New insights on Bjerkandera (Phanerochaetaceae, Polyporales) in the Neotropics with description of Bjerkandera albocinerea based on morphological and molecular evidence

1Núcleo de Pesquisa em Micologia, Instituto de Botânica, Av. Miguel Stefano 3687, 04301-902, São Paulo, Brazil 2Centro de Investigación y Extensión Forestal Andino Patagónico, C.C. 14, 9200 Esquel, Chubut, Argentina 3Laboratorio de Micología, Fundación M. Lillo, Miguel Lillo 251, T4000JFE San Miguel de Tucumán, Argentina 4Instituto Multidisciplinario de Biología Vegetal-CONICET, Universidad Nacional de Córdoba, CC 495, CP 5000, Córdoba, Argentina 5BioTecA3 – Centro de Biotecnología Aplicada al Agro y Alimentos, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Ingeniería Agrícola Félix Aldo Marrone 746, Planta Baja CC509, CP 5000, Ciudad Universitaria, Córdoba, Argentina 6CONICET, Consejo Nacional de Investigaciones Científicas y Técnicas 7Fundación FungiCosmos, https://fungicosmos.org/, Córdoba, Argentina *Corresponding author: gerardo.robledo@agro.unc.edu.ar REGULAR PAPER


INTRODUCTION
The genus Bjerkandera (Polyporales, Basidiomycota) was described by Karsten (1879) and typified with Bjerkandera adusta (Willd.) P.Karst. The genus has been traditionally established based on morphological characters and mating system characteristics, differing from other polyporoid genera by the combination of the pale cream to smoky or mouse grey hymenophore becoming greyish to blackish when bruised or dried; two-layered context with a white, fibrous upper layer and a brown to black ceraceous layer at the base of the tubes; a monomitic hyphal system, generative hyphae with abundant clamps, thin-to thick-walled, subglobose to ellipsoid basidiospores; heterocytic nuclear behaviour, and bipolar mating system (biological data from B. adusta, Rajchenberg 2011). These features were used by some authors to include B. adusta (and, therefore, the genus as a whole) within Gloeoporus Mont. (Pilát 1937;Corner 1989). However, phylogenetic analyses have shown that these two genera belong to independent lineages with Bjerkandera species grouped within the family Phanerochaetaceae Jülich and Gloeoporus species grouped within the family Irpicaceae Spirin & Zmitr. (Binder et al. 2013;Miettinen et al. 2016;Justo et al. 2017).
According to MycoBank (http://www.mycobank.org/), there are several names associated with Bjerkandera. Nonetheless, morphological and phylogenetic studies have traditionally accepted only two species in the genus, B. adusta and B. fumosa (Pers.) P.Karst. Both species have been originally described from temperate Europe, growing mainly on dead hardwood logs and rarely on conifers (Bondartseva et al. 2014;Ryvarden & Melo 2017). In addition, both species have been widely recorded in different biomes of the world (Gilbertson & Ryvarden 1986;Ryvarden & Gilbertson 1993;Núñez & Ryvarden 2001;Bernicchia 2005;Robledo et al. 2006;Dai et al. 2007;Rajchenberg & Robledo 2013;Zmitrovich et al. 2016). Morphologically, B. adusta and B. fumosa are rather similar, differing by the size of their pores (6-7 per mm in the former and (1-)2-4(-5) per mm in the latter) and the presence of paler tubes separated from the context by a greyish dark line in B. fumosa. Zmitrovich et al. (2016) provided a morphological revision of B. adusta and B. fumosa specimens in Eastern Europe and Northern Asia, highlighting their high intraspecific morphological variability, substrate specialization and recognizing several morphotypes that have not been phylogenetically tested.
The genus Bjerkandera was first reported from the Neotropics by Spegazzini (1919), with B. fumosa recorded from Northwest (NW) Argentina. However, the record was considered uncertain by Robledo & Rajchenberg (2007) because there was no reference herbarium material. From then on, B. fumosa and other species in the genus have been extensively recorded in Brazil, Chile, Costa Rica, Cuba, Jamaica, and Venezuela (Rick 1960;Ryvarden 2000;Ryvarden & Iturriaga 2001;Baltazar & Gibertoni 2009;Ţura et al. 2010;Westphalen & Silveira 2013). Currently, based on morphological examination of type specimens and phylogenetic analyses, Westphalen et al. (2015) proposed the combination of the Brazilian species Tyromyces atroalbus (Rick) Rajchenb. in Bjerkandera and described a new species from Mexico, B.
centroamericana Kout, Westphalen & Tomšovský, expand-ing the knowledge of the genetic and morphological diversity of the genus. Additionally, B. mikrofumosa Ryvarden was recently described from Venezuela (Ryvarden 2016) based on morphological data without evidence of its phylogenetic relationships. It is known so far from the type specimen.
While studying polypores from the Yungas forests of NW Argentina and the Atlantic Forest of Southeast (SE) Brazil, we made several collections of specimens identified as Bjerkandera spp. A comparative morphological study between the collected specimens and examination of molecular evidence revealed that some of our collections represent a new species of Bjerkandera, here described. Also, a species-level phylogenetic hypothesis for the genus based on the internal transcribed spacer (ITS) and the large subunit of ribosomal RNA genes (nLSU), morphological descriptions, comments, illustrations, and an identification key are presented.

Morphological analysis
This study was based on morphological examination of specimens collected from 2012 to 2017 in the Yungas of NW Argentina and the Atlantic Forest of SE Brazil (Morrone 2014). Specimens from the herbaria CORD, ICN, LIL, NY, O, PACA and SP were studied. Herbarium names are abbreviated according to Thiers (continuously updated). For microscopic analysis, free-hand sections of basidiomata prepared in cotton blue with lactic acid, indicator of cyanophilic (CB+) or acyanophilic (CB−) reactions, were mounted on microscope slides and observed using phase-contrast objectives and immersion oil. Melzer's reagent was used to determine the presence of amyloid or dextrinoid reaction or negative reaction (IKI−). Additionally, 3% of KOH was used for microscopy. In KOH and to a lesser extent also in Melzer's reagent, the hyphal walls swell inward, and our measurements of hyphal wall thickness and width, and size of basidiospores are not valid for these reagents (Miettinen et al. 2018). All microscopic structures were measured using an eyepiece micrometre and 30 measurements were taken from each structure. When presenting spore size, 5% of the measurements at each end of the range are given in parentheses. Drawings of microstructures were made using a camera lucida with the exception of spores, which were drawn freehand. Statistics were calculated with R v.3.2.2 (R Core Team 2013). Abbreviations and codes used for the measurements are as follows: L = mean length, W= mean width, Q = L/W (average length divided by average width), Q' = length/width ratio of individual spores, n = number of spores measured for a given number of specimens. Fresh spore prints were obtained and used for preparation of polysporic cultures. Cultures were grown in malt extract agar (MEA) or potato dextrose agar (PDA) at 25°C and described following Nobles (1965).

DNA extraction, PCR amplification and sequencing
Total DNA was extracted from small pieces of dried basidiomata ground with a pestle in a porcelain mortar containing liquid nitrogen. The powder was transferred to centrifuge microtubes and mixed with lysis buffer consisting of 2% CTAB, 1.4 M NaCl, 0.10 M Tris-HCl, 0.1% mercaptoethanol, 20 mM EDTA, and incubated at 65°C for at least 2 h. After one round of chloroform extraction, DNA was precipitated with isopropyl alcohol (Doyle 1987). The following primers were used for both PCR amplification and sequencing: ITS1-ITS4 (including ITS1, 5.8S, and ITS2) for the ITS region and LR0R-LR7 for the nLSU region (White et al. 1990;Gardes & Bruns 1993;Hopple & Vilgalys 1999). PCR reactions for ITS and nLSU genes were performed in a 25 µL volume reaction and conducted on a thermal cycler (C1000 Touch™ Thermal Cycler Bio-Rad) following the cycling parameters described by Oghenekaro et al. (2014).
PCR products were visualized with 1.5% agarose gel electrophoresis. Amplified products were purified and sequenced in both directions on an Applied Biosystem 3730xl DNA analyzer (MacroGen Ltd., South Korea).

Taxon sampling and phylogenetic inference
Sequencing chromatograms were visualized, assembled and edited using the Consed/PhredPhrap package Gordon et al. 1998;Gordon & Green 2013). Once assembled, consensus sequences were queried against the GenBank database using BLAST (http:// blast.ncbi.nlm.nih.gov/) and their pairwise identity was recorded. All newly generated consensus sequences were deposited in GenBank. For this study, eleven ITS and seven nLSU sequences were generated. The additional 30 ITS and 21 nLSU sequences were retrieved from GenBank (https:// www.ncbi.nlm.nih.gov/genbank/). Phlebia radiata Fr. was   (Darriba et al. 2012) based on the corrected Akaike Information Criterion (AICc). The best-fit models for ITS and nLSU were TPM3uf+I+G and TIM3+I+G respectively. For the analysis, the substitution models were set as unlinked.Bayesian inference (BI) was implemented by four Markov chain Monte Carlo (MCMC) independent runs, each starting from random trees and with four simultaneous independent chains, performing 20 million generations, and sampling every 1000 th generations until the average standard deviation of the split frequencies dropped below 0.01. The programme Tracer v.1.6 (Rambaut et al. 2014) was used to check if the Markov chains had reached stationarity by examining the effective sample size values and to determine the correct number of generations to discard as burn-in for the analyses. The first 20% of the sampled trees was discarded whereas the remaining ones were used to reconstruct a 50% majority-rule consensus tree. Clade robustness was expressed as posterior probabilities.

Species/specimen L' (length variation) L W' (width variation) W Q' (L'/W' ratio)
We also analysed the previous dataset using maximum likelihood (ML) within the context of static homology to evaluate whether our results were sensitive to different optimality criteria. Tree searches were performed using the parallel implementation of GARLI (v.2.0;Zwickl 2006Zwickl -2011. For each partition model and selected substitution model, we conducted a total of 1000 independent search replicates and remaining default parameters from the GARLI configuration file. Bootstrap frequencies were calculated from 1000 pseudo-replicate analyses using default search parameters. Bootstrap results were compiled using SUMTREES (v.3.1.0; Sukumaran & Holder 2010). All phylogenetic analyses were run remotely at the CIPRES Science Gateway (Miller et al. 2010).

Phylogenetic analysis
The combined dataset included 41 ITS sequences with 630 characters and 28 nLSU sequences with 941 characters including gaps. The dataset resulted in an aligned length of 1571 characters, of which 1177 characters were constant, 89 were uninformative variable, and 305 were parsimony-informative characters. We show the topology recovered from BI analysis with PP and BS support values given in the nodes ( fig. 1).
The average standard deviation of split frequencies in the four independent BI runs was 0.005982, and the posterior inspection of the runs' log files in Tracer showed that the 20% of trees discarded as burn-in to construct the consensus tree was a suitable value.
The topology recovered in our phylogenetic analyses ( fig.  1) was overall consistent with previous results reported by Westphalen et al. (2015) and Justo et al. (2017).

albocinerea).
A full description of B. albocinerea and a more detailed description of B. mikrofumosa based on a larger number of recent collections are presented below. Morphological comparisons of the species currently accepted in the genus are presented in tables 2 and 3. The morphological characters of B. adusta and B. fumosa, for the construction of the comparative morphological tables mentioned above, followed data collected in this study and data published by Ryvarden & Gilbertson (1993), Zmitrovich et al. (2016), and Ryvarden & Melo (2017). Etymology -"albocinerea" -albo (Latin) white; cinerea (Latin) grey; refers to the colour combination of the basidiomata. Description -Basidiomata annual, sessile, adnate, pileated to effused-reflexed, effused up to 8 cm long × 3 cm wide × 0.5 cm thick at base; pileus 0.5-2.5 cm long × 0.8-2.0 cm wide, waxy and juicy when fresh to leathery when dry, often fused together; pilear surface sordid white when fresh to pale cream when dry, azonate, finely velvety; pileus margin entire, thin to slightly blunt, sterile, velutinous, white, mostly narrow up to 0.1 cm broad, constricting upon drying and tending to roll inwards. Context divided into two layers, the upper layer slightly fibrous, white when fresh to ochraceous yellow when dry, 90-100 µm thick, lower layer clearly contrasting at the base of the tubes, waxy and dark grey, 45-55 µm thick. Pore surface shallow, dark brownish grey, changing to a darker colour, almost black, in bruised parts or when dried, pores round, 8-11 per mm; dissepiments slightly lacerated, up to 14-18.2 µm thick; tube layer not stratified, up to 0.4 cm deep. No change observed in KOH. Odour faintly fungoid or absent.

DISCUSSION
In our phylogenetic analysis, we focus on the evolutionary relationships of the genus Bjerkandera, one of the few polyporoid genera of the Phanerochaetaceae family. The overall topology recovered of the Bjerkandera clade was widely consistent with previous studies ( fig. 1) (Floudas & Hibbett 2015;Westphalen et al. 2015;Miettinen et al. 2016;Justo et al. 2017). The relationship between Bjerkandera and Ceriporiopsis carnegieae as sister taxa was recovered with strong support (PP = 1.0, BS = 100) in accordance with results shown by Justo et al. (2017).
Ceriporiopsis carnegieae was originally described as Poria carnegieae by Baxter (1941) from southern Arizona, as an important agent of decay on the saguaro cactus [Carnegiea gigantea (Engelm.) Britton & Rose)]. Macromorphologically, C. carnegieae is characterized by the resupinate and effused basidiomata, poroid surface, deeply cracked in older specimens, margin finely fimbriate or fibrillose with very delicate mycelial strands, and subiculum less than 1 mm thick. Micromorphologically, it is characterized by the monomitic hyphal system, thin-walled clamped generative hyphae and oblong to short-cylindric basidiospores. In studies of pure cultures, Gilbertson & Canfield (1972) found that the decay caused by this species typically corresponds to white rot fungi but gives no oxidase reaction on gallic and tannic acid media or with gum guaiac solution; additionally, mating tests revealed a heterothallic nuclear behaviour and bipolar type of mating system. The authors discussed that the key code based on Nobles (1965) places the species in the same group of B. adusta; however, they suggested that due to morphological differences between them, these species should be placed in different genera. Our analysis showed that C. carnegieae is phylogenetically closer to Bjerkandera species than to C. gilvescens (Bres.) Domański (the type species of Ceriporiopsis Domański), which is nested in the family Meruliaceae. Although these data support the hypothesis that Ceriporiopsis is not a suitable genus for the species, we prefer to keep C. carnegieae separate from Bjerkandera until a more detailed study can be made including a larger taxon sampling and additional morphological and molecular evidence.
In this study, we recognized Bjerkandera as a monophyletic genus (PP = 1.0, BS = 99) based on the phylogenetic analysis of sequences from the ribosomal ITS and nLSU regions. Bjerkandera atroalba is recovered within clade 1 ( fig.  1) as sister taxon of B. centroamericana (PP = 0.99, BS = 95), in accordance to the topology reported by Westphalen et al. (2015). Both species are very similar morphologically. They are distinguished by the size of the pores (2-5 per mm in the former and 7-11 per mm in the latter) and, microscopically, by the basidiospore shape (slightly wider spores in B. centroamericana, table 2).
Bjerkandera mikrofumosa, the third member of clade 1, was recovered as a monophyletic lineage (PP = 1.0, BS = 100). This is the first time that the species has been included in a phylogenetic analysis. Although the type specimen of B. mikrofumosa was not sequenced, comparative morphological analyses of the type and the sequenced specimens from Argentina and Brazil showed no differences. Additionally, we studied several Neotropical specimens from different herbaria, mainly collected in the Atlantic Forest of SE Brazil and of NW Argentine Yungas, that were identified as B. fumosa. We came to address all these examined specimens as B. mikrofumosa. Bjerkandera fumosa has been reported with a high intraspecific morphological variability, which is probably the cause of erroneous identifications. In our study we use different lines of evidence (morphological, molecular and cultural) and we find that both species form independent, not closely related lineages within Bjerkandera. Through rigorous examination and comparison, both species can be morphologically differentiated by the basidiomata, pores and basidiospores size and shape, which are bigger in B. fumosa (tables 2, 3). Bjerkandera mikrofumosa can be differentiated from B. centroamericana by the pilear and poroid surface colour (table 3). Microscopically, the basidiospores of B. centroamericana are slightly wider, subglobose to broadly ellipsoid (Q' = 1.20), whereas in B. mikrofumosa are mainly ellipsoid (Q' = 1.55, table 2).
All species in clade 1 have a Neotropical distribution. Some specimens of B. atroalba have been recorded in the SE of the United States (Westphalen et al. 2015); however, more molecular data on these specimens is necessary to clarify if B. atroalba has a wider distribution on the American continent or if the North American specimens represent a different lineage.
In clade 2 ( fig. 1), the conventionally accepted evolutionary relationship of B. adusta and B. fumosa as sister taxa has changed due to the inclusion of Bjerkandera albocinerea, the new Neotropical species described here. The new species forms a highly supported lineage as the sister taxon of B. adusta (PP = 1.0, BS = 100). Both species can be easily differentiated by the slightly bigger pores (6-7 per mm, table 2) and basidiospores of B. adusta (table 3). Bjerkandera adusta, originally described from temperate Europe, has been extensively recorded in the Neotrop-ics (Ryvarden 2000;Ryvarden & Iturriaga 2001;Robledo et al. 2003;Baltazar & Gibertoni 2009). Westphalen et al. (2015) included in their phylogenetic analysis sequences of B. adusta from temperate Argentina and from different specimens of an endophytic fungus isolated from Hevea brasiliensis leaves in Peru (Martin et al. 2015;Yuan et al. 2010). The authors reported that these sequences are almost identical to those of European specimens and form a strongly supported clade with B. adusta. So far, it seems that B. adusta is the only species of the genus with a worldwide distribution. However, it is still necessary to obtain additional molecular data from Neotropical specimens identified as B. adusta to properly assess their identity.
Bjerkandera fumosa was recovered at the base of clade 2 with full support (PP = 1.0, BS = 100). Superficially, the species is similar and easily confused with B. adusta, especially when basidiomata are immature. Nevertheless, experts have already documented several useful morphological characters to differentiate both species (see tables 2 and 3). Recent studies have shown that the morphological diversity across the whole distributional area of both species may be high and worth studying in detail. Jung et al. (2014) studied the intraspecific variation between ITS sequences from Korean specimens of B. adusta and B. fumosa. The authors reported that both species show little intraspecific variation (0.0-0.55% in the former and 0.0% in the latter), whereas interspecific variation is high (5.15-5.89%), indicating that DNA data are useful to distinguish B. adusta from B. fumosa. This information is also useful for accurate identification, especially when morphological characters are not suitable for analysis (e.g., cultures or fragments from dried specimens).
As documented above, B. fumosa and some of its synonyms have been widely recorded from Neotropical biomes. Recently, Zmitrovich et al. (2016) listed two forms of B. fumosa from Cuba recorded by Murrill (1907). The authors suggested that B. terebrans (Berk. & M.A.Curtis) Murrill is probably a form of B. fumosa with a stipe-like base whereas B. subsimulans Murrill may actually be an Abortiporus Murrill species. Unfortunately, sequences of B. fumosa available at GenBank are mostly from temperate regions; and although the data shown in our study do not allow to define the specific limits for B. fumosa, there is a strong evidence, at least in the Neotropics, pointing out that this species may encompass a species complex, as clearly exemplified by B. mikrofumosa. Future studies should invest efforts to re-evaluate the diagnostic characters of B. fumosa, to examine and recollect Neotropical specimens in type localities, to compare type specimens, to obtain new molecular data and to test the evidence under the light of phylogenetic inference.
This study provided the most extensive documentation of the species diversity within Bjerkandera to date. Also, it contributes to the expansion of the species-level phylogeny of the genus in the Neotropics and worldwide with morphological and molecular evidence, providing a phylogenetic backbone for the interspecific relationships within the genus. In addition, our results indicate that the number of taxa in Bjerkandera has been underestimated by morphological evidence, and may actually be greater than traditionally accepted.