Fungal Biology 125 (2021) 447e458 Contents lists available at ScienceDirect Fungal Biology journal homepage: www.elsevier.com/locate/funbio Crystallicutis gen. nov. (Irpicaceae, Basidiomycota), including C. damiettensis sp. nov., found on Phoenix dactylifera (date palm) trunks in the Nile Delta of Egypt * Hoda M. El-Gharabawy a, Caio A. Leal-Dutra b, Gareth W. Griffith c, a Botany and Microbiology Department, Faculty of Science, Damietta University, New Damietta, 34517, Egypt b Section for Ecology and Evolution, Department of Biology, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark c Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Adeilad Cledwyn, Penglais, Aberystwyth, Ceredigion, SY23 3DD, Wales, UK article info abstract Article history: The taxonomy of Polyporales is complicated by the variability in key morphological characters across Received 28 July 2020 families and genera, now being gradually resolved through molecular phylogenetic analyses. Here a new Received in revised form resupinate species, Crystallicutis damiettensis sp. nov. found on the decayed trunks of date palm (Phoenix 12 January 2021 dactylifera) trees in the fruit orchards of the Nile Delta region of Egypt is reported. Multigene phyloge- Accepted 13 January 2021 netic analyses based on ITS, LSU, EF1a, RPB1 and RPB2 loci place this species in Irpicaceae, and forming a Available online 23 January 2021 distinct clade with Ceraceomyces serpens and several other hitherto unnamed taxa, which we also incorporate into a new genus Crystallicutis. We name two of these species, Crystallicutis huangshanensis Keywords: Brown rot sp. nov. and Crystallicutis rajchenbergii sp. nov. The distinctive feature of Crystallicutis gen. nov. is the White-rot presence of crystal-encrusted hyphae in the hymenium and subiculum. Basidiomes are usually honey- Insect vector yellow with white margins but there is variability in the presence of clamp connections and cystidia, Corticioid fungi as noted for other genera within Irpicacae. C. damiettensis is hitherto consistently associated with date Agaricomycetes palms killed by the red palm weevil Rhynchophorus ferrugineus, a highly damaging and invasive pest, Phylogeny recently spread to the Mediterranean region. C. damiettensis causes rapid wood decay by a potentially PolyPEET unusual white-rot mechanism and may play a role in the damage caused by R. ferrugineus. © 2021 British Mycological Society. Published by Elsevier Ltd. All rights reserved. 1. Introduction currently comprises 12 genera (Byssomerulius, Ceriporia, Cytidiella, Efibula, Emmia, Flavodon, Gloeoporus, Hydnopolyporus, Irpex, Lep- Resupinate or corticioid fungi can be difficult to classify even to toporus, Meruliopsis and Trametopsis)(Justo et al., 2017). Within family level based on morphological characteristics (Larsson, 2007; multigene phylogenies of Irpicaceae, it is also apparent that several Larsson et al., 2004). The deployment of molecular phylogenetics clearly delineated clades within this family remain to be named has led to substantial revision of the placement of many taxa within (Justo et al., 2017). Polyporales, coordinated through focused initiatives such as the The family Irpicaceae also illustrates the fundamental problems PolyPEET project (https://wordpress.clarku.edu/polypeet/)(Binder associated with the classification of Polyporales based on et al., 2013; Floudas and Hibbett, 2015; Justo et al., 2017). Howev- morphological traits. In terms of macromorphology, three forms of er, multigene phylogenies now provide a robust backbone at family basidiomes (pileate [Trametopsis], resupinate [Gloeoporus] and level but there is an urgent need to reclassify many taxa which have stipitate [Hydnopolyporus fimbriatus]) and four states for hymeno- been attributed to incorrect genera based on morphological data. phore configuration (poroid, daedaleoid/lamellate, smooth, hyd- This problem is well-illustrated by the family Irpicaceae Spirin noid) are found (Justo et al., 2017; Sjokvist€ et al., 2012). A similar and Zmitr. 2003 (Spirin, 2003), a well-supported clade which variation is evident in the diversity of hyphal systems (mostly monomitic but some dimitic [Flavodon/Irpex/Trametopsis]), which affects the consistency and longevity of fruiting bodies. Decay mode in Polyporales is one of the most stable characters that has been * Corresponding author. used as the basis for segregating genera (Gilbertson and Ryvarden, E-mail address: [email protected] (G.W. Griffith). https://doi.org/10.1016/j.funbio.2021.01.004 1878-6146/© 2021 British Mycological Society. Published by Elsevier Ltd. All rights reserved. H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458 1986; Ryvarden, 1991) but within Irpicaceae, whilst most are (Hitachi, Tokyo, Japan) with the following emission settings: 10 mA/ white-rotting, a single genus [Leptoporus] exhibits brown rot decay 1500V and using Mixed (M) or upper (U) detectors. (Justo et al., 2017). With regard to other microscopic traits, most members of the family are rather nondescript, with consistently 2.2. DNA extraction, PCR amplification and sequencing smooth hyaline spores but variation both within and between genera with regard to the presence of cystidia and clamped septa Genomic DNA was extracted from pure cultures using a CTAB- (e.g. cystidia in Irpex, Emmia and others; clamp-connections in based method (Griffith and Shaw, 1998). PCR amplifications of Gloeoporus and others). several parts of DNA were carried out using different primer pairs. These various morphological transitions have occurred repeat- PCR reactions were performed in a volume of 25 ml(13ml of Dream edly within this lineage (Floudas and Hibbett, 2015; Miettinen et al., Taq [Green PCR Master mix] and 2 ml (ca. 1e10 ng) genomic DNA) 2016), making the construction of any dichotomous key based on using a thermal cycler (PTC-100 Techne, UK). PCR cycling condi- morphological characteristics very unwieldy. Recognizing smaller, tions were as follows: one cycle of initial denaturation stage at well-supported clades as independent families would not result in 96 C for 5 min, 35 amplification cycles [Denaturation at 95 C for a more straightforward morphological grouping of these taxa. 30 s, annealing at 48-58 C (see below) for 30 s, extension at 72 C In this study we describe a new resupinate fungus found on for 45 s], then final extension at 72 C for 5 min. The following decaying trunks of Phoenix dactylifera trees killed by red palm primer pairs were used for different loci: ITS1, 5.8S, ITS2 (55 C weevil (Rhynchophorus ferrugineus). Based on the genetic and anneal) (ITS1F [CTT GGT CAT TTA GAG GAA GTA A] and ITS4 [TTC morphological similarities of this new species to Ceraceomyces TTC GCT TAT TGA TAT GC]) (Schoch et al., 2012); ITS2 and LSU (D1/ serpens and several other ‘orphan’ resupinate species, we include D2 region (53 C anneal) (ITS3 [GCA TCG ATG AAG AAC GCA]/ all four taxa in a new genus which we name Crystallicutis gen. nov. HyglonR1 [TAA AGC CAT TAT GCC AGC ATC]) (Detheridge et al., 2016; El-Gharabawy et al., 2016); LSU (55 C anneal) (LR0R [ACCCGCTGAACTTAAGC]/LR5 [TCCTGAGGGAAACTTCG]); RPB1 2. Methods (58 C touchdown anneal, with 1 C drop in annealing temperature per cycle to 48 C, then 30 cycles at 48 C for both amplicons) 2.1. Sampling and morphological studies (rpb1:aAf [GAG TGT CCG GGG CAT TTY GG]/rpb1:i2.2f [CGT TTT CGR TCG CTT GAT] and rpb1:aCr [ARA ART CBA CHC GYT TBC CCA T]/ Basidiome samples were collected during a survey for wood- rpb1:940R [CTT CGT CYT TCG AAC GYT TRT A]) (Binder et al., 2010); inhabiting fungi across orchards, and gardens of the North Nile EF1a (58 C anneal) (EF1-1018F [GCY CCY GGH CAY CGT GAY TTY Delta region of Egypt (2013e2020). Isolation was conducted from AT]/EF1-1620R [ACH GTR CCR ATA CCA CCR ATC TT]) (Rehner and basidiome tissues (Stalpers, 1978) at the Microbiological laboratory Buckley, 2005). of Faculty of Science, Damietta University. Pure cultures were ob- PCR-DNA products were purified using spin column PCR puri- tained on Potato dextrose agar (PDA) and 3% Malt extract agar fication kit (NBS Biologicals Ltd., Huntingdon, UK) and visualized on (MEA), routinely incubated at 28 C. Cultures were stored at 4 C, on 1.5% agarose gel by gel electrophoresis system. The samples were agar slopes and frozen at À80 C in 10% glycerol. Radial growth rate then sent for Sanger sequencing at the IBERS Translational Geno- was quantified on MEA in 90 mm petri dishes, with mycelial plugs mics Facility (Aberystwyth University). of actively growing cultures placed at the edge of the dish, ac- DNA sequences and chromatograms were curated and assem- cording to the method of (Adaskaveg and Gilbertson, 1986). bled using Geneious R10 (Kearse et al., 2012). The sequences Optimal growth temperature was investigated across a range of generated in this study have been submitted to GenBank (Table 1). temperatures (20e41 C). Culture compatibility tests were carried In addition to sequences submitted to GenBank from previous out for different isolates on MEA at 30 C for 3 weeks (Worrall, phylogenetic studies of Irpicaceae (Table 1), sequences from Cyti- 1997). Vouchers from the samples were deposited at Aberystwyth diella melzeri (FP 102339) and Hydnopolyporus fimbritatus (CBS University Herbarium (ABS). Herbarium acronyms follow Index 384.51) were extracted from next-generation sequencing data Herbariorum (http://sweetgum.nybg.org/science/ih/). deposited on NCBI database (SRX3006953-