the first time and two novel species were described as Ceratocystiopsis lunata sp. nov. and Raffaelea promiscua sp. nov.

A new species in the Mycosphaerellaceae from Cecidomyiidae leaf galls on Avicennia marina in South Africa.

During studies to investigate the health of mangrove trees in South Africa, high numbers of Avicennia marina were found with leaf galls caused by unidentified adults and larvae of midges (Cecidomyiidae). Fungal fruiting structures were commonly observed on the abaxial areas of the galls. To determine the identity of the fungi associated with the gall midges, phylogenetic analyses using multigene sequence data were used. The nuclear large subunit (LSU), internal transcribed spacer (ITS), and a portion of the actin gene region (ACT), were amplified and analyzed. The results revealed that the fungal fruiting structures represent a new taxon in the Mycosphaerellaceae described here as Zasmidium mangrovei sp. nov. This is the first report of a species in the Mycosphaerellaceae associated with cecidomyiid leaf galls on A. marina.

Ophiostomatoid fungi including a new species associated with Asian larch bark beetle Ips subelongatus, in Heilongjiang (Northeast China).

Ips subelongatus (Coleoptera, Scolytinae) is an important bark beetle species that infests Larix spp. in Asia. Individuals of this beetle are vectors of ophiostomatoid fungi, on their exoskeletons, that are transmitted to infested trees. In this study, the symbiotic assemblage of ophiostomatoid fungi associated with I. subelongatus in Northeast China was studied. Fungal isolates were identified based on their morphological characters and sequences of ITS, beta-tubulin, elongation factor 1-alpha and calmodulin gene regions. In total, 48 isolates were collected and identified, residing in six taxa. These included a novel species, described here as Ophiostoma gmelinii sp. nov.

Screening for Susceptibility of Macadamia to Euwallacea fornicatus and its Fungal Symbiont Fusarium euwallaceae.

The polyphagous shothole borer (Euwallacea fornicatus, PSHB), an ambrosia beetle, with its fungal symbiont, Fusarium euwallaceae, is responsible for Fusarium dieback (FD) in a wide range of woody hosts. In 2019, the first suspected case of E. fornicatus was reported in macadamia in South Africa. The aims of this study were to confirm the E. fornicatus report and thereafter to assess the susceptibility of commercially planted macadamia cultivars to FD caused by F. euwallaceae. The identities of the beetle and associated fungal symbionts were confirmed by means of DNA sequence analysis of the 28S ribosomal large subunit gene for beetles and the internal transcribed spacer region for fungi. Isolates identified as Fusarium species were further characterized by phylogenetic analysis of the translation elongation factor 1α and the ß-tubulin gene regions. Thereafter, Koch's postulates regarding F. euwallaceae were fulfilled on a mature Macadamia integrifolia tree planted at the experimental farm of the University of Pretoria. In order to determine susceptibility against FD, additional cultivar screening was conducted on nine commercially planted cultivars by means of pathogenicity trials using sterilized or inoculated toothpicks inserted into detached branches. Detached branch inoculations showed no significant lesion development six weeks post inoculation, except for cultivar 816. The restricted growth of F. euwallaceae observed in macadamia tissues therefore suggests that macadamia may not be a suitable host for F. euwallaceae and that the threat of FD in macadamia in the event of E. fornicatus infestation is less than for other E. fornicatus hosts. Future work on beetle attraction to macadamia is recommended for a more comprehensive understanding of the interaction between E. fornicatus and its fungal symbionts and macadamia.