How mushrooms tend to break through the genetic dead end

Genes, genetics, genomics, and the roles of mushrooms and toadstools in the global carbon cycle (GCC) are reviewed here. The literature survey is a tribute to the contributions made by Hungary and Hungarian scientists to fungi and mushroom research. For this reason, the names of the fungi discussed are also given in Hungarian. Fungi – like wood eating insects – are the main decomposers (a type of consumers, syn.: heterotrophs) and consequently recycle the biomass produced by photosynthetic organisms (i.e., the producers, syn.: autotrophs). Photosynthesis is driven by the solar energy day by day (by photo-autotrophs) (i.e., primary producers of chlorophyllous plants), and primary production night by night is performed by chemo-autotroph prokaryotes. Only autotrophic organisms can produce organic materials in the Earth to supply food and feed the hetero-trophs (e.g., animals, including Human), and sapro-trophs (i.e., decomposers) including fungi and bacteria. The crucial excess oxygen from the oxygenic photosynthesis supports diverse life on Earth. Mushrooms were found to have 100-1000 times smaller genomes than plants or animals, however, enormous genome expansions e.g., of Armillarias (Eng./Hung.: honey mushrooms / tuskógombák) have indicated recently that fungi continue to expand their genome. Comparative genome analyses of Polyporales mushrooms have recently identified an ongoing transitioning from white-rot (WR) towards brown-rot (BR) life style with loss of genes encoding enzymes to decay cell wall components of plants (and woody plants, the trees) including cellulose, hemicellulases, lignin (the three together are also called lignocelluloses), and pectin. In the case of lignin, genes of ligninase enzymes, which are capable of digesting lignin only, developed only in wood-decay fungi which underscore their role in GCC. Symbiosis between fungi and green algae or cyanobacteria created a new phylum the Lichens (Mycophycophyta) in evolution. A tripartite symbiosis among achlorophyllous (i.e., parasitic) mycoheterotrophic plants ↔ mycorrhizal fungi ↔ and autotrophic green plants were re-discovered recently. Here we review the achievements of research of Di-caria true fungi (Eu-mycota) of both Asco-mycota (Eng./Hung.: Sac fungi / Tömlős gombák) and Basidio-mycota (Eng./Hung.: Club fungi / Bazidiumos gombák) with special emphasis on genes, genetics and genomic and evolutionary relationships. In brackets, the commercial mushroom names of English (Eng.) and Hungarian (Hung.) are given.


Introduction
Photosynthesis (i.e., Global Carbon Cycle, GCC) drives the fundamental basis of life on Earth (Calvin, 1961;Szalay et al., 1967;Mitchell, 1978;Lehoczki et al., 1992).It goes through the 'auto-troph' organisms of both chemo-autotroph bacteria, and photo-autotroph plants by fixing CO 2 from the atmosphere and returning back O 2 into the atmosphere, and, by means of producing organic materials ('biomass') they supply organisms of 'heterotrophs', and 'sapro-trophs'.These cycles have been ongoing day by day (in photo-auto-troph chlorophyllous plants), and night by night (in chemo-auto-troph bacteria) since the emergence of these processes (Field et al., 1998;Nealson and Conrad, 1999;Gyulai et al., 2019).
Photoautotrophic organisms are comprised of (i) photosynthetic bacteria, cyanobacteria and protists; and (ii) chlorophyllous plants of seedless algae, moss, and ferns, and seed plants of Gymnosperm trees (all Gymnosperms are woody plants; there are no 'herbaceous Gymnosperm' species), and Angiosperm plants and trees (Calvin, 1961;Mitchell, 1978).
The GCC cycle is driven by physical sunlight radiational energy (i.e., APAR -the Absorbed Photosynthetically Active solar Radiation within the range of 400 to 700 nm), which is converted into chemical energy ('biomass') by photosynthesis, which is about 1.0 -1.15 x 10 11 metric tons of carbon (taken from CO 2 ) per year with roughly equal contributions from land and oceans.The sunlight energy captured by photosynthesis is of approximately 130 terawatts globally (i.e., net primary production -NPP), which is about three times bigger than the power consumption of human civilization 'currently' (Field et al., 1998).
The 'excess' oxygen that oxygenic photosynthesis produces is the single and unique process to oxygenate and maintain diverse life on Earth.The remains and 'leftovers' of heterotrophs are used up further by saprotrophs (i.e., decomposers) including bacteria and fungi of both micro and higher fungi.
Of both Basidiomycota and Ascomycota, about 1,500 mushroom species were reported to grow naturally in Japan (Mizuno, 1995); about 3,487 species were recorded in Hungary to date (Hungarian Mycological Society); and approximately 5,500 known species (estimated total at 9,000) were recorded in Ireland (Cullen and Fox, 2010).
In evolutionarily speculations, fungi were assumed to be more closely related to animals than plants based on the chemical structure of hyphal cell walls; the composition of mitochondrial and nuclear DNA, and ribosomal RNA; and the nutritional and metabolic pathways (Burnett, 1987).
An early study of amino acid composition of fungal elongation factor 3 (EF-3), and its gene (ycf3) was also found to be highly homologous to mammalian myosin (Beltfield, 1995).However, these speculations have not been confirmed by the current molecular methods, e.g., by the sequence analyses of genes of the evolutionarily highly conserved GAPDH enzyme proteins (Gyulai et al., 2019).An important biochemical difference was found in the storage form of sugar (i.e., carbon storage) as in glycogen of fungi, bacteria, protozoa and animal cells; compared to starch in plant cells.The first speculative hypothesis of the algal origin of fungi assumed the loss of chlorophyll (and consequently the chloroplasts) from the ancient algal cells that evolved to fungi (Martin, 1955).However, it is believed currently that fungi descended from protozoanlike ancestors (De Clerck et al., 2012).Nevertheless, chlorophyll loss is not rare among parasitic plants, e.g., the tropical Cathaya argyrophylla (NCBI# NC_014589), and the temperate zone Cuscuta obtusiflora (Eng./Hung.: dodder / aranka) (family Orobanchaceae) (NCBI# NC_009949).Interestingly, a new invasive mushroom species in Hungary, the Chlorophyllum molybdites (Eng./Hung.: green-spored parasol / mérgező őzlábgomba) (Massee, 1898), has chlorophyll green spores, unlikely from chlorophyll.
Phylogenetics (Frank, 1885;Heckman et al., 2001;James et al., 2006), and ontogenesis of fungi also shows unique characters: spores grow to haploid (n) hyphae, and when hyphae meet, they mate and fuse, and produce dikariotic hypha (2n) (hence the name Dicaria).Hyphae later grow a very large (up to square kilometers) net of fungal mycelium (Stanosz et al., 1987;Smith et al., 1992), which produces the fruiting bodies known as mushrooms.
Similar to the clonally propagated, largest and oldest giant flowering plants like the aquatic Posidonia oceanica (monocot, Alismatales) (Telesca et al., 2015) with 100,000 year age and over 8 km long growing areas in Mediterranean Sea; and the trembling giant (Populus tremuloides) with 80, 000 year age (Utah, U.S.A.) (Grant, 1993), there are also giant mushrooms.
In the U.S. forests, the "giant" honey mushroom Armillaria bulbosa showed how the mycelium grown from a single fungal spore cell can colonize hectares (up to 15 ha) of forests and live 1,500 -2,400 years with a final total weight of at least 10 tones to date (Smith et al., 1992).This prolonged capacity to grow is attributable to the white-rot (WR) life style of Armillaria species, which are 'fully armed' with WR enzyme genes ready to degrade all woody plant cell wall components such as cellulose, hemicellulose, lignin, and pectin (Kusano, 1911;Lan et al., 1994).A similar species of Desarmillaria tabescens (syn.: Armillaria t.) (Eng./Hung.: ringless honey fungus / csoportos tuskógomba) also can colonize hectares in the forests.
(3) Small eukaryotic fungal genome condensed in small chromosomes Not only the transposon research, but also fungal cytogenetics started when Barbara McClintock (1945) studied and succeeded in visualizing the extremely small chromosomes of the fungus Neurospora crassa (the 'today boring' but) the former model organism of early geneticists (McClintock, 1945).Later, the invention of pulsed field gradient gel electrophoresis (PFGE) (Schwartz et al., 1982;Schwartz and Cantor, 1984) opened a new era of fungal cytogenetics (Zolan, 1995;Lai et al., 1989;Fekete et al., 1993).Currently, genome sequencing projects revealed fundamental correlations between fungal genomes and functions (Floudas et al., 2015;Sipos et al., 2017).Similarly to fungi chromosome sizes, genome sizes of fungi (Fig. 2a,b) were found to be ten times smaller than the genome of the smallest known grass genome of diploid ancient grass genus Oropetium (245 x 10 6 bp DNA; encoding for 28,466 protein-coding genes; and with chloroplast DNA of 0.135324 x 10 6 bp) (VanBuren et al., 2015).Wood decaying fungilike wood eating insectsare the main decomposers and consequently recyclers of biomass produced by photosynthesis.
One classification divides fungi into two main groups such as saprotrophs, which degrade non-living organic substrates, and biotrophs, which obtain carbon (i.e., sugar) from living hosts.The ecological lifestyles of fungi include dung decay (DG), wood decay (WD), and mycorrhiza (MR) fungi (Reynolds et al., 2018).
Genome sequencing of the EM Tuber melanosporum (Eng./Hung.: black truffle / fekete szarvasgomba) also showed an extreme genome expansion (0.125 x 10 9 bp) as a result of the proliferation of transposable elements accounting for about 58% of the genome (Fig. 2a).In contrast, this large genome only contains approximately 7,500 protein-coding genes with very rare multigene families (Martin et al., 2010).
The structure and composition of hemicellulose and lignin components of wood cell walls are different in softwood (SW) (Hung.: puhafa) (Gymnosperm coniferous trees), which are rich in galactoglucomannan and guaiacyl lignin) compared to hardwood (HW) (Hung.: keményfa) (deciduous trees) rich in glucuronoxylan, syringyl and guaiacyl lignins).The SW trees are predominant of land plant biomass in the Northern hemisphere; however, they are more recalcitrant to wood decaying fungi than HW trees.WR basidiomycete (Polyporales) fungus Phanerochaete carnosa decays strictly only coniferous SW trees.
Another WR fungus Phanerochaete chrysosporium mainly infects hardwood trees.The genome sequencing of the two fungi revealed, that WR-SW P. carnosa genome is enriched (nearly doubled) with genes that encode cytochrome-P450-monooxygenases (a group of hemoproteins with plant heme cofactor) with 266 copies of P450s (CYPs) that participate in extractives degradation, and manganese peroxidases involved in lignin degradation.About 33% of the P450 genes in the P. carnosa genome were found tandemly duplicated.This high P450 gene number is higher than in the third species of BR P. placenta (236 copies of P450s) (Suzuki et al., 2012).
Further genome analyses are organized by the '1,000 Fungal Genomes Projects' launched by the US Department of Energy (DOE) Joint Genome Institute (JGI) (Nordberg et al., 2014) (Fig. 2a,b).One of the most current results revealed how a dry rot (DR) fungus Serpula (syn.: Merulius) lacrymans (Boletales) (Eng./Hung.: dry rot / könnyező házigomba) became a very effective brown rot (BR) fungus compared to its wild relatives and became a successful invader of timbers of pine, fir, and spruce in houses (Balasundaram et al., 2018).

(6) Fungi parasitized by Plants
Myco-heterotrophy is a form of tripartite symbiosis among (i) achlorophyllous mycoheterotrophic parasitic plants, which parasitizes (ii) mycorrhizal fungi that are attached to roots of (iii) photosynthetic plants.In this way mycoheterotrophic plants get all or part of their mineral food from photosynthetic plants through the bridge of mycorrhizal fungi (the haustrorial parasite plants are different).

(7) Transition from SAP to EM life form of Mycorrhiza fungi
EM fungi colonize the root surface of host plants and grow between the host plant cells (but do not penetrate cell walls).Mycorrhiza literally means 'fungi-roots'.Most of the 500 species of genus Amanita, the iconic group of mushroom-forming fungi, engage in autonomous saprotrophic (SAP) ectomycorrhizal (SAP-EM) symbioses with plants by a transition from saprotrophic (SAP) decomposition of dead organic matters to biotrophic (BT) dependence on the host plants for carbon (i.e., sugar) source (Wolfe et al., 2012).Saprotrophic (SAP) (i.e., free living) fungi efficiently decay celluloses of dead plant materials into sugars by producing three classes of enzymes: the endoglucanases (encoded by, e.g., eg1 gene), cellobiohydrolases (encoded by cbhI), and beta-glucosidases (encoded by bgl), as it was detected in all investigated SAP species of Amanita genus of Amanita inopinata, A. manicata, A. thiersii, and two Volvariella (Eng./Hung.: rosegill / bocskorosgombák), Volvariella volvacea, and V. bombycina.
These enzyme genes were found to absent from most EM types of the other Amanita genomes, e.g., A. citrina (Eng./Hung.: false deathcap / citromgalóca), and A. muscarina (Eng./Hung.: fly agaric / légyölő galóca).This gene loss suggests that EM Amanita species can no longer function as free-leaving saprotrophs (SAP) (Wolfe et al., 2012).These results also confirmed the Martin et al.'s (2010) hypothesis of different 'molecular tool kits used by different mushroom species to form symbioses.
Cultivation of Flammulina velutipes started in China from the 8 th Century the Agaricus bisporus was cultivated in Europe from the 17 th Century, and oyster mushrooms (Pleurotus spp.) are reported to have commenced in 1917 on tree stumps and wood logs (in Mamiro et al., 2014).

(9) Medicinal mushrooms
Many bioactive compounds have been isolated not only from fungi (as antibiotic penicillin first by Fleming, 1945) but also from higher mushrooms (Blagodatski et al., 2018).There are four main groups based on chemical structure: lectins, terpenoids, proteins, and polysaccharides.
Microfungus Saccharomyces cerevisiae, the common food grade brewer's and baker's yeast also produce immunopotentiating glucans found in the cell wall.S. cerevisiae is also the industrial producer of zymosan, an immunomodulating cell wall proteoglucan (Giavasis, 2014).

(11) Mycoremediation
Unlike plants, fungal cell walls are made up of mostly polysaccharides including chitin, which is the most characteristic polysaccharide found in protozoa, insects and fungi compared to plant polysaccharides which lack chitin (Ruiz-Herrera, 1992).However, unlike animal cells; plants, fungi and bacteria (except Mycoplasmas) have solid cell walls.
Mineral content of fungi has been compared to plants, and showed major differences with significantly low contents of Ca, due probably to fungal cell wall structure; and low Mg, due to the achlorophyllous fungal cells (Vetter, 2003).Low Ca and Fe content of the mushroom Pleurotus compared to leaf vegetables were also found by Mamiro et al. (2014).

Conclusion
Genes, genetics and genomics of the systematically still mysterious mushrooms and fungi have revealed several changes in the genome expansions, which makes mushrooms (and fungi) a crucial part of the global carbon cycle.

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Genome evolution: Gene loss.Fungal wood decay -Evolutionarily transition from White-rot (WR) toward Brown-rot (BR) fungi with gene loss The comparative genome sequencing of two Polyporales species of white-rot (WR) and brown-rot (BR) mushrooms of Cylindrobasidium torrendii (WR) compared to Fistulina hepatica (BR) revealed that C. torrendii (WR) has been at a transitioning intermediate stage from WR towards BR life style through gene loss (Floudas et al., 2015).