bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206029; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Flavoceraceomyces (NOM. PROV.) (Irpicaceae, Basidiomycota), encompassing 2 Ceraceomyces serpens and Ceriporia sulphuricola, and a new resupinate 3 species, F. damiettense, found on Phoenix dactylifera (date palm) trunks in the 4 Nile Delta of Egypt 5 6 Hoda M. El-Gharabawy1, ([email protected]) 7 Caio A. Leal-Dutra2 8 Gareth W. Griffith2* (ORCID iD:0000-0001-6914-3745) 9 10 1 Botany and Microbiology Department, Faculty of Science, Damietta University, New 11 Damietta, 34517, EGYPT. 12 2 Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, 13 Adeilad Cledwyn, Penglais, Aberystwyth, Ceredigion SY23 3DD, WALES. 14 * Corresponding author ([email protected]) 15 16 Keywords: Brown rot; white rot; insect vector; polypore; Agaricomycetes; Phylogeny; 17 PolyPEET 18 19 20 Abstract 21 The taxonomy of Polyporales is complicated by the variability in key morphological 22 characters across families and genera, now being gradually resolved through 23 molecular phylogenetic analyses. Here a new resupinate species, Flavoceraceomyces 24 damiettense (NOM. PROV.) found on the decayed trunks of date palm (Phoenix 25 dactylifera) trees in the fruit orchards of the Nile Delta region of Egypt is reported. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206029; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 26 Multigene phylogenetic analyses based on ITS, LSU, EF1a, RPB1 and RPB2 loci place 27 this species in Irpicaceae, and forming a distinct clade with Ceraceomyces serpens 28 and Ceriporia sulphuricolor, which we also incorporate into a new genus 29 Flavoceraceomyces (NOM. PROV.). The honey-yellow basidiomes with white margins 30 and presence of crystal-encrusted hyphae in the hymenium and subiculum are 31 distinctive features of Flavoceraceomyces (NOM. PROV.), despite variability in 32 hymenium morphology and presence of clamp connections and cystidia, as noted for 33 other genera within Irpicacae. F. damiettense is hitherto consistently associated with 34 date palms killed by the red palm weevil Rhynchophorus ferrugineus, a highly 35 damaging and invasive pest, recently spread to the Mediterranean region. F. 36 damiettense causes rapid wood decay by a potentially unusual white-rot mechanism 37 and may play a role in the damage caused by R. ferrugineus. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206029; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 38 Introduction 39 The taxonomic placement of many taxa within Polyporales has undergone substantial 40 revision in light of molecular phylogenetic evidence, coordinated through focused 41 initiatives such as the PolyPEET project (https://wordpress.clarku.edu/polypeet/) 42 (Binder et al. 2013; Floudas and Hibbett 2015; Justo et al. 2017). It is by now clear that 43 resupinate or corticioid fungi can be difficult to classify even to family level based on 44 morphological characteristics. However, multigene phylogenies now provide a robust 45 backbone at family level but there is an urgent to reclassify many taxa which have been 46 attributed to incorrect genera based on morphological data. 47 48 This problem is well-illustrated by the family Irpicaceae Spirin & Zmitr. 2003 (Spirin 49 2003), a well-supported clade which currently comprises 12 genera (Byssomerulius, 50 Ceriporia, Cytidiella, Efibula, Emmia, Flavodon, Gloeoporus, Hydnopolyporus, Irpex, 51 Leptoporus, Meruliopsis, Trametopsis) (Justo et al. 2017). Within multigene phylogenies 52 of Irpicaceae , it is also apparent that several clearly delineated clades within this family 53 remain to be named (Justo et al. 2017). 54 55 The family Irpicaceae also illustrates the fundamental problems associated with the 56 classification of Polyporales based on morphological traits. In terms of 57 macromorphology, three forms of basidiomes (pileate [Trametopsis], resupinate 58 [Gloeoporus] and stipitate [Hydnopolyporus fimbriatus]) and four states for 59 hymenophore configuration (poroid, daedaleoid/lamellate, smooth, hydnoid) are found 60 (Justo et al. 2017; Sjökvist et al. 2012). A similar variation is evident in the diversity of 61 hyphal systems (mostly monomitic but some dimitic [Flavodon/Irpex/Trametopsis]), 62 which affects the consistency and longevity of fruiting bodies. Decay mode of 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206029; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 63 Polyporales, is one of the most stable characters that has been used as the basis for 64 segregating genera (Gilbertson and Ryvarden 1986; Ryvarden 1991) but within 65 Irpicaceae, whilst most are white rotting, a single genus [Leptoporus] exhibits brown 66 rot decay (Justo et al. 2017). With regard to other microscopic traits, most members of 67 the family are rather nondescript, with consistently smooth hyaline spores but variation 68 bother within and between genera with regard to the presence of cystidia and clamped 69 septa (e.g. cystidia in Irpex, Emmia and others; clamp-connections in Gloeoporus and 70 others). 71 72 These various morphological transitions have occurred repeatedly within this lineage 73 (Floudas and Hibbett 2015; Miettinen et al. 2016), making the construction of any 74 dichotomous key based on morphological characteristics very unwieldy. Recognizing 75 smaller, well-supported clades as independent families would not result in a more 76 straightforward morphological grouping of these taxa. 77 78 In this paper we describe a new resupinate fungus found on decaying trunks of Phoenix 79 dactylifera trees killed by red palm weevil (Rhynchophorus ferrugineus). Based on the 80 genetic and morphological similarities of this new species to two other ‘orphan’ 81 resupinate species (Ceriporia sulphuricolor, Ceraceomyces serpens), we include all 82 three taxa in a new genus which we name Flavoceraceomyces (NOM. PROV.). 83 84 Methods 85 1. Sampling and Morphological studies 86 Basidiome samples were collected during a survey for wood-inhabiting fungi across 87 orchards, and gardens of the North Nile Delta region of Egypt (2013-2020). Isolation 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206029; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 88 was conducted from basidiome tissues (Stalpers 1978) at the Microbiological 89 laboratory of Faculty of Science, Damietta University. Pure cultures were obtained on 90 Potato dextrose agar (PDA) and 3% Malt extract agar (MEA), routinely incubated at 91 28°C. Cultures were stored at 4°C, on agar slopes and frozen at -80°C in 10% glycerol. 92 Radial growth rate was quantified on MEA in 90 mm petri dishes, with mycelial plugs 93 of actively growing cultures placed at the edge of the dish, according to the method of 94 (Adaskaveg and Gilbertson 1986). Optimal growth temperature was investigated 95 across a range of temperatures (20-41°C). Culture compatibility tests were carried out 96 for different isolates on MEA at 30°C for 3 weeks (Worrall 1997). Vouchers from the 97 samples were deposited at Aberystwyth University Herbarium (ABS). Herbarium 98 acronyms follow Index Herbariorum (http://sweetgum.nybg.org/science/ih/). 99 100 The basidiome surface was observed with a dissecting microscope (Prior model 101 29362) at 50x. Basidiome sections were investigated by light microscopy (Olympus 102 BX51M) mounted in 5% KOH, cotton blue or Melzer’s reagent at 1000x magnification. 103 Photomicrographs recorded with a Nikon Coolpix 995 digital camera. Measurements 104 were taken using an objective micrometer or calibrated ocular. 105 106 Colony characters as colour, shape and size of hyphae and type of septa were checked 107 after 1,2,4 weeks of incubation on MEA plates at 28°C (Stalpers 1978). Hyphae were 108 mixed with 20 µl of Calcofluor solution (200 µg/ml [w/v] in distilled water to stain the 109 chitin cell walls), then visualized by epifluorescence microscopy (Olympus BX51). The 110 spore shape index (Q; length/diameter) was calculated for 10 spores (Wu 1990). 111 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.16.206029; this version posted July 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 112 Scanning electron microscopy was performed at IBERS, Aberystwyth University. 113 Sections of air-dried samples were mounted directly on the surface of carbon stubs 114 and coated with platinum and palladium (Pt/Pd; 4 nm thick layer) mixture using a High 115 Resolution Sputter Coater (Agar Scientific Ltd, UK) at 20 mA under vacuum, and the 116 thickness was monitored with a quartz crystal micro-balance thickness controller. SEM 117 was undertaken with a Hitachi S-4700 field emission scanning electron microscope 118 (Hitachi, Tokyo, Japan) with the following emission settings: 10 µA/1500V and using 119 Mixed (M) or upper (U) detectors.