1 Identity and Chemical Composition of an Albino Mutant Chanterelle
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Identity and Chemical Composition of an Albino Mutant Chanterelle R. Greg Thorn ( [email protected] ) Western University Alicia Banwell Western University Thu Hong (Nicole) Pham Memorial University of Newfoundland Natalia P. Vidal Memorial University of Newfoundland Charles F. Manful Memorial University of Newfoundland Muhammad Nadeem Memorial University of Newfoundland Alexander G. Ivanov Western University Beth Szyszka Mroz Western University Michael B. Bonneville Western University Norman Peter Andrew Hüner Western University Michele D. Piercey-Normore Memorial University of Newfoundland Raymond Thomas Memorial University of Newfoundland Research Article Keywords: White chanterelles (Basidiomycota), chemical composition, albino mutant, Cantharellus enelensis Posted Date: February 24th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-244471/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 Identity and chemical composition of an albino mutant chanterelle 2 3 R. Greg Thorn1*, Alicia Banwell1, Thu Huong (Nicole) Pham2, Natalia P. Vidal2, Charles Felix 4 Manful2, Muhammad Nadeem2, Alexander G. Ivanov1,3, Beth Szyszka Mroz1, Michael B. 5 Bonneville1, Norman Peter Andrew Hüner1, Michele D. Piercey-Normore2, and Raymond 6 Thomas2. 7 8 1Department of Biology, University of Western Ontario, 1151 Richmond St. N., London, ON 9 N6A 5B7, Canada 10 2School of Science and the Environment, Grenfell Campus, Memorial University, 20 University 11 Drive, Corner Brook, NL A2H 5G4, Canada 12 3Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. 13 Bonchev str. Bl. 21, 1113 Sofia, Bulgaria 14 15 *Corresponding author: [email protected] 1 16 Abstract 17 White chanterelles (Basidiomycota), lacking the orange pigments and apricot-like odour of 18 typical chanterelles, were found recently in the Canadian provinces of Québec (QC) and 19 Newfoundland & Labrador (NL). Phylogenetic analyses confirmed the identification of all 20 white chanterelles from NL and QC as Cantharellus enelensis; we name these forma 21 acolodorus. We characterized carotenoid pigments, lipids, phenolics, and volatile compounds 22 in these and related chanterelles. White mutants of C. enelensis lacked detectable b-carotene, 23 confirmed to be the primary pigment of wild-type, golden-orange individuals, and could also be 24 distinguished by their profiles of fatty acids and phenolic acids, and by the ketone and terpene 25 composition of their volatiles. We detected single base substitutions in the phytoene desaturase 26 (Al-1) and phytoene synthase (Al-2) genes of the white mutant, which are predicted to result in 27 altered amino acids in their gene products and may be responsible for the loss of b-carotene 28 synthesis in that form. 2 29 Introduction 30 Chanterelles (Cantharellus: Basidiomycota) are widely distributed and highly prized 31 edible mushrooms with an estimated annual international export market of over $1.5 billion 32 US1,2. Chanterelles are ectomycorrhizal, growing in a mutualistic association with host trees, 33 and thus cannot be cultivated readily for commercial sale but are wild-harvested in the forest by 34 both amateur enthusiasts and commercial mushroom pickers3–6. A large part of the culinary 35 appeal of chanterelles is their brilliant golden-orange colour (Fig. 1A), derived from carotenoid 36 pigments7–8, their apricot-like odour, and firm texture. Chanterelles are famous for their long- 37 lasting fruiting bodies, which can persist in the woods in good condition for weeks or months, 38 often without undergoing decay or being consumed by slugs, fly larvae, or other 39 invertebrates4,9. The chemical components that prevent microbial decay or invertebrate 40 consumption are largely unknown, but have been suggested to be associated with the colour and 41 odour4. 42 Until recently, most golden-orange chanterelles were referred to as Cantharellus 43 cibarius Fr., and this name is still widely used in commerce and by mushroom enthusiasts. 44 Studies using DNA sequence data have shown that C. cibarius is restricted to Eurasia, and have 45 delimited multiple species of golden-orange chanterelles around the world10. In the Canadian 46 province of Newfoundland and Labrador (NL), the common species of golden-orange 47 chanterelles was recently described as Cantharellus enelensis, and differentiated from two 48 other, less common golden species C. camphoratus and C. amethysteus11, the latter now 49 separated as a new species C. betularum12. Soon after that publication, scattered fruitings of 50 pure white chanterelles were reported across the island of Newfoundland (Fig. 1B), often 51 occurring mixed within normally pigmented individuals of C. enelensis13. Fruiting bodies of the 3 52 white chanterelles differed not only in colour but in the absence of the apricot-like odour of the 53 typical golden-orange specimens13. While investigating the NL white chanterelles, we were sent 54 white chanterelles from Québec (QC), Canada, and specimens of commercially harvested pale 55 chanterelles from Minnesota (MN) in the USA. 56 Golden chanterelles get their colour from carotenoid pigments. Carotenoid analyses 57 have been performed on C. cibarius, but not extensively studied because carotenoids can be 58 difficult to analyze since they degrade over time and rapidly with drying14. The principal 59 carotenoid in C. cibarius is β-carotene, which is responsible for its golden appearance, followed 60 by lycopene, as well as some α-carotene and γ-carotene8,15–16. Carotenoid synthesis has been 61 studied in the filamentous ascomycete Neurospora crassa, and the genes producing the key 62 enzymes have been named for the albino phenotype of their mutant alleles. The 20-carbon 63 precursor is formed by geranylgeranyl pyrophosphate synthetase, encoded by the gene referred 64 to as albino-3 (Al-3). Dimerization to form the 40-carbon colourless phytoene is carried out by 65 the phytoene synthase activity of the dual-function gene product of albino-2 (Al-2). Phytoene 66 desaturase (encoded by albino-1, or Al-1) converts phytoene to lycopene through a series of 67 cyclic reactions, which is then converted to the coloured product β-carotene by the lycopene 68 cyclase function of Al-217. 69 The purpose of this study was to determine the species identity of the white and pale 70 chanterelles from NL, QC and MN using phylogenetic analyses of nuclear ribosomal DNA 71 (internal transcribed spacer, or ITS, and large subunit, LSU) and the translation elongation 72 factor gene (Tef-1). The presence of a pigmentless chanterelle with altered odour profile raised 73 the questions of the genetic underpinning of the apparent albinism, and of how the chemical 4 74 composition of these variants compared to typical golden-orange specimens of C. enelensis, C. 75 camphoratus, and C. betularum. 76 77 Results 78 New ITS, LSU and Tef-1 sequences were generated from fifteen specimens in this study 79 (Table 1) and were aligned with sequences downloaded from GenBank. The maximum 80 likelihood tree produced in MEGA X, with node support from 1000x bootstrap replicates and 81 from Baysesian posterior probabilities of an analysis with 5 million trees in MrBayes is shown 82 (Fig. 2A). White chanterelles from NL and QC cluster phylogenetically with the NL golden 83 species, C. enelensis, with strong bootstrap support and well separated from C. roseocanus, a 84 golden species from the Pacific Coast of North America, C. cibarius, a golden species from 85 Europe and C. cascadensis, a golden to white species found on the Pacific Coast of North 86 America. We provide a name for this white variant at the rank of “forma”. 87 Cantharellus enelensis f. acolodorus, Voitk & Thorn, forma nova. Fig. 1B MycoBank 88 MB835379. 89 Typification: CANADA. NEWFOUNDLAND AND LABRADOR: Gambo, Mint 90 Forest resource road, in spruce forest mixed with birch, among moss and duff (48°43'02.3" N, 91 54°34'56.5" W; 143 m above sea level), 11 Aug 2017, Eugene Kean, A. Voitk coll. no. 92 17.08.11.av06 (holotype UWO-F730, isotype TU117603). GenBank: ITS = MN206912. 93 Etymology: acolodorus is a contracted combination adjective from Latin (a=none), to 94 indicate without colour or odour. 95 Diagnosis: A white chanterelle found among golden specimens of C. enelensis and 96 resembling them in every regard except for the lack of golden colour and the characteristic 5 97 “apricot note” to the odour. Briefly described and illustrated in Thorn et al.13; known from the 98 Island of Newfoundland and Québec (Table 1). 99 A pale specimen from the US Midwest clustered phylogenetically with the US Midwest 100 golden to whitish species, C. phasmatis, with strong bootstrap support (Fig. 2A), and well 101 separated from C. tenuithrix, a golden to white chanterelle from the Southeastern US, C. flavus, 102 a golden species from the US Midwest, C. deceptivus, a golden to white chanterelle from the 103 US Midwest, C. pallens, a golden-white species from Europe, C. subalbidus, a white 104 chanterelle from the Pacific Coast of North America, and C. cascadensis, a golden chanterelle 105 with white hymenophore from the Pacific Coast of North America. Sequences of LSU and Tef- 106 1 of a Chinese specimen identified as C. cibarius, for which a draft genome was recently 107 published18, placed this taxon in the unresolved clade of C. pallens – C. phasmatis, i.e, outside 108 the core C. cibarius clade (Fig. 2A). 109 Newly designed primers were used to amplify and sequence portions of the phytoene 110 desaturase gene Al-1 (1381 bases) and dual-function phytoene synthase / lycopene cyclase gene 111 Al-2 (1752 bases) in the white and gold variants of C. enelensis. The Al-1 gene in the white 112 chanterelle differed from that of the gold one by a 3-base deletion, resulting in the loss of a 113 phenylalanine in the predicted gene product, and 5 single nucleotide substitutions, of which 4 114 were determined to be synonymous, but a fifth was predicted to result in the replacement of 115 valine with phenylalanine in the gene product.