Fungal Biology 125 (2021) 447e458
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Fungal Biology
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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 80 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- SRX3006967 and The basidiome surface was observed with a dissecting micro- SRX2124808-SRX2124809, respectively) using an in-house devel- scope (Prior model 29362) at 50x. Basidiome sections were inves- oped pipeline. tigated by light microscopy (Olympus BX51M) mounted in 5% KOH, cotton blue or Melzer’s reagent at 1000x magnification. Photomi- 2.3. Phylogenetic analyses crographs were recorded with a Nikon Coolpix 995 digital camera. Measurements were taken using an objective micrometer or cali- A dataset containing 169 sequences from 54 samples, including brated ocular. members of Irpicaceae and three Phanerochaetaceae as outgroup, Colony characters as colour, shape and size of hyphae and type was created including ITS (54 sequences), LSU (54 sequences), EF1a of septa were checked after 1,2,4 weeks of incubation on MEA (6 sequences), RPB1 (35 sequences), and RPB2 (20 sequences). plates at 28 C(Stalpers, 1978). Hyphae were mixed with 20 mlof GenBank accession numbers and voucher details for the sequences Calcofluor solution (200 mg/ml [w/v] in distilled water to stain the used in these analyses are provided in Table 1. chitin cell walls), then visualized by epifluorescence microscopy For all the following analyses the ITS region was divided into (Olympus BX51). The spore shape index (Q; length/diameter) was three subsets ITS1, 5.8S and ITS2. Only the CDS from the protein- calculated for 10 spores (Wu, 1990). coding genes were used except for the third intron from RPB1 Scanning electron microscopy was performed at IBERS, Aber- that was included in a separate subset. Thus, eight subsets were ystwyth University. Sections of air-dried samples were mounted created: ITS1, 5.8S, ITS2, LSU, EF1a, RPB1, RPB2 and RPB1_I3. Each directly on the surface of carbon stubs and coated with platinum subset was aligned separately with MAFFT v7.311 (Katoh and and palladium (Pt/Pd; 4 nm thick layer) mixture using a High- Standley, 2013) using the EeINSei algorithm for ITS1, ITS2 and Resolution Sputter Coater (Agar Scientific Ltd, UK) at 20 mA un- RPB1_I3, and LeINSei for the remaining subsets. The alignments der vacuum, and the thickness was monitored with a quartz crystal were curated manually with AliView v1.5 (Larsson, 2014) and micro-balance thickness controller. SEM was undertaken with a trimmed to remove uneven ends. With ITS1, ITS2 and RPB1_I3 Hitachi S-4700 field emission scanning electron microscope alignments, morphological matrices were constructed coding the
448 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Table 1 Details of sequences used in the phylogenetic analyses. Sequence data generated in the present study is indicated in bold font. Sequence sources are indicated as follows: CH (Chen et al., 2020); FL (Floudas and Hibbett, 2015); GA (Garzoli et al., unpublished); GO (Gomez-Montoya et al., 2017); JGI (Joint Genome Institute); JI (Jia et al., 2014); JN (Jung et al., 2018); JS (Justo and Hibbett, 2011); VU (Vu et al., 2019); WU (Wu et al., 2017); YO (Li et al., 2015); YU (Yuan et al., 2016).
Taxon Collection FAMILY nrITS nrLSU rpb1 rpb2 ef1a
Bjerkandera adusta a HHB-12826-Sp Bjerkanderaceae KP134983 KP135198 KP134784 KP134913 d FL Bjerkandera adusta MUT
*** sequences extracted from genome sequencing a Type species indels as morphological characters using the simple indel coding position (EF1a_1, RPB1_1, RPB2_1, EF1a_2, RPB1_2, RPB2_2, (Simmons and Ochoterena, 2000) implemented in SeqState EF1a_3, RPB1_3, RPB2_3). (Müller, 2005) and the nucleotide alignments were then trimmed The separately aligned markers were combined into a single with trimAl v1.4.rev22 (Capella-Gutierrez et al., 2009) with the data matrix prior to phylogenetic reconstruction. Maximum- option -gappyout to remove unalignable regions. Moreover, the likelihood tree reconstruction was performed with IQ-TREE v2 protein-coding sequences were analysed in three partitions ac- (Minh et al., 2020). The best-fit evolutionary models and parti- counting for each codon position. Therefore, 17 partitions were tioning scheme for this analysis were estimated by the built-in used for the phylogenetic estimations: five nucleotide partitions ModelFinder (option -m MF þ MERGE) allowing the partitions to (5.8S, LSU, ITS1, ITS2, RPB1_I3), three morphological (ITS1_indel, share the same set of branch lengths but with their own evolution ITS2_indel, RPB1_I3_indel) and nine accounting for each codon rate ( p option) (Chernomor et al., 2016; Kalyaanamoorthy et al., 2017). Branch supports were assessed with ultrafast bootstrap
449 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
(UFBoot; 1000 replicates) (Hoang et al., 2018), allowing resampling assuming a dark or brownish colour with addition of KOH solution. partitions and then sites within these partitions to reduce the No pores were observed on the hymenium. Fresh basidiomes have a likelihood of false positives on branch support (option –sampling strong but pleasant mushroom-like odour. No mycelial cords or GENESITE) (Gadagkar et al., 2005). Additionally, ultrafast jackknife rhizomorphs were observed (Fig. 2). (1000), SH-aLRT test (1000) (Guindon et al., 2010) and non- parametric bootstrap (100) were performed. 3.3. Cultural characteristics Bayesian Inference (BI) was conducted with MRBAYES v3.2 (Ronquist et al., 2012) with two independent Markov Chain Monte Cultures were obtained from eight basidiomes collected from Carlo (MCMC) runs, each one with four chains and starting from Damietta and Kafr El-Sheikh. In axenic culture (MEA), the mycelium random trees. The best-fit evolutionary models and partitioning is yellowish or creamy white to pale buff coloured, sparse scheme for these analyses were estimated as for the ML analysis but adpressed mycelium, with slow radial growth rate (1.6e2.2 mm/ restricting the search to models implemented on MRBAYES (op- day) (Fig. 3A). On PDA growth is more cottony/downy, with occa- tions -m TESTMERGEONLY -mset mrbayes). Chains were run for 10 sionally denser growth on the inoculum plug. Maximal growth rate million generations with tree sampling every 1000 generations. was obtained at 30 C and no growth was observed above 35 C. The burn-in was set to 25% and the remaining trees were used to In older cultures (>4 weeks), following growth at ambient light calculate a 50% majority consensus tree and Bayesian Posterior levels, yellowish brown pigmentation developed as a waxy- Probability (BPP). The convergence of the runs was assessed on leathery thin layer at the edges of colonies (Fig. 3B). Thus, there TRACER v1.7 (Rambaut et al., 2018) to ensure the potential scale was some differentiation of hymenial structures but basidia and reduction factors (PSRF) neared 1.0 and the effective sample size basidiospores were not observed. The pigmentation and differen- values (ESS) were sufficiently large (>200). tiation did not occur when cultures were incubated in the dark. Nodes with BPP 0.95 and/or UFBoot 95 were considered Hyphae from pure cultures are hyaline, thin-walled (3e4 mm strongly supported. Alignment and phylogenetic trees are available diameter; Fig. 4A/B/C), with large clamps abundant. Chlamydo- in Figshare (DOI: 10.6084/m9.figshare.13574255). spores, both intercalary and terminal (Fig. 4B/C), were present in older cultures (after 3e4 weeks of growth); these, are numerous, 3. Results globose or fusiform (7.5e9.5 mm diam) with encrustation by small irregularly shaped crystals visible on some hyphae (Fig. 4D, inset). 3.1. Field investigation and sample collection Amorphous areas of brown resinous material were also visible in some areas but it was not clear whether these were associated with A resupinate fungus with honey-yellow basidiomes and white the crystals (Fig. 4D). margins was initially collected as a single infection on the dead Pairings of isolates obtained from different trees at Kafr El- stump of a date palm tree (P. dactylifera) in February, 2014 in a fruit Sheikh revealed the formation of zone lines due to somatic farm at Baltim city, Kafr El-Sheikh (31.5764 N, 31.0796 E; a gover- compatibility (Fig. 3 C,D). This result is suggestive of an outcrossing norate of Nile Delta of Egypt). In more detailed surveys, basidiomes breeding strategy (Griffith and Hedger, 1994). were found to be more widespread and present on dead date palm stumps and logs in several adjacent orchards (2017; five infected 3.4. Basidiome micromorphology of voucher UN63A stumps in area of 200e250 m2). In 2018e2020, basidiomes were also found in orchards 60 km to the east in El- Sinaniah, Damietta Basidia, rarely observed, are clavate (6.0e7.5 12.0e15.0 mm), (31.4429 N, 31.7798 E), always on dead date palm stumps or fallen smooth, thin-walled, 4-spored (Fig. 5A; SuppFig1G). Basidioles are trunks (10 different trees across an area of about ca. 400 m2). In all similar in shape but smaller (4e5 mm diameter) (Fig. 5A; cases, the date palm stumps were in dense orchard with dense SuppFig1G). Basidiospores are short, ovoid to ellipsoid, tear- cover of planted trees (mainly mango, lemon, and date palm). shaped, smooth, sometimes thick-walled, 3.0e3.5 4.0e5.0 mm Basidiomes were present throughout the year (Winter temp in size (Q ¼ 1.42e1.66), colourless, non-cyanophilous (not staining 10e20 C [RH 60e80%], Summer temp 25e35 C [RH 40e45%]). with Cotton Blue), with no colour change associated with KOH or Some of the basidiomes were formed on the bark (Fig. 1A/D) and Melzer’s reagent (i.e. non-amyloid and non-dextrinoid) (Fig. 5A; others on decorticated areas of exposed wood (Fig. 1C/E/F) and the SuppFig1H). Cystidia are abundant, long, smooth (not encrusted), basidiomes were associated with advanced white-rot decay. The hyaline, thin-walled, septate (2-celled), spear-shaped or with a cause of death of the host trees was R. ferrugineus (red palm weevil), sharply pointed apex (4.0e5.0 22e25 mm) (Fig. 5B; SuppFig1J/K). whose boreholes were readily visible in some stumps (Fig. 1B/C; Cystidioles are fusiform, 3.0e4.0 18e22 mm(Fig. 5C; SuppFig1L). arrowed). The subhymenial layer is composed of tightly packed hypha (SuppFig1E/F), with clamps present on all primary septa, (subicular, 3.2. Basidiome morphology of voucher UN63A subhymenial and sub-basidial). The hyphal system is monomitic; generative hyphae bear abundant clamps (Fig. 5F; SuppFig1F) and The basidiomes are annual, effuse-resupinate, comprising a thin frequent stumpy branches (hyaline and thin-walled, 3.0e4.5 mm layer of patchy of tissue tightly attached to the host. When young diam) (Fig. 5A/B/C). Some generative hyphae are thick-walled to and fresh, basidiomes are lemon to olive/honey-yellow coloured nearly solid (pseudo-skeletoid, sparsely branched, fewer clamps, with a crenulate/papillate/tuberculate hymenium surface, up to 6.5e8.0 mm diam) (Fig. 5G). 20 45 cm in extent, up to 0.3 mm thick (Fig. 2). Sometimes, Large irregularly-shaped hyaline crystals (10e30 20e50 mm) basidiomes appear as a thicker velvety mat, deep yellow-coloured, are visible on the hymenium surface (Fig. 4E) and also in the sub- warty (bullate or phlebioid) irregularly crenulated surface with hymenium and subiculum (SuppFig1C/D/E). More widespread, in waxy (ceraceous) appearance. Basidiomes have white, thin, loose the hymenium, subhymenium and subiculum were smaller cobweb-like fibrous margins. With age, the flesh of the basidiome (1e4 mm) rhomboid-shaped hyaline crystals (Fig. 4F; SuppFig1F), becomes thinner and more compressed, with a pinky-buff or found mainly forming encrustations of hyphae. The latter were ochraceous to pale brown or olive brown colour, smoother surface, similar to those observed on hyphae in pure cultures. The larger and dull (not waxy) appearance. The flesh is yellowish white, soft, crystals and also the smaller crystals when not forming hyphal loose when fresh, more fragile with small cracks when dry, encrustations were soluble in KOH but the hyphal encrustations of
450 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Fig. 1. Crystallicutis damiettensis infecting date palm trunks. Honey-yellow basidiomes with white margins are visible emerging from the bark (A), often associated with bore holes of red weevil (B, arrowed) and batches of white-rot decay (C). Basidiome sometimes formed in long decay columns (D), also at trunk ends (E) and inside hollowed dead stumps (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) smaller crystals remained intact in KOH. Brown, resinous agglom- DNA sequence was obtained for the full ITS region of all seven erations, similar to those observed in pure cultures, were also isolates and all were identical (KX428470). Additionally, for isolate visible (SuppFig1A) but it was not clear how these were associated UN63A, the D1/D2 region of the large (28S) ribosomal subunit with the crystalline deposits. No chlamydospores were seen within (GenBank MW508515), the EF1a gene (GenBank MW523002) and basidiome tissues. also the RPB1 gene (GenBank MW523003) were sequenced. In all cases, the sequences obtained were quite distinct from any other 3.5. Phylogenetic reconstruction sequences present on GenBank but more detailed phylogenetic analysis placed the fungus at the base of the Byssomerulius clade, as fi The final alignment consisted of 54 sequences with 4291 char- de ned by Floudas and Hibbett (2015) and intermediate between Meruliopsis sp. and Ceriporia spp. (Fig. 6). acters and 2133 parsimony-informative sites. The BI analysis converged in both runs as indicated by the effective sample sizes Sequences were also extracted from two genome sequence projects (C. melzeri and H. fimbriatus). Alignment and phylogenetic (ESS) of all parameters above 4500 and the potential scale reduc- fi tion factors (PSRF) equal 1.000 for all the parameters according to trees are available in Figshare (DOI:10.6084/m9. gshare.13574255). The clades recovered here within Irpicaceae correspond well the 95% highest posterior density interval. Details about evolu- tionary models and partitioning schemes used and all trees with those recovered by Justo et al. (2017) (Fig. 4). Clade/crystal- licutis (BS ¼ 98; BPP ¼ 1) is strongly supported in our phylogeny generated in the analyses can be found in SuppData1.
451 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Fig. 2. Detailed view of resupinate basidiomes of Crystallicutis damiettensis in the field. The resupinate basidiomes are honey yellow coloured with a granulated (papillate) surface and white thin fibrous margin when young (A). Thicker velvety mats with a warty (bullate) surface, irregularly crenulated and a waxy appearance are also found (B). Older basidiomes are buff to pale brown with white margins and cracked (C), with the surface becoming smoother with a dull (not waxy) appearance, concolorous flesh and soft dense texture (D). Scale bar indicates 1 cm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) and occupies a similar position in the phylogeny presented by Justo Etymology. Derived from the Latin words crystallus ¼ crystal and et al. (2017), basal to the subclade containing Trametopsis, Efibula, cutis ¼ rind/surface, due to the presence of crystals in the hyme- Byssomerulius and Irpex. Within clade/crystallicutis, UN63 occupies nium and subiculum of the basidiomes. a basal position, with the other samples forming two distinct pairs. Diagnosis. Differs from other members of family Irpicaceae due The first of these contains the well-established and widely to the presence of hyphae encrusted with crystals in the subiculum distributed C. serpens (Bernicchia and Niemela,€ 1998; Ginns, 1975) and usually yellow colour of the hymenial surface when fresh. alongside Dai6090, a specimen collected from Huangshan Moun- Description. Basidiomes resupinate with smooth, tuberculate, tain, Anhui, China and which was named as Ceriporia sulphuricolor papillate, merulioid or sometimes poroid hymenophores, usually by Cui and Jia (2011). honey-yellow (not bright yellow) in colour (but occasionally rosy, The type specimen of C. sulphuricolor Bernicchia & Niemela€ reddish or greenish), waxy with white margin. Hyphae of sub- originates from Italy (Bernicchia and Niemela,€ 1998; Ginns, 1975) iculum and sometimes hymenium/subhymenium encrusted with but the type specimen (voucher 6591 [HUBO fungarium]) was crystals associated with darker (brown) resinous granules. Hyphal recently moved to the University of Oslo, Natural History Museum system is monomitic, usually with abundant clamp connections Fungarium (O) following retirement of the HUBO curator Dr. present. Basidia cylindric-clavate, basidiospores hyaline, smooth, Annarosa Bernicchia. Sequencing of the ITS spacer region and ellipsoid, and non-amyloid and non-dextrinoid. Found forming partial LSU of HUBO- 6591 (now named O-F-76351; GenBank: white-rot on heavily decayed trunks of woody monocot and dicot MW508516) revealed it to be identical to several specimens of angiosperms, as well as coniferous trees. Rhizomorphs and mycelial Ceriporia alachuana, and therefore unrelated to Dai6090 and other cords absent. members of /crystallicutis (SuppData2). Notes. Crystallicutis is also defined as the least inclusive clade Also falling within/crystallicutis are sequences derived from containing the following pairs of sequences: ITS1-2 spacer regions voucher MR-4310, labelled in GenBank as Phlebia cf. griseo- rDNA (KX428470, JX623934); D1eD3 region of 28S rDNA flavescens. We obtained this voucher and microscopic examination (MW508515, JX644066). Chlamydospores abundant in old agar revealed the presence of hyphae encrusted with small and large cultures of C. damiettensis and sometimes in C. serpens (Ginns, crystals, as detailed below (SuppFig2L). 1975).
Crystallicutis damiettensis El-Gharabawy, Leal-Dutra and G.W. 3.6. Taxonomy Griff., sp. nov. IndexFungorum. IF557790. Crystallicutis El-Gharabawy, Leal-Dutra and G.W. Griff., gen. nov. IndexFungorum. IF557789. Etymology. The species epithet “damiettensis” refers to Damietta University (North Nile Delta, Egypt), close to the location where the Type species. Crystallicutis damiettensis El-Gharabawy, Leal- fungus was first discovered. Dutra and G.W. Griff.
452 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Fig. 3. Crystallicutis damiettensis in pure culture on PDA. Floccose appearance of young colonies of isolate UN63A (2 weeks old), with slight yellow colouration (A). Colonies incubated in the light exhibited pigmentation similar to that seen in basidiomes (B). Assessment of somatic incompatibility between isolates from Kafr El-Sheikh (UN63A, UN63B, UN63C). Yellow pigmentation after incubation in the light for 4 weeks is visible on the upper surface (C), with zone lines between genetically different cultures (B vs A/C), more clearly visible from beneath (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Diagnosis. Basidiome resupinate, honey-yellow, tuberculate to (31.5764 N, 31.0796 E), AugeDec 2017; vouchers UN63B, UN63C, papillate-warty, with waxy texture, white margin. Basidia clavate UN63X, UN63Y; coll. HM El-Gharabawy, held at the Faculty of Sci- (6.0e7.5 12.0e15.0 mm), smooth, thin-walled, 4-spored. Basi- ence at Damietta University. Ten basidiomes, on trunks and stumps dioles similar in shape but smaller (4e5 mm diameter). Basidio- of fallen date palms killed by R. ferrugineus, at El-Sinaniah, Damietta spores short, ovoid to ellipsoid, tear-shaped, smooth, sometimes (31.4429 N, 31.7798 E), Jan 2018eFeb 2020; vouchers UN1-UN10; thick-walled (3.0e3.5 4.0e5.0 mm), non-amyloid and non- coll. HM El-Gharabawy, held at the Faculty of Science at Damietta dextrinoid. Cystidia abundant, long, smooth, hyaline, thin-walled, University. septate (2-celled), spear-shaped (4.0e5.0 22e25 mm). Cys- Distribution. Nile Delta region of Egypt. Hitherto only found on tidioles are fusiform, 3.0e4.0 18e22 mm. Monomitic hyphal fallen and heavily-rotted trunks or stumps of P. dactylifera system, with generative hyphae bearing abundant clamps and (datepalm). frequent stumpy branches. Brown, resinous agglomerations and Notes: White-rot decay mechanism with secretion of Mn- large irregularly-shaped hyaline crystals (10e30 20e50 mm) are dependent and Mn independent peroxidases, but only low and present on the hymenium surface, subhymenium and subiculum. transient secretion of laccase (El-Gharabawy et al., 2016). Smaller (1e4 mm) rhomboid-shaped hyaline crystals are present forming encrustations of hyphae. Differs from other members of Crystallicutis serpens (Tode ex Ginns) El-Gharabawy, Leal-Dutra this genus in having abundant cystidia and cystidioles. and G.W. Griff., comb. nov. Typification. The holotype (Figs. 2 and 5) was collected from: IndexFungorum: IF557791. EGYPT. Kafr El-Sheikh, Baltim (31.5764 N, 31.0796 E; North Nile Delta), growing on fallen trunk of date palm (P. dactylifera) killed by Basionym. Merulius serpens Tode, Abh. Hallischen Naturf. Ges. 1: R. ferrugineus, 14 Feb 2014, coll. HM El-Gharabawy, Voucher UN63A 355 (1783). is held at
453 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Xylomyzon crustosum Pers., Mycol. eur. (Erlanga) 2: 34 (1825). Description (Ginns, 1975): 147. Notes. A widespread and common species in Europe and North America. The species was placed in the genus Ceraceomyces by Ginns (1975). Ginns (1975) distinguished this white-rot forming species from its ‘relative’ C. borealis on the basis that the latter formed a brown rot. However, he noted that whilst C. serpens formed a white-rot of angiosperm hosts, on conifers this was less clear (“incipient brown rot”). The type species of Ceraceomyces is C. tessulatus (Amylocorticiales), only very distantly related to C.serpens which Ginns placed in Meruliopsis. Ginns (1975) also noted that the subhymenial area of basidiomes was “often heavily impregnated with fine granules or a resin-like substance”.
Crystallicutis huangshanensis El-Gharabawy, Leal-Dutra and G.W. Griff., sp. nov. IndexFungorum. IF557792
Etymology. The species epithet “huangshanensis” refers to Huangshan Mountain, Anhui Province China where the type specimen was found. Diagnosis (Cui and Jia, 2011): 534e535. Honey-yellow to olivaceous-buff pore surface, margin thin, cream. Subiculum cream, cottony, up to 0.5 mm thick with tubes concolorous with pore surface, Hyphal system monomitic, generative hyphae with simple septa. Generative hyphae in subiculum hyaline, coarsely encrusted with small crystals (2e4 mm across). Typification. The holotype (Figs. 3 and 4 in (Cui and Jia, 2011)) Fig. 4. Micromorphology of Crystallicutis damiettensis. In pure culture, abundant clamp was collected from Huangshan Mountain Anhui Province, China connections are visible in brightfield (A) and under epifluorescence microscopy with (30.139 N; 118.164 E) on rotten angiosperm wood, 12th December Calcofluor B staining (B,C; arrowed). Spherical-ellipsoid chlamydospores, both inter- 2004 by Y.C. Dai (Dai 6090). calary and terminal, are abundant (BeD), alongside brown resinous agglomerations (D) and encrustation by small crystals of individual hyphae (D, inset). In basidiomes Additional specimens examined. None examined by scanning electron microscopy, large crystals are visible, embedded in the Notes. Specimen Dai6090 was incorrectly named as matrix of the hymenium surface (E; arrowed) and smaller crystals encrusting the C. sulphuricolor by Cui and Jia (2011) based on its morphological surface of hyphae in the subhymenium and subiculum (F). Scale bar indicates 10 mm. fi similarity to the description of C. sulphuricolor (HUBO6591/O-F- (For interpretation of the references to colour in this gure legend, the reader is € referred to the Web version of this article.) 76351), which was described from Italy (Bernicchia and Niemela, 1998). Our genetic analysis reveals the two samples to be
Fig. 5. Diagram of microscopic structures of Crystallicutis damiettensis basidiome. Hymenium layer with clavate 4-spored basidia, thick-walled basidiospores, smaller basidioles and basal clamps (A). Cystidia are spear-shaped (B) or fusiform (C), with cystidioles also present (D). Large and small hyaline crystals (E) and other encrustation on hyphae were observed (F). Generative hyphae with abundant clamps (G) and pseudo-skeletoid subicular hyphae (H) are present. Scale bar indicates 10 mm.
454 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Fig. 6. Multigene maximum-likelihood tree of Irpicaceae. Support values are presented as numbers (UFBoot/BPP) on branches and shown only for UFBoot 70 and BPP 0.70. Type species for other genera within Irpicaceae are represented by a star. Outgroups are Bjerkandera adusta and Terana caerulea (Phanerochaetaceae). Crystallicutis gen. nov. is boxed in red. Asterisks (*) represent maximum UFBoot/BPP values, dashes ( ) represent values below the cut-off threshold (70%), and dots (.) represent ML clades that were not recovered in the BI tree Scale bar indicates number of substitutions per site. More details on supporting values can be found in SuppData1. unrelated, with the type specimen of C. sulphuricolor being very hymenium. Unlike HUBO6591/O-F-76351, Dai 6090 lacks hyphoid similar to C. alachuana (SuppData2). Jia et al. (2014) undertook DNA cystidia and the tramal hyphae are interwoven, not parallel. The sequencing of the ITS/and partial LSU region of Dai 6090, placing hyphae of the subiculum are heavily encrusted, with small (2e4 mm the specimen basal to the main Ceriporia clade, with Chen et al. diameter) hyaline crystals (Cui and Jia, 2011) but the larger free (2020) later placing it close to C. serpens in their analyses. crystals seen in C. damiettensis and C. serpens are not reported. Cui The description of Dai 6090 by Cui and Jia (2011) differs from et al. (2011) noted the similarity of voucher Dai 6090 to Ceriporia that of Bernicchia (Bernicchia and Niemela,€ 1998) in that the basi- subspissa, collected in Guyana (Aime et al., 2007) but the latter diome is “honey-yellow to ochraceous buff” (Fig. 3 in (Cui and Jia, species is reported to bear cystidia in the hymenium and have a 2011)) rather than bright yellow (Fig. 3 in (Cui and Jia, 2011)). Un- deep reddish brown hymenium/subiculum. like other members of genus Crystallicutis, pores are present on the
455 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458
Crystallicutis rajchenbergii El-Gharabawy, Leal-Dutra and G.W. However, following more detailed phylogenetic analysis using Griff., sp. nov. RPB1 and EF1a locus, UN63A was placed close to C. serpens and IndexFungorum. IF556941 voucher Dai6090 (labelled as C. sulphuricolor), with strong statis- tical support. Previous phylogenetic reconstructions including Etymology. The species epithet “rajchenbergii” in honour of the these two species, also placed them in the same clade (Chen et al., mycologist Mario Rajchenberg who collected this specimen. 2020; Justo et al., 2017), and it was already apparent that the Diagnosis. Fruitbody resupinate, adnate, effused-elongate, generic names attributed to these species were incorrect, since the 0.1e0.3 mm thick, smooth continuous surface, non-ceraceous type species of Ceraceomyces is in Amylocorticiales, whilst Ceriporia with small cracks when old/dry, honey-yellow coloured with tan viridans (type species for this genus, also in Irpicaceae) is placed to brownish compact centre, bright yellow to whitish fibrous close to Meruliopsis. Sequence data was obtained for the type thinner margins. Hyphal system monomitic hyphae, generative specimen of C. sulphuricolor (Bernicchia 6591, from Italy (Bernicchia hypha thin-walled, 2.5e3.0 mm, thinner (1.5e2 mm) and more and Niemela,€ 1998)). Our phylogenetic analyses showed it to be densely branched in subhymenial hyphae (SuppFig2G/). Some unrelated to voucher Dai6090 from China (Cui and Jia, 2011; Chen distinct thick-walled, smooth (pseudo-skeletoid) hyphae with et al., 2020) and to be identical to C. alachuana (SuppData2). larger diameter (5e6 mm) are visible. Hyphae (3e4 mm) in the hy- As has been found to be the case with other genera within menium and subiculum are heavily encrusted with small crystals Irpicaceae and other families within Polyporales (Floudas and (0.5e1 mm), Large regular rhomboid crystals (25e37 mm across) and Hibbett, 2015; Justo et al., 2017), morphological comparisons of some irregular-shaped crystals (3x4-30 35 mm) are present the four species which we now place within Crystallicutis, revealed throughout the subhymenium and subiculum. Cystidia (4e5x relatively few consistent features but all four form basidiomes with 25e30 mm) are frequent, two-celled and clavate with a sharp apex, crystalline encrustations of the hymenium, subhymenium and mostly non-encrusted, thick walled. Cystidioles are fusiform subiculum. Basidiomes are usually yellow-coloured with a white (3 10 um), thick-walled, with a basal swelling. Basidia/basidioles margin, though C. serpens basidiomes are sometimes pinkish or are 4-spored, ovoid to clavate (5e6x8e10 mm). Basidiospores greenish-coloured. Other features such as presence of cystidia and subellipsoid with a small rounded apiculus (3e6x10e15 (occa- clamp connections, as well as the macromorphology of the hyme- sionally 6 22 mm), smooth, thick-walled, IKI-, non-amyloid/ nophore surface, are not conserved within the genus. dextrinoid but cyanophilous (CBþ). Habitat. On decayed, decidu- Ginns (1975) (p. 147) observed that C. serpens exhibited some ous wood, USA: North Carolina. unusual traits in wood decay, “with a white-rot of angiosperms and Typification. The holotype (voucher MR-4310; SuppFigs2,3)is what, visually, appears to be an indistinctive white or incipient kept at the Forest Disease Herbarium, USDA Northern Research brown rot of conifers”. In pure culture, he observed it to oxidise Station. It was collected by Dr. Mario Rajchenberg (12th July 1988) gallic acid strongly but did not detect laccase activity using gum from Rustic Falls Area (off horse Cove Road), south-east of High- guaiac substrate. We have previously examined the ligninolytic lands, Macon County, North Carolina (35.039 N;-83.156 W) and capability of isolate UN63A using the model substrate ABTS (2,20- originally named as Phlebia cf. griseoflavescens. azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) (El-Gharabawy Additional specimens examined. None but a BLAST search using et al., 2016). It was found to secrete Mn-dependent and Mn- the C. rachjenbergii ITS and LSU sequences revealed a strong match independent peroxidase, as well as laccase. Despite its slow to and unidentified culture (isolate code 6/1e30) isolated from leaf growth rate and low enzymatic activity relative to the other Poly- litter in Sussex County, New Jersey (ITS: AF241321 [99.5%ID]; LSU: porales with which it was compared, it caused rapid loss of sub- AF241355 [99.9% ID]) (Polishook et al., unpublished). strate dry weight in ash sawdust culture. In nature, C. damiettensis Notes. This specimen was subject to DNA barcoding and causes a white-rot on decaying datepalm trunks but enzymatic phylogenetic analysis (KY948797 [ITS]; KY948888 [LSU]; evidence suggests that it may have an unusual mechanism of wood KY948963 [RPB1]) by Justo et al. (2011), who place it in a well- decay. C. huangshanensis (Dai 6090) was reported to produce a supported clade alongside C. serpens and Ceraceomyces sp. white-rot (Cui and Jia, 2011) but to our knowledge, no pure cultures Miettinen-16854.3. Our attempts to obtain voucher Miettinen- of this species exist. However, a culture derived from voucher MR- 16854.3 from H have hitherto been unsuccessful. Mario Rajchen- 4310 (C. rajchenbergii) is available at CFRM. The presence of crystal- berg originally named as Phlebia cf. griseoflavescens. Phlebia gri- encrusted hyphae in several of the species in this clade may relate seoflavescens was recently renamed as Antrodia griseoflavescens to the unusual mechanism of wood decay alluded to above. (Runnel et al., 2019). Although known only from the single voucher There have been very few previous studies of macrofungi MR-4310, we consider that the detailed phylogenetic analyses associated with datepalm logs. Of these, Rattan et al. (1980) was the conducted by Justo et al. (2011) and ourselves (which show it to be most detailed with regard to resupinate fungi but none of the or- distinct), combined with our microscopic analysis of the voucher ganisms they describe (from Iraq) matches the features of Crystal- merit its formal naming. licutis damiettensis. Egypt is globally the largest grower of date palm and the arrival of red palm weevil in 1993 has caused a drop in 4. Discussion production exceeding 30% (Al-Dosary et al., 2016). It is possible that this fungus has only recently arrived in the Mediterranean region, Resupinate fungi are among the most important wood-decay following the spread of the red palm weevil R. ferrugineus (since the fungi. Not only do they contribute to nutrient cycling by decom- 1980’s). This highly invasive pest attacks more than 40 palm species posing wood debris, but they are also a potentially valuable source and has spread from Asia widely across the Mediterranean region in of novel pharmacologically-active biomolecules (Sto si c-Gruji ci c recent decades (Al-Ayedh, 2008; Al-Dosary et al., 2016). et al., 2011; Zapora et al., 2016). During a survey of basidiomy- The consistent association of C. damiettensis with R. ferrugineus. cetes growing on North Nile Delta of Egypt, resupinate basidiomes suggests that a mutualistic interaction between these organisms, as with unusual morphology were found growing on fallen, dead has been found in other woodeboring insects (Geib et al., 2008; trunks of P. dactylifera in a fruit farm. Kasson et al., 2016; Skelton et al., 2019a; Son et al., 2011) and that Following initial DNA barcoding, generic placement of this taxon the presence of C. damiettensis exacerbates the damaging effects of could not be determined readily from its morphological features the weevil. For example, the white-rot fungus Donkioporia expansa because it possessed characters assignable to several genera. (Polyporaceae) is found in consistent association with the
456 H.M. El-Gharabawy, C.A. Leal-Dutra and G.W. Griffith Fungal Biology 125 (2021) 447e458 deathwatch beetle (Xestobium rufovillosum)(Belmain et al., 2002; Binder, M., Larsson, K.-H., Matheny, P.B., Hibbett, D.S., 2010. Amylocorticiales ord. Campbell and Bryant, 1940). Similarly, Flavodon ambrosius (also nov. and Jaapiales ord. nov.: early diverging clades of Agaricomycetidae domi- nated by corticioid forms. Mycologia 102, 865e880. Irpicaceae) is found as mycosymbiont of Ambrosiodmus ambrosia Campbell, W.G., Bryant, S.A., 1940. A chemical study of the bearing of decay by beetles (Li et al., 2015), being transported by their vector in speci- Phellinus cryptarum Karst. and other fungi on the destruction of wood by the alised mycangia (Simmons et al., 2016) and suppressing the activity death-watch beetle (Xestobium rufovillosum de G.). Biochem. J. 34, 1404. Capella-Gutierrez, S., Silla-Martínez, J.M., Gabaldon, T., 2009. trimAl: a tool for of other wood decay fungi (Skelton et al., 2019b). There is hitherto automated alignment trimming in large-scale phylogenetic analyses. Bioinfor- no direct evidence at present that C. damiettensis contributes to the matics 25, 1972e1973. tree damage caused by R. ferrugineus (subfamily Dryophthorinae). Cui, B., Jia, B.-S., 2011. Wood-rotting fungi in eastern China 7. Polypores from huangshan mountain, Anhui Province. Mycosystema 30, 529e535. However, it lies within the same family (Curculionidae; order Chen, C.-C., Chen, C.-Y., Lim, Y.W., Wu, S.-H., 2020. Phylogeny and taxonomy of Coleoptera) as Ambrosia beetles (subfamilies Platypodinae and Ceriporia and other related taxa and description of three new species. Myco- Scolytinae) and bark beetles (subfamily Scolytinae) so the coinci- logia 112, 64e82. Chernomor, O., Von Haeseler, A., Minh, B.Q., 2016. Terrace aware data structure for dence in the distribution of R. ferrugineus and C. damiettensis raises phylogenomic inference from supermatrices. Syst. Biol. 65, 997e1008. the possibility that the former may be a targeted vector of the latter Detheridge, A.P., Brand, G., Fychan, R., Crotty, F.V., Sanderson, R., Griffith, G.W., (Jacobsen et al., 2017). Marley, C.L., 2016. The legacy effect of cover crops on soil fungal populations in a cereal rotation. Agric. Ecosyst. Environ. 228, 49e61. El-Gharabawy, H.M., Detheridge, A.P., El-Fallal, A.A., El-Sayed, A.K.A., Griffith, G.W., Author contributions 2016. Analysis of wood decay and ligninolysis in Polyporales from the Nile Delta region of Egypt. Mycosphere 7, 392e404. fi Floudas, D., Hibbett, D.S., 2015. Revisiting the taxonomy of Phanerochaete (Poly- HME - undertook eldwork and labwork; writing of the paper; porales, Basidiomycota) using a four gene dataset and extensive ITS sampling. CALD - Phylogenetic analyses; writing of the paper; GWG - Fungal Biology 119, 679e719. conceived the paper; analysis of data; drafted the manuscript. Gadagkar, S.R., Rosenberg, M.S., Kumar, S., 2005. Inferring species phylogenies from multiple genes: concatenated sequence tree versus consensus gene tree. J. Exp. Zool. B Mol. Dev. Evol. 304, 64e74. Acknowledgements Geib, S.M., Filley, T.R., Hatcher, P.G., Hoover, K., Carlson, J.E., del Mar Jimenez- Gasco, M., Nakagawa-Izumi, A., Sleighter, R.L., Tien, M., 2008. Lignin degrada- tion in wood-feeding insects. Proc. Natl. Acad. Sci. Unit. States Am. 105, We are grateful to Alan Cookson, IBERS, for assistance with 12932e12937. Scanning electron microscopy and also to IBERS HPC and Super- Gilbertson, R.L., Ryvarden, L., 1986. North American Polypores, vol. I. Abortiporus- computing Wales (SCW project, is part-funded by the European Lindtneria. Fungiflora A/S, Oslo, Norway. Ginns, J.H., 1975. Merulius: ss and sl, taxonomic disposition and identification of Regional Development Fund via Welsh Government) for computing species. Can. J. Bot. 54, 100e167. support. We thank Prof. Amira El-Fallal at the Faculty of Science, Gomez-Montoya, N., Drechsler-Santos, R.E., Ferreira-Lopes, V., Tom sovský, M., Damietta University for her help during forays for fungal samples Urcelay, C., Robledo, G.L., 2017. New insights on Trametopsis Tom sovský (Poly- porales Gaum)€ based on phylogenetic evidences and morphological analyses of collection, to Dr. Paul Kirk (RBG, Kew) and Dr. Shaun Pennycook neotropical species. Phytotaxa 311, 155e167. (Landcare Research, Auckland) for taxonomic advice, and also to Griffith, G.W., Hedger, J.N., 1994. Spatial distribution of mycelia of the liana (L-) and Dr. Andrew P. Detheridge (IBERS) for assistance with DNA biotype of the agaric Crinipellis perniciosa (Stahel) Singer in tropical forest. New e sequencing. We are grateful to Katriina Bendiksen at the Natural Phytol. 127, 243 259. Griffith, G.W., Shaw, D.S., 1998. Polymorphisms in Phytophthora infestans: four History Museum, University of Oslo and Dr. Beatriz Ortiz-Santana at mitochondrial haplotypes are detected after PCR amplification of DNA from the USDA Northern Research Station for provision of voucher pure cultures or from host lesions. Appl. Environ. Microbiol. 64, 4007e4014. specimens, to Dr. Anarrosa Bernicchia of Bologna for useful dis- Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: cussions and to anonymous reviewers for helpful suggestions. We assessing the performance of PhyML 3.0. Syst. Biol. 59, 307e321. thank the Egyptian Government for the funding of a Joint-mission Hoang, D.T., Chernomor, O., Von Haeseler, A., Minh, B.Q., Vinh, L.S., 2018. UFBoot2: e PhD programme (2013-2016) and also a postdoctoral fellowship to improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518 522. Jacobsen, R.M., Kauserud, H., Sverdrup-Thygeson, A., Bjorbækmo, M.M., visit Aberystwyth in 2020. The Institute of Biological, Environ- Birkemoe, T., 2017. Wood-inhabiting insects can function as targeted vectors for mental, and Rural Sciences receives strategic funding from the decomposer fungi. Fungal Ecology 29, 76e84. BBSRC. Jia, B.-S., Zhou, L.-W., Cui, B.-K., Rivoire, B., Dai, Y.-C., 2014. Taxonomy and phylogeny of Ceriporia (Polyporales, Basidiomycota) with an emphasis of Chinese collec- tions. Mycol. Prog. 13, 81e93. Appendix A. Supplementary data Jung, P.E., Lee, H., Wu, S.-H., Hattori, T., Tom sovský, M., Rajchenberg, M., Zhou, M., Lim, Y.W., 2018. Revision of the taxonomic status of the genus Gloeoporus (Polyporales, Basidiomycota) reveals two new species. Mycol. 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