Studies of Neotropical tree pathogens in Moniliophthora: a new species, M.mayarum, and new combinations for Crinipellisticoi and C.brasiliensis

Abstract The crinipelloid genera Crinipellis and Moniliophthora (Agaricales, Marasmiaceae) are characterized by basidiomes that produce long, dextrinoid, hair-like elements on the pileus surface. Historically, most species are believed to be saprotrophic or, rarely, parasitic on plant hosts. The primary morphological diagnostic characters that separate Crinipellis and Moniliophthora are pliant vs. stiff (Crinipellis) stipes and a tendency toward production of reddish pigments (ranging from violet to orange) in the basidiome in Moniliophthora. Additionally, most species of Moniliophthora appear to have a biotrophic habit, while those of Crinipellis are predominantly saprotrophic. Recently, several new neotropical collections prompted a morphological and phylogenetic analysis of this group. Herein, we propose a new species and two new combinations: Moniliophthoramayarumsp. nov., described from Belize, is characterized by its larger pileus and narrower basidiospores relative to other related species; Moniliophthoraticoicomb. nov. (= Crinipellisticoi) is recollected and redescribed from biotrophic collections from northern Argentina; and M.brasiliensiscomb. nov. (= Crinipellisbrasiliensis), a parasite of Heteropterysacutifolia. The addition of these three parasitic species into Moniliophthora support a hypothesis of a primarily biotrophic/parasitic habit within this genus.


Introduction
The crinipelloid genera Crinipellis Pat. and Moniliophthora H.C. Evans, Stalpers, Samson & Benny are characterized by basidiomes that produce thick-walled, dextrinoid, hair-like terminal cells on the pileus surface (Kerekes and Desjardin 2009). These belong to the Marasmiaceae in a lineage that includes Marasmius Fr. and Chaetocalathus Singer (Aime and Phillips-Mora 2005;Antonín 2013). Crinipellis and Moniliophthora appear to be most speciose in the Neotropics (Singer 1976;Kerekes and Desjardin 2009). Only a few authors have studied these genera in the Neotropics, primarily Singer (1942Singer ( , 1976, who described 41 neotropical species of Crinipellis. Moniliophthora was described by Evans et al. (1978) as an incertae sedis, monotypic genus of basidiomycetes, with M. roreri (Cif.) H.C. Evans, Stalpers, Samson and Benny, a parasitic fungus of T. cacao, as the type. Aime and Phillips-Mora (2005) (Takahashi 2002), M. marginata Kerekes, Desjardin & Vikinesw., and M. (=Crinipellis) nigrilineata (Corner) Desjardin & Kerekes (Kerekes and Desjardin 2009). The primary morphological diagnostic characters that separate Crinipellis and Moniliophthora are pliant vs. stiff (Crinipellis) stipes, and a tendency toward production of pink to orange pigments in the basidiome, that do not change to green or olive when treated with KOH or NaOH (Moniliophthora). Additionally, many Moniliophthora species appear to have a biotrophic habit, including important pathogens of tropical crops such as cocoa (Theobroma cacao L.), while those of Crinipellis are primarily saprotrophic.
Recent collecting efforts in northern Argentina and within the Mayan Mountains of Belize included two crinipelloid species. One, an orange fungus fruiting copiously from living roots and trunks of three different species of living trees in Argentina was identified as Crinipellis ticoi. The other, an orange fungus fruiting gregariously on a dead root in Belize was determined to represent a new species of Moniliophthora. Herein we provide updated descriptions, as well as phylogenetic analyses supporting the placement of these and one other former species of Crinipellis within Moniliophthora as: M. ticoi comb. nov., M. brasiliensis comb. nov., and M. mayarum sp. nov., bringing the total number of known species of Moniliophthora to 11.

Morphological studies
The specimens studied here were collected in Belize (deposited at BRH and CFMR) and from northern Argentina (deposited at CTES). Specimens were described macroscopically according to Largent (1986). Kornerup and Wanscher (1978) colors are followed by chart numbers and letters in parentheses. Capitalized color names are from Ridgway (1912) as reproduced by Smithe (1975), except for Spectrum Orange which was created by Smithe (1975) to fill a gap. Microscopic characters were examined by light microscopy (LM) on a Leica model CME or an Olympus BH-2. All LM images were made with a Leica EC3 incorporated camera from material mounted in 5% KOH and Phloxine (1%), and Melzer's reagent. The measurements were made directly in the LM or through the photographs taken using the software IMAGEJ (Schneider et al. 2012). Microstructures (length and width of spores, basidia, hyphae, pileipellis) were measured using LM. The following notations were used for spore measurement: x = arithmetic mean of the spore length and width, with standard deviation (+/-); Q = quotient of length and width indicated as a range of variation; Q x = mean of Q values; n = number of spores measured, N = number of analyzed basidiomes. All GPS readings were taken on a Garmin eTrex 10, hand-held unit using WGS84 standard. Herbarium abbreviations follow Index Herbariorum (Thiers 2019) and authors' abbreviations follow Kirk and Ansell (1992).

DNA extraction, amplification, and sequencing
Extraction, amplification and sequencing of the new species at CFMR in Madison, WI followed Lindner and Banik (2009). For the other specimens, DNA was extracted from dried basidiomes using the Promega Wizard Genomic DNA Purification Kit (Promega Corp., Madison, WI, USA). Amplification of the internal transcribed spacer (ITS) and large subunit (28S) of the ribosomal DNA repeat follow the methods of Aime and Phillips-Mora (2005). Sequencing of PCR products was conducted at GeneWiz (South Plainfield, NJ, USA). Sequences were manually edited with Sequencher 5.2.3 (Gene Codes Corp., MI, USA) and confirmed via BLAST queries of the NCBI databases (National Center for Biotechnology Information, Bethesda, MD, USA). Collection data and GenBank accession numbers of the specimens used in this study are detailed in Table 1.

Phylogenetic analysis
Initially, sequences derived for this study were analyzed within a dataset (Aime unpubl.) of 612 published and unpublished Marasmiaceae sequences inclusive of all genera in the family (data not shown). Results from preliminary phylogenetic and blast analyses indicated that the Argentina and Belize material both belong within Moniliophthora, as does Crinipellis brasiliensis -the sister species to M. perniciosa (Arruda et al. 2005). Datasets were then trimmed to include: 1) all species of Moniliophthora for which ITS and/or 28S sequence data exist (only ITS data were available for M. canescens, M. aurantiaca, and M. brasiliensis); 2) newly generated sequences of the material from Argentina and Belize and from the type of C. ticoi; 3) exemplar sequences from the other related Marasmiaceae genera -Crinipellis, Marasmius, and Chaetocalathus -for context (Aime and Phillips-Mora 2005; Antonín et al. 2014;Koch et al. 2018). Individual datasets for each locus were aligned in GENEIOUS 9.1.5 (Biomatters Ltd., Auckland, NZ) using the MUSCLE algorithm (Edgar 2004). Individual alignments were then concatenated in Geneious, and analyzed by maximum likelihood (RAXML; Stamatakis 2006) methods using the CIP-RES Science Gateway (Miller et al. 2010), following the methods of Koch et al. (2018).

Phylogenetic analyses
Based on our Maximum Likelihood (ML) analysis of ITS and 28S rDNA, Marasmiaceae is comprised of two clades, Marasmius + Crinipellis + Moniliophthora +  Chaetocalathus and Tetrapyrgos + Brunneocorticium. Moniliophthora is the sister genus to Crinipellis (96 BS). The newly sequenced material (M. mayarum and M. ticoi) are strongly supported as members of Moniliophthora, as is C. brasiliensis based on previously sequenced material (Arruda et al. 2005) (Fig. 1). The collected material in Argentina shared 100% identity with the type specimen of Crinipellis ticoi, collected and described from Bolivia. Description. Pileus 7-20 mm, convex with an inrolled margin when young, broadly convex with age, some slightly depressed at center, some with a papillate umbo, color Chrome Orange (Plate II, 11, -), with center Scarlet (Plate I, 5, -) to Flame Scarlet (Plate II, 9, -), surface moist or slightly viscid when wet but not gelatinized, smooth, rarely sparsely minutely pubescent on umbo when dry, margin translucent-striate to disc, some sulcate-striate with age. Lamellae subdistant, 2 per mm on margin and halfway to margin, adnate or slightly adnexed, 2-4 mm broad, regular, 1 or more lengths of lamellulae inserted, Spectrum Orange with a coral tint, margin even, concolorous. Stipe central, 12-27 × 0.8-1.2 mm, equal or slightly clavate, some flared at apex, pale Spectrum Orange, pale Orange-Yellow (Plate III,17, f ) at apex, surface dry, densely minutely pubescent, dense Warm Buff (Plate XV, 17´, d) mycelial pad at base. Annulus absent. Spore-print not observed, presumably white. Context pale orange in pileus and stipe, odor none, taste sweet. KOH and NaOH reactions on pileus surface negative.
Distribution. Know only for the type locality. Ecology. Gregarious, putatively parasitic on roots of a tree, possibly Ceiba pentandra (L.) Gaertn.
Etymology. mayarum -of the Maya people in the region where the fungus was found.  The two Neotropical species are more robust, reaching 20 mm in diameter (or more in M. ticoi), compared to the two Paleotropical species, 6-11 mm in C. hygrocyboides and 3-15 mm in M. aurantiaca. Antonín (2007) published a type revision of C. hygrocyboides based on study of an isotype that included microscopic meas- urements and observations of spores and cheilocystidia as neither Hennings (1902) nor Singer (1989) included these details and Halling (1993) reported he could not find spores or cystidia in the type. The spores of M. mayarum are distinctly narrower (3.2-4.2 µm) than those of C. hygrocyboides [4.5-6(-7) µm]. While Antonín's description of the cheilocystidia in C. hygrocyboides notes they are ornamented with apically branched obtuse projections, cheilocystidia shape seems to be highly variable and therefore unreliable for distinguishing species in this group. The basidiospores are longer and broader in both M. aurantiaca 7.5-11 × 4-6, and M. ticoi (9.5-)10.5-13.7 × (3.8-)4.5-6.3 µm, than in M. mayarum 6.5-8.5(-10.5) × 3.2-4.2 µm. Only the larger spores of M. mayarum are used in the preceding comparison, and it is not clear why there is a cohort of smaller basidiospores also present. Although, different spore sizes are often observed in the presence of bisporic basidia, lacking clamp-connection and bearing larger spores that are mixed with tetrasporic basidia bearing smaller spores, in repeated examination of the material specifically looking for 2-sterigmate basidia and absence of basal clamps, we observed only 4-sterigmate basidia, and all hymenial elements with clamp-connections. Furthermore, one of the illustrated 4-spored basidia (Fig. 3C), shows a large spore attached to a 4-sterigmate basidium with a basal clamp connection, which negates the hypothesis of a bisterigmate origin for the large spore cohort in M. mayarum. Although small spores observed on the hymenium could have been immature and thus smaller, basidiospores of similar size and shape were observed on the pileipellis surface that must have been released from basidia, which indicates they were mature. A similar case occurs in Crinipellis trinitatis Dennis. Dennis (1951) in the original description reported smaller basidiospores (5-7 × 2-4 µm) than the revised description of the type by Pegler (1983) (7-9 × 4.1-5.1 µm), so there may be something unusual in the phenology of spore production in this group that leads to two size classes of spores depending on when they are formed and released. (Arruda, G.F.Sepúlveda, R.N Distribution. This species is known from Minas Gerais, Brazil (Arruda et al. 2005).

Moniliophthora ticoi
Distribution. This species is known from Bolivia (Halling 1993) and northern Argentina (Yungas and Chaco region). Observations. This species was described by Halling (1993) from Bolivian specimens. It is characterized by its relatively large, bright orange basidiomes, covered with scattered dextrinoid setiform hairs. The most similar species is M. mayarum, which shares morphological characters such as the large basidiomes with bright orange coloration. These two species, however, differ clearly by the smaller spores and by the presence of ornamented cheilocystidia in M. mayarum. Another similar species is M. aurantiaca from American Samoa (Kropp and Albee-Scott 2012). Both share the orange colored pileus surface with a narrow light yellowish margin. However, they differ in that M. aurantiaca has the smaller pileus (3-15 mm broad), smaller basidiospores (7.5-11 × 5-8 µm) and numerous cheilocystidia with several irregular apical appendages resembling fingers (Kroop and Albee-Scott 2012). Another similar species is C. hygrocybioides (Henn.) Singer from Africa (Singer 1989), however this is a smaller fungus (pileus 6-11 mm broad) with an umbilicate to papilate pileus that is pilose at the margin (Halling 1993). Based on its morphological characters such as the bright orange pileus surface, C. hygrociboides could be included in the genus Moniliphthora, but new collections are needed to elucidate its habitat and to obtain sequences and corroborate this hypothesis (currently there are not sequences available for C. hygrocybioides).
Crinipellis siparunae is a widely distributed species that is microscopically similar to M. ticoi, especially regarding the range of spore size. However, C. siparunae is distinguished by its lilac to brownish lilac pileus surface and by its appendiculate cheilocystidia (Singer 1942(Singer , 1976. A taxon thought to be closely related to C. siparunae, C. eggersii Pat. var. lilaciceps Singer and described from Amazonian Ecuador shares unornamented cheilocystidia with C. ticoi, but it differs from the latter in having a violet to lilac vs orange pileus, and broader basidiospores 6-6.5 × (3.8-)4.5-6.3 µm (Singer 1976;Kerekes and Desjardin 2009). Crinipellis eggersii var. eggersii, which includes the facultative synonym Marasmius vinosus Speg. described from Argentina, has similar spore dimensions (mostly 11-13 × 5.5-6.3 µm) but differs in having a purple to violet purple pileus and a variety of cheilocystidia shapes (ampullaceous, fusiform, cylindrical or clavate and mostly forked, obtuse or mucronate, sometimes with a subcapitate or capitate apex).
Of the three recent collections in northern Argentina, the specimens of the Yungas forest (Niveiro et al. 2249) closely resemble the original description of M. ticoi, with specimens not exceeding 40 mm broad and having a bright red pileus surface (Halling 1993). However, the specimens of the Chaco region differ in having larger basidiomes up to 60 mm broad, and a paler coloration (orange with a yellowish margin), differ-ences that may be due to the drier weather conditions in the Chaco region. Another important difference observed in the Argentinean specimens is the habitat. Halling (1993) found this species growing on rotten wood, however, the new specimens examined were growing on tree trunks and roots of living trees, confirming a biotrophic habit for this species during at least part of its life history.
Key to striking bright orange Moniliophthora and Crinipellis species

Discussion
The addition of these three parasitic species into Moniliophthora support a hypothesis of a primarily biotrophic/parasitic habit in this linage of Marasmiaceae. However, nutritional strategies for several species not studied in the present work remain to be definitively ascertained: M. aurantiaca was found on woody debris ( Purple, violet, and red pigments in the pileus combined with a negative (not greenish) reaction with KOH distinguish Crinipellis section Iopodinae (Singer 1976), and these characters are shared with the known basidiome-producing species of Moniliophthora. Although no recent collections or sequences are available for other species of Crinipellis section Iopodinae, Kerekes and Desjardin (2009) did show that a specimen identified as C. aff. iopus Singer (the type species of Crinipellis sect. Iopodinae) belonged in Moniliophthora, although due to a lack of data they were unable to confirm this placement. The current study adds striking orange pigmentation to the suite of characteristics for Moniliophthora, as well as confirming a biotrophic habit for the majority of species.