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Taxonomy of Pseudolagarobasidium (Polyporales, Basidiomycota)

Pseudolagarobasidium (Polyporales, Basidiomycota) is a small, monophyletic genus of crustose, wood-inhabiting fungi with spines and a saprobic, endophytic, or parasitic habit. Seven species are accepted in the genus including two new species, P. belizense from Belize and P. pusillum from Australia. Sequence analysis of the internal transcribed spacer of the ribosomal RNA gene places P. belizense in a monophyletic clade with P. acaciicola and an undescribed foliar endophyte. New combinations proposed include P. modestum for Irpex modestus Berk., P. pronum for Hydnum pronum Berk. & Broome which is an earlier name for P. calcareum, and P. venustum for Radulodon venustus Hjortstam & Ryvarden. Irpex colliculosum Berk. & Broome from Sri Lanka is conspecific with P. subvinosum. Two species, Sistotrema ochroleucum and Radulum concentricum are not accepted in Pseudolagarobasidium. Pseudolagarobasidium is compared with Radulodon and similar genera. A key to the species of Pseudolagarobasidium is provided.

An unusual crustose species with small spines was collected from Doyle's Delight in 2004 by the junior author. Doyle's Delight Peak, the highest peak in the Maya Mountains of Belize, is a mycologically productive site that has yielded a number of new taxa (Baroni et al. 2007; Baroni et al. 2008;Ginns et al. 2010;Lindner et al. 2011;Ryvarden et al. 2009). DNA sequence analyses indicated that the crustose species belonged in Pseudolagarobasidium, and morphological examination confirmed that it was an undescribed species.
In this paper, we describe and illustrate two new species of Pseudolagarobasidium from Belize and Australia and propose the transfer of Irpex modestus Berk. and Radulodon venustus Hjortstam & Ryvarden to Pseudolagarobasidium. Type specimens of Hydnum pronum Berk. & Broome and Irpex colliculosus Berk. & Broome were examined and found to be congeneric with Pseudolagarobasidium. An emended description of Pseudolagarobasidium and key to the seven accepted species are presented.

Morphological methods
Thin, freehand sections from basidiomata were mounted in Melzer's reagent (Kirk et al. 2008) or 1% (weight/volume) aqueous phloxine and 1% (w/v) aqueous potassium hydroxide. Cyanophily of basidiospore and hyphal walls was observed in 0.1% cotton blue in 60% lactic acid (Kotlaba and Pouzar 1964;Singer 1986). Line drawings were made with a camera lucida attachment on an Olympus BH2 compound microscope. Photographs were taken with an Olympus DP12 camera attached to an Olympus SZH stereomicroscope. Q values were obtained from dividing average basidiospore length by width (Kirk et al. 2008). Basidiospores were often scarce in specimens, thus Q values based on less than 30 basidiospores are approximate and indicated with an asterisk (*). Color names are from Kornerup and Wanscher (1978), although capitalized names are from Ridgway (1912). Herbarium designations follow that of Index Herbariorum (Thiers 2012).
DNA sequence methods DNA sequences of the internal transcribed spacer region (ITS) of the ribosomal RNA were obtained from cultures following methods detailed in Greslebin et al. (2004) and from basidiomata as described in Palmer et al. (2008).

Taxon selection and phylogenetic analyses
Initial BLAST searches placed DCL04-31 near Pseudolagarobasidium in the Polyporales. Taxa chosen for the phylogenetic analyses were selected after consulting Hallenberg et al. (2008), Lee and Lim (2010), and results from BLAST searches. In addition to newly generated sequences, Pseudolagarobasidium belizense (JQ070173, holotype), Radulodon americanus Ryvarden (JQ070174, JQ070175), and R. casearius Twenty ITS sequences were aligned with ClustalW in MEGA5 (Tamura et al. 2011) then manually adjusted. In the phylogenetic analyses, maximum likelihood (ML) method based on the Tamura 3-parameter model (Tamura 1992) with gamma distribution was employed, following results from a best-fit substitution model test. As implemented in MEGA5, initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method (Gascuel 1997) with Markov cluster distance matrix was used. A discrete Gamma distribution modeled evolutionary rate differences among sites in five categories (+G, parameter0 0.4593). All positions containing gaps and missing data were eliminated from the analyses. Ten thousand ML bootstrap replicates were conducted with the same parameters. In addition, maximum parsimony (MP) analyses were performed in MEGA5 using close-neighbor interchange algorithm (Nei and Kumar 2000) in which the initial trees were obtained by random addition of sequences (10 replicates). Finally, 10000 MP bootstrap replicates were conducted employing the same parameters. Sequence alignment and ML and MP trees are deposited in TreeBase (accession number 12351).

Phylogenetic analysis
There were 20 nucleotide sequences in the ITS dataset and 657 base pairs in the full alignment with 308 variable and 200 parsimony informative sites. Sequence divergence between sequences was calculated in which ambiguous positions were removed for each sequence pair (data not shown). Within the Pseudolagarobasidium clade ( Fig. 1), sequence divergence ranged from 0 to 12 %. ITS sequence of P. acaciicola AM849050, isolated from soil in India, was the most divergent in the clade, differing by 10% from P. acaciicola DQ517883 and 9% from DQ517882, both from South Africa, and 12% from P. belizense and Basidiomycota RCBC XG8D.
After all positions containing gaps and missing data were eliminated, there remained 498 positions in the final ITS dataset. In both ML and MP analyses, P. belizense always clustered with P. acaciicola and Basidiomycota RCBC XG8D. The ML tree with the highest log likelihood (−2315.2104) is shown in Fig. 1. With the MP method, 47 most parsimonious trees were recovered with a consistency index of 76.7%, retention index of 84.5%, and composite index of 64.8%. The MP consensus tree is similar to the ML tree except that the Spongipellis Pat. and Radulodon clades were not joined together. In the ML bootstrap tree, the Pseudolagarobasidium clade is supported at 86% and slightly higher with the MP bootstrap analysis at 92%.
The features that distinguish this genus are the softtextured basidiomata, non-agglutinated hyphae throughout the aculei and context (except in one species), the distinct, even, clamped hyphae of the aculei trama, the numerous tramal and hymenial cystidia with homogenous contents that stain deeply in phloxine and cotton blue, and the small, globose to ellipsoid basidiospores. Oil-like particles are produced throughout the basidioma but are observed only in water mounts because they readily dissolve in KOH and other mounting media. Because the basidioma context is not agglutinated, hyphae and microscopic elements are readily observed in squash mounts. Microbinding hyphae are easily overlooked but are usually present in the subiculum adjacent to the substrate or in mycelial pockets in the woody substrate beneath the basidiomata. Also noteworthy is the variable hymenophore, ranging from spinose to reticulate. Microscopically, the species included in the genus are remarkably consistent with respect to aculei microstructure and shape and size of hymenial elements but differ in texture, color, and aculei morphology. Bifactorial incompatibility system (tetrapolar mating system) was reported for P. pronum (Maekawa and Hasebe 2002) and P. subvinosum (Chang and Chen 1984).
Species of Pseudolagarobasidium are primarily saprobes although several are endophytes or facultative or opportunistic pathogens. Pseudolagarobasidium subvinosum causes a stem canker or root rot (Jang and Chen 1985;Petch 1923;Sankaran and Sharma 1986;Wood and Ginns 2006) whereas P. acaciicola and P. pronum are associated with a dieback disease (Wood and Ginns 2006). In addition, molecular analyses of the nuclear large subunit ribosomal DNA placed undescribed endophytic fungal isolates from healthy stems of cacao and leaves of mangrove in the monophyletic Pseudolagarobasidium clade (Chokpaiboon et al. 2010;Hallenberg et al. 2008).
Habitat and distribution: Saprobic on wood, also pathogenic, attacking root crowns of Acacia cyclops; known from South Africa.
Pseudolagarobasidium acaciicola is characterized by brown basidiomata with flattened, fused aculei, dimitic hyphal system, and ellipsoid basidiospores. It is most similar to P. modestum which has slightly smaller basidiospores; see discussion under P. modestum. Another morphologically similar species, P. subvinosum has slightly larger basidiospores and more prominent hymenial cystidia compared to P. acaciicola. Arthroconidia are produced in cultures and basidiomata of P. subvinosum and P. pronum also. For a complete description with illustrations of basidiomata and cultures see Wood and Ginns (2006). The specimens of van der Bijl cited above were originally identified as Irpex modestus; however, the basidiospores were more similar in size to those of P. acaciicola than to P. modestum. Molecular analyses of the nLSU sequences show that P. acaciicola has a basal position in the genus (Hallenberg et al. 2008), although with ITS data ( Fig. 1) P. belizense is basal to P. acaciicola.
Habitat and distribution: Saprobic on bark and wood of angiosperms; known only from the type locality, Belize.
Pseudolagarobasidium belizense is characterized by grayish tan-colored basidiomata, small, subceraceous aculei <500 μm long, and agglutinated subicular hyphae. It is the only species in Pseudolagarobasidium in which the ascending subicular hyphae are agglutinated. Microbinding hyphae occur randomly in the subiculum and substrate. Because of its unique color and small aculei, P. belizense is readily distinguished from other species in the genus with basidiospores of similar size. Pseudolagarobasidium venustum, also known from the Americas, has pale yellow to pale orange basidiomata. ITS sequence analyses place P. belizense basally in the monophyletic Pseudolagarobasidium clade (Fig. 1).
Habitat and distribution: Saprophytic, rarely pathogenic, on wood and bark of angiosperms; known from mainland China (Wu 2008), Taiwan, Japan, Sri Lanka, Malaysia, Australia, and Sierra Leone. Pseudolagarobasidium pronum is characterized by soft, brittle, cretaceous or chalky aculei, microbinding hyphae, and small basidiospores. It has the softest texture in the genus, and unlike other species, the microbinding hyphae are easily observed. Although Maekawa and Hasebe (2002) reported the presence of arthroconidia in many specimens of P. pronum from China and Japan, we observed arthroconidia in only one specimen from Japan, TFM-F-16917. Pseudolagarobasidium pronum is mostly likely to be confused with P. subvinosum because of an overlap in basidiospore size. However, the lighter colored, softer, chalky basidiomata and microbinding hyphae of P. pronum can be used to distinguish it from the darker colored P. subvinosum. Sequence analyses of the nLSU show a close relationship between P. pronum and P. subvinosum (Hallenberg et al. 2008). Pseudolagarobasidium pusillum, also from Western Australia, has much smaller basidiospores.
The hymenium of the holotype and isotype specimens of H. calcareum is poorly preserved; no basidia and few basidiospores were observed. The holotype of H. pronum is in better condition with dark-colored areas on the aculei which indicate bruising or improper drying. Maas Geesteranus (1974) provides a brief description of the H. pronum holotype, but basidiospores were not observed. Despite the condition of the types, there can be no doubt that H. pronum and H. calcareum are conspecific. Shivas and Brown (1989) reported that Pirex subvinosum is associated with a dieback of L. leucocephala in Western Australia; however, we identified their specimen, PERTH 00734527, as P. pronum based on basidioma texture and presence of microbinding hyphae in the subiculum. Although no basidia or basidiospores were observed, another specimen collected in 1993 from the same area and host species, PERTH 02340968, has basidiospores typical for P. pronum.
Basidioma resupinate, widely effuse, thin, up to 225 μm thick between aculei, spinose with distinct, smooth areas between aculei, occasionally with large, knobby structures bearing aculei, Buffy Brown, greyish orange (5B4), at first dark brown in KOH or water then fading; cracks scattered; hymenial surface composed of fragile, subceraceous to membranous aculei, up to 3 aculei per mm, up to 3 mm long, mostly terete to conical, sometimes compressed or fused laterally, smooth, occasionally with warty outgrowths, gradually tapering to a subacute, sterile apex, apices creamcolored or concolorous with base of aculeus; margin not observed.
Habitat and distribution: Saprobic on Acacia in Western Australia.
Pseudolagarobasidium pusillum is characterized by grayish brown basidiomata, long, terete aculei, and the smallest basidiospores in the genus. Pseudolagarobasidium pronum is reported from Western Australia also but has chalky basidiomata, larger basidiospores, and arthroconidia.
Habitat and distribution: Saprobic or parasitic, on bark and wood of various angiosperms; known from Taiwan, Sri Lanka, India, and Zaire. Pseudolagarobasidium subvinosum can be distinguished from other species in the genus by its large, ellipsoid basidiospores. Although microbinding hyphae were found in the substrate, not in the basidioma, of the holotype collection, it is probable that these hyphae occur in other specimens, too. Herbarium specimens are uniformly a shade of brown, but fresh basidiomata were described as deep purple, pale lavender, and blue by Petch (1923) and deep violet or purplish by Jang and Chen (1985). Although P. subvinosum is often confused with P. pronum, the basidiomata of the former are usually darker brown and lack microbinding hyphae whereas in the latter they are softer, chalky, off-white to light brown, with microbinding hyphae in the subiculum. Basidiospores of P. subvinosum, on average, are larger than those of P. pronum although there is an overlap in size.
The holotype of H. subvinosum at K is in better condition than the isotype at BPI, but basidiospores were abundant in both specimens. Microbinding hyphae were found in pockets of white tissue in the substrate directly beneath the basidioma of the holotype. The holotype of Irpex colliculosus is in good condition although few basidiospores were observed. The average size, 6.1×4.2 μm (n014), Q01.4, is typical for P. subvinosum. See Maas Geesteranus (1974) for brief descriptions of holotypes of H. subvinosum and I. colliculosus.
See Jang and Chen (1985), Petch (1923), Sankaran and Sharma (1986), and Wu (1990) for additional descriptions and illustrations of P. subvinosum. Culture descriptions are provided by Jang and Chen (1985) and Sankaran and Sharma (1986). Chang and Chen (1984) report that P. subvinosum has a tetrapolar mating type system. Pseudolagarobasidium subvinosum is pathogenic on L. leucocephala causing a stem canker in India (Sankaran and Sharma 1986) and a stem and root rot in Taiwan (Jang and Chen 1985). Earlier, Petch (1923) described P. subvinosum on diseased roots of Acacia decurrens Willd., Tephrosia candida DC., and Camellia sinensis (L.) Kuntze in Sri Lanka. Although Pirex subvinosus, PERTH 734527, was reported from Australia by Shivas and Brown (1989) to cause a stem canker of L. leucocephala, we re-identified their specimen as P. pronum; for further discussion see P. pronum.
Because of its coriaceous texture and trimitic hyphal system, S. ochroleucum is not congeneric with Pseudolagarobasidium. The isotypes examined were fragments, so it was not possible to determine the true form and structure of the basidioma. We follow Ryvarden (1981) who examined the holotype specimen of S. ochroleucum at PC and placed it in synonymy with L. acutus.

Discussion
Pseudolagarobasidium is established as a monophyletic genus based on morphological and molecular data. The seven accepted species are distributed widely in Africa, Asia, Australia, and Central and South America. Most species are known from fewer than three collections; only P. pronum and P. subvinosum can be considered not uncommon. The varied nutritional modes associated with species of Pseudolagarobasidium, from saprobic, parasitic, and endophytic, are noteworthy. Species of Pseudolagarobasidium treated herein are saprobes although some can be described as facultative pathogens. Endophytic taxa are known from DNA sequences obtained from healthy stems of Theobroma cacao from Brazil and Cameroon (Crozier et al. 2006;Hallenberg et al. 2008) and leaves of Xylocarpus granatum König from Thailand (Chokpaiboon et al. 2010).
The ITS sequence of P. acaciicola UDSC-RCK, AM849050, isolated from soil in India, differs significantly from other P. acaciicola sequences and may be a different species. Our phylogenetic results also suggest that Antrodiella albocinnamomea (FJ613650) and the unidentified taxon 'Fungal sp. ref 1' (EU834826) should be classified in Cerrena.
Both nLSU and ITS phylogenetic analyses provide strong evidence that Pseudolagarobasidium is a monophyletic genus in a Polyporales clade that includes Radulodon, Cerrena Gray, and Spongipellis (Hallenberg et al. 2008;Lee and Lim 2010;Moreno et al. 2011). The four genera are distinct and readily recognizable displaying a wide range in habit (effuse to pileate), hymenophore configuration (odontioid to poroid), and texture (soft and fragile to tough and cartilaginous). They have in common basidia with four sterigmata and hyaline basidiospores that do not react in Melzer's reagent.
Pseudolagarobasidium is most similar to Radulodon based on morphology and molecular data. Stalpers (1998) considered Pseudolagarobasidium a synonym of Radulodon based on overall similarities of the generic types. Later, Nakasone (2001) restricted Radulodon to species with a dimitic hyphal system. In this study, we discovered that most species of Pseudolagarobasidium are dimitic also, developing microbinding hyphae in addition to clamped generative hyphae. Microbinding hyphae, however, are scarce or apparently absent in most specimens of P. subvinosum and P. venustus. Although there is some overlap, Radulodon basidiomata are ceraceous or cartilaginous with mostly agglutinated, hyaline hyphae whereas in Pseudolagarobasidium they are soft to subceraceous and fragile or brittle with mostly non-agglutinated, hyaline to light brown hyphae in the aculeus trama and subiculum. At the microscopic level, Radulodon species develop only hymenial cystidia, hyphidia maybe present or absent, and the acyanophilous, slightly thick-walled basidiospores usually contain an oil-like globule. In contrast, Pseudolagarobasidium species develop abundant tramal and hymenial cystidia and lack hyphidia. Basidiospores in Pseudolagarobasidium lack oil-like particles and may have thin or slightly thickened walls that are acyanophilous or weakly cyanophilous.
The placement R. cirrhatinus and R. revolubilis remain unresolved. Radulodon cirrhatinus from Malaysia appears to straddle two genera. It has non-agglutinated tissues, long aculei, and small, subglobose basidiospores with cyanophilous walls, as in Pseudolagarobasidium, but oil-like globules in the basidiospores which are characteristic of Radulodon. It has a monomitic hyphal system (Hjortstam et al. 1990), for the skeletal hyphae described by Nakasone (2001) are probably better characterized as sclerified generative hyphae. Interestingly, it occurs in southeast Asia where several Pseudolagarobasidium species are present. Radulodon revolubilis from Venezuela has a resupinate, cartilaginous basidioma with a sublamellate to irpicoid or lacerate hymenophore, monomitic hyphal system, and small, subglobose basidiospores with slightly thickened, cyanophilous walls. This taxon appears to be congeneric with Spongipellis but more study is required before a formal transfer is proposed.
Radulomyces M.P. Christ. and Sarcodontia Schulzer are similar to Pseudolagarobasidium in developing resupinate basidiomata, aculei, clamped hyphae, and globose to ellipsoid basidiospores. Radulomyces, however, is distinguished by its ceraceous, hygrophanous basidiomata, large basidia and basidiospores, hyphidia, and lack of cystidia. In addition, its hyphae, basidia and basidiospores are filled with oillike particles. Molecular analyses of the nLSU sequences show that Radulomyces is closely related to Typhula (Pers.) Fr. and Macrotyphula R.H. Petersen in the Pterulaceae Corner (Larsson et al. 2004). The generic type of Sarcodontia, S. crocea (Schwein.) Kotlaba, has a distinctive, nearly fleshy basidioma bearing ceraceous, slender, yellow to brown aculei 5-15 mm long. At the microscopic level, hyphae in the subiculum are thick-walled and terminally inflated, cystidia are absent, and the slightly thick-walled basidiospores contain oillike particles. It is associated with cultivated apple trees and other angiosperms. Molecular sequence analyses place it in the Meruliaceae Rea (Larsson 2007).