Bjerkandera carnegieae comb. nov. (Phanerochaetaceae, Polyporales), a wood-decay polypore of cactus

Poria carnegieae was described from Arizona growing on the woody ribs of the saguaro cactus, Carnegiea gigantea, and was transferred to Ceriporiopsis due to morphological evidence. Posterior phylogenetic studies showed a relationship of Poria carnegieae with Bjerkandera. New sequence data and morphologic evidence are presented to support the transfer of Ceriporiopsis carnegieae to Bjerkandera.


Introduction
Poria carnegieae was described from Arizona growing on the woody ribs of the saguaro cactus, Carnegiea gigantea (Baxter 1941). Cultural characters, decay studies, and sexuality of the species were described and studied by Gilbertson and Canfield (1972) and Lindsey and Gilbertson (1977). Canfield (1972: 1309) noted that the bipolar mating system and negative phenol oxidase reaction placed P. carnegieae with Bjerkandera adusta (syn. Polyporus adustus) based on Nobles' 1965 key pattern of wood-decay fungal cultures. Because of morphological features such as an effused basidiome, light-colored pores, monomitic hyphal system with thinwalled, clamped generative hyphae, lack of cystidia, and thin-walled basidiospores, P. carnegieae was transferred to Ceriporiopsis by Gilbertson and Ryvarden (1985). In a multigene phylogenetic study of the order Polyporales by Justo et al. (2017), C. carnegieae was recovered in a clade with two species of Bjerkandera in the Phanerochaetaceae. Subsequent phylogenetic studies confirmed and supported this relationship (Chen et al. 2018;Motato-Vásquez et al. 2020;Wang et al. 2021). Due to differences in morphological features of the basidiome, such as its resupinate and effused habit and uniform, light-colored context lacking a dark brown zone or black line between the tube layer and subiculum, researchers refrained from transferring C. carnegieae to Bjerkandera pending more data (Motato-Vásquez et al. 2020;Wang et al. 2021).
The purpose of this study is to provide additional phylogenetic and morphological evidence to support the transfer of Poria carnegieae to Bjerkandera. We also review additional biological information relating to this taxon.

Morphological study
Specimens from the Center for Forest Mycology Research (CFMR) fungarium were studied. For microscopic analysis, free-hand sections of basidiomes were mounted in 2% (w/v) aqueous potassium hydroxide (KOH) and 1% (w/v) aqueous phloxine or Melzer's reagent. Cyanophily of hyphal and basidiospore walls was observed in 1% (weight/volume) cotton blue in 60% (w/v) lactic acid. Basidiospores were measured in KOH and phloxine mounts under oil immersion with at 100× magnification. Q values were calculated from average spore length divided by average spore width of at least 30 spores. Color codes and names follow Kornerup & Wanscher (1978). Micrographs of basidiomes were taken with an Olympus DP27 camera attached on an Olympus BX43 compound microscope.
DNA extraction, PCR amplification and sequencing DNA extraction and amplification were performed from cultures at CFMR following a standard CTAB protocol (Mercado & Ortiz-Santana 2018). Sequencing was

Phylogenetics analyses
New DNA sequences generated in the present work were combined with sequences retrieved from GenBank (NCBI) to construct two datasets. Scientific names and GenBank Accession Numbers of sequences are listed in  (Do et al. 2005), whereas LSU, rpb1, rpb2 and tef1-α were individually aligned using MAFFT 7 (Katoh et al. 2017) using the G-INS-i alignment method. Alignments were manually inspected and adjusted using MEGA 6 (Tamura et al. 2013). ModelFinder (Kalyaanamoorthy et al. 2017) as implemented in the IQ-Tree software (Nguyen et al. 2015) was used to estimate the best-fit partitioning strategy and the best-fit model of nucleotide evolution for the dataset using 16 data blocks (ITS1; 5.8S; ITS2; LSU; rpb1 codon positions, 1stpos, 2ndpos, and 3rdpos; rpb1 introns; rpb2 codon positions, 1stpos, 2ndpos, and 3rdpos; rpb2 introns; tef1-α codon positions 1stpos, 2ndpos, and 3rdpos and tef1-α introns). Models were restricted for those implemented in MrBayes 3.2 (Ronquist et al. 2012). Bayesian inference (BI) and maximum likelihood (ML) phylogenetic analyses were applied to the concatenated datasets using the partition scheme and evolutionary models defined by ModelFinder. BI was performed following Robledo et al. (2020)   likelihood searches were conducted with IQ-TREE. The analyses initially involved 100 ML searches, each one starting from one randomized stepwise addition parsimony tree. Branch supports were calculated using the UFBoot (ultrafast bootstrap approximation) (Hoang et al. 2018) implemented in IQ-TREE with 1000 replications. A node was considered strongly supported with BPP ≥ 0.95 or BS ≥95% (Hyde et al. 2013;Minh et al. 2020).
Remarks. Descriptions and illustrations of the basidiome are readily available (see above), and our observations generally agree except as follows: (1) The subicular trama is composed primarily of slightly thick-to thick-walled subicular hyphae 3-5.5 µm diam with walls thin to 1.5 µm thick.
Basidiomes of B. carnegieae are entirely effused and adnate with nearly white to ivory-white pores when fresh that darken slightly to light brown or buff, and a uniform, cream-colored context. The pore layer is very fragile and brittle when dried. These characters differ from most species of Bjerkandera which are pileate, effuse-reflexed, except the resupinate species B. resupinata. In addition, most species in the genus have dark gray to buff-colored pores that typically darken to black when bruised in contrast to the light-colored pores in B. carnegieae that darken to light brown. Furthermore, the context in B. carnegieae is uniformly light-colored, whereas other species of Bjerkandera have a tan, brown or black zone or line between the base of the tubes or pores and context. Motato-Vásquez et al. (2020) and Wang et al. (2021) have summarized some critical morphological characters of accepted species in Bjerkandera and included keys.
Despite these macromorphological differences with other species in the genus, B. carnegieae shares important characters such as a monomitic, clamped hyphal system of thin-to thick-walled generative hyphae with thick-walled hyphae dominating in the subiculum and trama (Fig. 3C-D), and basidia and basidiospores that are similar in shape and size. Furthermore, cultures of B. carnegieae, B. adusta, and B. fumosa share some important biological features, such as developing arthroconidia and a negative or weakly positive reaction on gallic acid agar with some mycelial growth and a negative or positive reaction on tannic acid agar, but no growth (Nobles 1948: 350;Gilbertson & Canfield 1972;Lombard et al. 1992). Finally, these three species have a heterocytic nuclear behavior and a bipolar mating system (Gilbertson & Canfield 1972;David 1988;Lombard et al. 1992). It is noteworthy that cultures of B. mikrofumosa and B. atroalba develop chlamydospores and not arthroconidia (Motato-Vásquez et. al. 2016.

Discussion
Our multi-gene phylogenetic tree of the phlebioid clade shown in Figure 1 is consistent with previous studies (Justo et al. 2017;Chen et al. 2018). Similarly, the ITS-LSU analysis of the genus Bjerkandera recovered a tree shown in Figure 2 that is congruent with that in Motato-Vásquez et al. (2020) and Wang et al. (2021). Phylogenetic studies showed that the current concept of Ceriporiopsis is polyphyletic with species recovered in several different phylogenetic clades (Tomšovský et al. 2010;Zhao & Cui 2014;Gómez-Montoya et al. 2017). The type of Ceriporiopsis, C. gilvescens, clusters in a lineage with Phlebia and Mycoacia (Binder et al. 2013;Zhao & Cui 2014;Zhao & Wu 2016;Justo et al. 2017).
The transfer of P. carnegieae to Bjerkandera requires a slight modification to the genus description to include species with effused basidiomes and uniform context without a dark line or dark zone separating the tube layer from the context. We believe that this is a better solution than the creation of a new genus for B. carnegieae that lacks strong phylogenetic, morphological, or biological characters. More studies of Bjerkandera sp. JV1512/13J (as Ceriporiopsis sp. in Wang et al. 2021) and Bjerkandera sp. L13104sp, both from Costa Rica, are required to see if sequence data are also supported by morphological and biological characters to describe it as a new taxon. Bjerkandera carnegieae was originally described by Baxter (1941) from southern Arizona as an important agent of decay in the saguaro cactus, Carnegiea gigantea. Most specimens of this species are from saguaro, but a few specimens are also known on other woody Cactaceae, such as Pachycereus sp and Lemaireocereus sp, from desert areas of Mexico (Lindsey & Gilbertson 1977; and data retrieved from MycoPortal, October 15, 2021). ITS BLAST searches in GenBank have recovered some environmental samples with 100% sequence identity with B. carnegieae, mostly from Arizona, but also from Puerto Rico and Brazil (Fröhlich-Nowoisky et al. 2012). Although the fungal diversity growing in saguaro has been recorded (Gilbertson et al. 1974;Lindsey & Gilbertson 1975), tree-like cacti are 'under sampled' in other parts of America. The biographical connection of desert areas from USA and Central Argentina has been previously reported, not only in similar physiognomic structure, spiny bush and trees and tree-like cacti, but in plant taxa, i.e. Prosopis spp., and polypores are not the exception. See for instance Inocutis texana, originally described from North America that has been registered in xerophitic areas of central Argentina (Robledo & Urcelay 2009;Rajchenberg & Robledo 2013). The only polypore so far registered in a tree-like cactus in Central Argentina has been Ceriporia xylostromatoides, growing inside a dead falling Stetsonia coryne (Robledo & Urcelay 2009). like to acknowledge the Center for Forest Mycology Research (CFMR) for making available culture and collections for this study. The assistance of Consejo Nacional de Investigaciones Científic as y Técnicas (CONICET) and Universidad Nacional de Córdoba, both of which supported the facilities used in this work, is also acknowledged.