Mycological Progress https://doi.org/10.1007/s11557-018-1416-3
ORIGINAL ARTICLE
Evolutionary relationships of Bresadolia (Basidiomycota, Polyporales) based on molecular and morphological evidence Viviana Motato-Vásquez 1 & Emmanuel Grassi 2 & Adriana M. Gugliotta 1 & Gerardo L. Robledo 3,4 Received: 6 February 2018 / Revised: 1 June 2018 / Accepted: 6 June 2018 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract In the last decades, several phylogenetic studies have shown that Polyporus is polyphyletic; accordingly, several genera have been newly described or reinstated. Nevertheless, the phylogenetic position of many species in the genus remains highly contentious, particularly those traditionally included in the Polyporus infrageneric group Polyporus s.s., i.e., P. austroafricanus, P. craterellus, P. radicatus, P. squamosus, P. tuberaster, and P. udus. Recently, based on morphological characteristics, P. udus, described from Indonesia, was synonymized with Bresadolia paradoxa, described from Paraguay, resurrecting Bresadolia as a good genus. In this study, the phylogenetic relationship of P. udus and its purported taxonomic synonym in South America was investigated. In addition, the phylogenetic positioning of Bresadolia within Polyporus s.l. and related genera was assessed, based on ITS and nLSU rDNA loci. Morphological revision of collections from the Atlantic Forest of Argentina, Brazil, and Paraguay; revision of type specimens; and the phylogenetic results showed that B. paradoxa and P. udus are not conspecific. Both species form independent lineages that cluster together within a monophyletic genus recognized here as Bresadolia. In this study, a complete description of B. paradoxa incorporating data of type specimens previously overlooked and characters from sequenced fresh specimens is provided, as well as comments on all species described or combined in Bresadolia. Keywords Atlantic Forest . Core polyporoid clade . Polyporus . Phylogeny . Taxonomy
Introduction Polyporus P. micheli ex Adans., as traditionally defined, accommodates ecologically and macro-morphologically variable species mainly characterized by stipitate basidiomata, dimitic hyphal system with skeleton-binding hyphae, and cylindrical, smooth basidiospores (Ryvarden and Johansen Section Editor: Yu-Cheng Dai * Gerardo L. Robledo
[email protected] 1
Núcleo de Pesquisa em Micologia, Instituto de Botânica, Av. Miguel Stefano 3687, São Paulo 04301-902, Brazil
2
Instituto Misionero de Biodiversidad (IMiBio), Puerto Iguazú, Misiones, Argentina
3
Instituto Multidisciplinario de Biología Vegetal - CONICET, Laboratorio de Micología, Universidad Nacional de Córdoba, CC 495, CP 5000 Córdoba, Argentina
4
Fundación FungiCosmos, Av. General Paz 154, 4° Floor, Office 4, Córdoba, Argentina
1980; Gilbertson and Ryvarden 1987). Núñez and Ryvarden (1995) accepted this broad concept of Polyporus and, in order to facilitate the study of the genus, they grouped 32 species into six infrageneric groups, based on morphological charact e r i s t i c s , v iz . A d m i r a b i l is , D e n d ro p o l y p o r u s [ = Dendropolyporus (Pouzar) Jülich], Favolus (= Favolus Fr.), Polyporellus (= Polyporellus P. Karst.), Melanopus (= Melanopus Pat.), and Polyporus s.s. In the last decades, several phylogenetic studies, based on molecular and morphological evidence, have shown that the above mentioned infrageneric groups are not monophyletic lineages. In addition, Polyporus has been recognized as polyphyletic, and accordingly, several genera have been newly described or reinstated (Ko and Jung 2002; Krueger 2002; Krueger and Gargas 2004; Sotome et al. 2008, 2013; Grand et al. 2011; Binder et al. 2013; Seelan et al. 2015; Zmitrovich and Kovalenko 2016; Zhou et al. 2016; Palacio et al. 2017). Despite the addition of several terminals and new molecular evidence, the phylogenetic position of many species remains highly contentious and, in particular, there are still open questions on how to properly delimit Polyporus as a
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monophyletic genus and to designate a type for the genus (Justo et al. 2017). Sotome et al. (2008) recognized the problems with the typification of Polyporus, but for practical purposes, they considered P. tuberaster as type species; since then, several modern authors have followed this recommendation (e.g., Dai et al. 2014; Zmitrovich and Kovalenko 2016; Palacio et al. 2017). Recently, Justo et al. (2017) highlighted that under Article 9 of the International Code of Nomenclature for Algae, Fungi, and Plant (McNeill et al. 2012), assuming that P. tuberaster is the type of Polyporus is not correct; they suggested that the conservation of Polyporus with a conserved type, preferably different from P. brumalis (Pers.) Fr., must be formally proposed. Nevertheless, they did not define what it should be. In this study, we are aware of the problems with the typification of Polyporus; therefore, for the moment, we refer to Polyporus s.s. clade only in the sense of the Polyporus infrageneric group Polyporus s.s. sensu Núñez and Ryvarden (1995), typified by P. tuberaster. Dai et al. (2014) suggested that a good starting point to define Polyporus as a monophyletic genus should be to assess the phylogenetic position of species traditionally included in the Polyporus infrageneric group Polyporus s.s., which comprises P. austroafricanus Núñez & Ryvarden, P. craterellus Berk. & M.A. Curtis, P. radicatus Schw., P. squamosus ( H u d s . ) F r. , P. t u b e r a s t e r, a n d P. u d u s J u n g h . Morphologically, this group was characterized by fleshy basidiomata, up to 5 cm thick, which were brittle upon drying, inflated up to 20 μm skeleton-binding hyphae, with large pores (< 2 per mm) and basidiospores (> 10 μm log) (sensu Núñez and Ryvarden 1995). Phylogenetically, P. squamosus, along with other species, was recovered as a monophyletic group, currently recognized as Cerioporus Quél. (Dai et al. 2014; Zhou et al. 2016; Zmitrovich and Kovalenko 2016). Polyporus craterellus and P. udus were recovered as distinctive species not closely related with P. tuberaster or P. squamosus; however, due to low taxon sampling, their phylogenetic position is considered highly uncertain (Krueger 2002; Sotome et al. 2008). Polyporus austroafricanus and P. radicatus have not been included, so far, in phylogenetic analysis to date. Therefore, P. tuberaster is generally considered as the only member of the so-called Polyporus s.s. clade (see Sotome et al. 2008; Dai et al. 2014; Zmitrovich and Kovalenko 2016; Palacio et al. 2017). On the other hand, Krueger (2002), Runnel and Ryvarden (2016), and Zhou et al. (2016) have shown, independently, that some other species, viz., Dichomitus campestris (Quél.) Domański & Orlicz and D. gunnii (Berk.) Ryvarden (= Hexagonia gunnii Berk.), P. hapalopus H.J. Xue & L.W. Zhou, P. minutosquamosus Runnel & Ryvarden, and P. umbellatus (Pers.) Fr., are closely related to P. tuberaster. However, an exhaustive morphological study that compares all of these species together and a phylogenetic
hypothesis that includes all of the molecular evidence and tests their phylogenetic relationships have not been done. Polyporus udus, originally described from Indonesia, has been widely recorded in the Neotropics (Rick 1960; Corner 1984; Ryvarden and Meijer 2002; Silveira and Wright 2005; Louza and Gugliotta 2007; Robledo and Rajchenberg 2007). Recently, based on morphological characteristics, it was synonymized with Bresadolia paradoxa Speg., described from Paraguay (Spegazzini 1883; Rajchenberg and Wright 1987), and the genus Bresadolia Speg. with P. udus as type species was resurrected (Audet 2017). Additionally, other species with similar morphology were combined in this genus, viz., P. craterellus, described from Cuba (Núñez and Ryvarden 1995), and P. cuticulatus Dai, Si & Shigel (Si and Dai 2016), and P. hapalopus (Xue and Zhou 2014), described from China. In this study, we investigate the phylogenetic relationships of P. udus and its purported taxonomic synonym in South America, based on morphological (e.g., new collections, examination of type specimens) and molecular evidence. In addition, we assessed the phylogenetic position of Bresadolia within Polyporus s.l. and related genera, based on ITS and nLSU rDNA loci, and discussed the phylogenetic relationship of Bresadolia with Polyporus s.s. (i.e., P. tuberaster) and with several species putatively related to P. tuberaster. Finally, we provided discussions on all species described or combined in Bresadolia.
Material and methods Morphological study This study was based on specimens collected from 2012 to 2017 in the Atlantic Forest (Morrone 2014) of Argentina, Brazil, and Paraguay and on the examination of specimens from the herbaria BAFC, DAOM, FCOS, LPS, HLB, K, S, TENN, and SP [herbarium abbreviations follow Thiers (n.d.) (continuously updated)]. The new specimens collected are now kept at SP and FCOS herbaria. Basidiomata colors are described according to Küppers (2002). For microscopic analyses, free hand sections of the basidiomata were mounted on microscope slides with a drop of 3% KOH and 1% aqueous phloxine solution, using phase contrast objectives and immersion oil. Cotton blue was used to check cyanophilic (CB+) or acyanophilic (CB−) reactions. Melzer’s reagent was used to verify amyloidicity or dextrinoidicity or a negative reaction (IKI−). All microscopic structures were measured with the aid of an eyepiece micrometer, and 30 measurements were taken from each structure. Drawings of the microstructures were created with the aid of a camera lucida. Abbreviations and codes used for the measurements are as follows: L = mean length × W = width mean, Q = L/W (average length divided by
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average width), Q′ = length/width ratio of individual spores, and n = number of spores measured from given number of specimens. The number of nuclei per basidiospores was observed with DAPI (Kapuscinski 1995).
DNA extraction, 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 Eppendorf tubes and mixed with lysis buffer consisting of 2% CTAB, 1.4 M NaCl, 0.10 M Tris-HCl, 0.1% mercaptoethanol, and 20 mM EDTA, at 65 °C for at least 2 h. Chloroform extraction was done once and DNA was precipitated with isopropanol (Doyle 1987). Amplification of the ITS and nLSU regions was performed using the ITS1/ ITS4 (including ITS1, 5.8S, and ITS2) primer combination for ITS and LR0R/LR7 for nLSU (White et al. 1990; Gardes and Bruns 1993; Hopple and Vilgalys 1999). PCR reactions for ITS and nLSU were carried out in a 25 μL volume reaction and conducted in a thermal cycler (C1000 Touch™ Thermal Cycler Bio-Rad) using the following parameters: an initial denaturation step at 94 °C for 2 min, followed by 35 cycles of 50 s denaturation at 94 °C, 1 min annealing at 55 °C, and 1 min extension at 72 °C; the reaction ended with a final extension of 5 min at 72 °C and cooling to 4 °C (Oghenekaro et al. 2014). PCR products were visualized with 1.5% agarose gel electrophoresis. Amplified products were purified and then sequenced in both directions. In all cases, the same primers used in the amplification were used for sequencing. The cycle sequencing was performed in an Applied Biosystems 3730xl DNA Analyzer (MacroGen Ltd., South Korea).
Taxon sampling, rooting, and phylogenetic inference Sequence chromatograms were assembled, visualized, and edited using the package Consed/PhredPhrap (Ewing and Green 1998; Ewing et al. 1998; Gordon et al. 1998; Gordon and Green 2013). Once assembled, consensus sequences were then queried against the entire 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. The concatenated dataset comprised 68 terminals representing the main recognized genera in Polyporus s.l. and allied genera. Of these, 57 terminals were chosen based on results of the phylogenetic analyses found by Krueger (2002), Krueger and Gargas (2004), Sotome et al. (2008, 2009, 2011, 2013), Binder et al. (2013), Dai et al. (2014), Li et al. (2014), Seelan et al. (2015), Runnel and Ryvarden (2016), Si and Dai (2016), Zhou et al. (2016), and Palacio et al. (2017) and through BLAST searches in the National Center for Biotechnology
Information (NCBI); the remaining nine correspond to the new sequences produced in this study. The final ITS and nLSU datasets were subsequently aligned using MAFFT v.7 under E-INS-i and Q-INS-i strategies, respectively (Katoh and Standley 2013), which were visualized and edited them in BioEdit (Hall 1999). The clade Trametes versicolor (L.) Lloyd [T. hirsuta (Wulfen) Pilát, T. villosa (Sw.) Kreisel] was defined as the root. All of the sequences used in this analysis are listed in Table 1. Automatic multiple alignment programs use the Needleman-Wunsch dynamic algorithm (Needleman and Wunsch 1970), which is guaranteed to optimally align two sequences. It is common practice to build fixed sequence alignments by incorporating gaps, which are subsequently subject to phylogenetic analyses. Gaps contribute a considerable portion of the potential phylogenetic information and are less prone to homoplasy (Simmons et al. 2001). However, considering gaps as a fifth character state is problematic if gaps are longer than a position, because these positions are treated as independent; to avoid overweighting contiguous gap positions, gaps were recoded as presence/absence characters using Simmons and Ochoterena’s simple code method (Simmons and Ochoterena 2000), as implemented in the program SeqState (Müller 2005). Two distinct analyses were performed: Bayesian inference (BI) and maximum likelihood (ML). Bayesian inference was conducted in MrBayes v.3.2.6 (Ronquist et al. 2012). The unlinked nucleotide substitution model was specified for each gene fragment based on corrected Akaike information criterion (AICc) generated in jModeltest v.2.1.4 (Darriba et al. 2012). BI was implemented by four MCMC independent runs, each starting from random trees and with four simultaneous independent chains, performing 10 million generations and sampling every 1000th generations until the average standard deviation of the split frequencies dropped below 0.05. The program Tracer v.1.6 (Rambaut et al. 2014) was used to check whether the Markov chains had reached stationarity by examining the effective sample size values and also to determine the correct number of generations to discard as burn-in for the analyses. The first 20% of the sampled trees were discarded while the remainder were used to reconstruct a 50% majority-rule consensus tree. Support values are expressed as posterior probabilities (BPPs). We also analyzed the previous dataset using ML within the context of static homology. Tree searches were performed using the parallel implementation of GARLI (version 2.0; Zwickl 2006–2011). 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 pseudoreplicate analyses using default search parameters. Bootstrap (BS) results were compiled using SUMTREES (v.3.1.0; Sukumaran and Holder 2010). All phylogenetic analyses were
Mycol Progress Table 1
Taxon sampling, specimen-voucher information, geographic origin, and GenBank accession numbers of sequences used in this study
Genus/group
Origin (ISO code)
Species, vouchers/culture
Accession no.
Reference
ITS
LSU
Atroporus Atroporus diabolicus, DS1266 Atroporus rufoatratus, DS1311
BR BR
KY631768 KY631769
KY631757 KY631758
Palacio et al. (2017) Palacio et al. (2017)
Bresadolia Bresadolia craterella, TENN59383
EC
–
AJ487944
Krueger (2002)
Bresadolia cuticulata, Dai 13101
CN
KP297863
KP297864
Si and Dai (2016)
Bresadolia paradoxa, MV23 Bresadolia paradoxa, MV161
BR BR
KY777230 KY777231
KY777235 KY777236
This study This study
Bresadolia paradoxa, MC4402 Bresadolia paradoxa, Robledo1958
BR AR
KY777232 KY777233
– KY777237
This study This study
Bresadolia paradoxa, Robledo1959
AR
KY777234
KY777238
This study
Bresadolia uda, FLAS-F-60005 Bresadolia uda, H6518
US IN
KY654718 AF518756
–
Unpublished Krueger (2002)
Bresadolia uda, WD1878 Cerioporus
JP
–
AB368108
Sotome et al. (2008)
Cerioporus squamosus, MUCL30721
BE
AB587630
AB368094
Sotome et al. (2008)
Cerioporus squamosus, Dai8082 Datronia Datronia mollis, RLG6304 sp. Datronia stereoides, Niemelä3020
CN
KC572030
KC572068
Dai et al. (2014)
US CA
JN165002 KC415178
JN164791 KC415195
Justo and Hibbett (2011) Li et al. (2014)
Datroniella Datroniella melanocarpa, Cui10646 Datroniella scutellata, RLG9584T Datroniella subtropica, Dai12881 Datroniella tibetica, Cui9486
CN US CN CN
KC415186 JN165004 KC415183 JX559265
KC415194 JN164792 KC415193 JX559299
Li et al. (2014) Justo and Hibbett (2011) Li et al. (2014) Li et al. (2014)
CN
KC415181
KC415189
Li et al. (2014)
JP JP EC
AB462321 AB462318 AF518754
AB462309 AB462306 –
Sotome et al. (2009) Sotome et al. (2009)
JP BR JP CA
AB735974 AB735977 AB587628 AB587629
AB735952 AB735953 AB587619 AB587620
Sotome et al. (2013) Sotome et al. (2013) Sotome et al. (2011) Sotome et al. (2011)
CN TH US CR DO TH BO CN US
KC572005 KP283480 KP283490 KP283495 GU207303 KP283487 KP283493 KP283482 KP283488
KC572044 KP283518 KP283519 KP283523 AY615984 KP283514 KP283510 KP283516 –
Dai et al. (2014) Seelan et al. (2015) Seelan et al. (2015) Seelan et al. (2015) Grand et al. (2011) Seelan et al. (2015) Seelan et al. (2015) Seelan et al. (2015) Seelan et al. (2015)
CN CN
JX559269 JX559272
JX559286 JX559283
Li et al. (2014) Li et al. (2014)
Datroniella tropica, Dai13147 Echinochaete Echinochaete brachypora, TFMF24996 Echinochaete russiceps, TFMF24255 Echinochaete sp., MN272 Favolus Favolus acervatus, TFMF_27345 Favolus brasiliensis, INPA:241452 Favolus emerici, WD2379 Favolus pseudobetulinus, TRTC51022 Lentinus Lentinus arcularius, Dai8159 Lentinus badius, DED07668 Lentinus brumalis, PB4 (EP4) Lentinus crinitus, DSH9243C Lentinus bertieri, TENN59773 Lentinus polychrous, KM141387 Lentinus sajor-caju, SNP24989 Lentinus squarrosulus, CUI6513 Lentinus tigrinus, DSH92D787 Neodatronia Neodatronia gaoligongensis, Cui 8055 Neodatronia sinensis, Dai 11921
Krueger (2002)
Mycol Progress Table 1 (continued) Genus/group
Origin (ISO code)
Accession no.
Reference
Neodictyopus Neodictyopus atlanticae, DS1285
BR
KY631773
KY631762
Palacio et al. (2017)
Neodictyopus dictyopus, TENN11501
BE
AF516561
AJ487945
Krueger (2002)
Neodictyopus dictyopus, GAS60 Neodictyopus gugliottae, GAS622
BR BR
KY631776 KY631772
KY631765 KY631761
Palacio et al. (2017) Palacio et al. (2017)
Neofavolus alveolaris, TUMH:50003 Neofavolus cremeoalbidus, TUMH50009
JP JP
AB735968 AB735980
AB735949 AB735957
Sotome et al. (2013) Sotome et al. (2013)
Neofavolus mikawai, TFMF27417 Neofavolus suavissimus, ADD7
JP US
AB735963 KP283501
AB735943 KP283527
Sotome et al. (2013) Seelan et al. (2015)
Mycobonia flava, TEN59088 Mycobonia flava, TEN57579
AR CR
AY513571 AY513570
AJ487933 AJ487934
Krueger (2002) Krueger (2002)
Picipes Picipes austroandinus, CIEFAP577 Picipes melanopus, H6003449
AR FI
AF516568 JQ964422
– KC572064
Krueger (2002) Xue and Zhou (2014)
Picipes tubiformis, Niemela 6855 Picipes badius, WD2341
CN JP
KC572036 AB587625
KC572073 AB368083
Dai et al. (2014) Sotome et al. (2008)
Picipes subtropicus, Cui2662
CN
KU189759
KU189791
Zhou et al. (2016)
Neofavolus
Mycobonia
Polyporus s.s. Polyporus tuberaster, WD2382
JP
AB474086
AB368104
Sotome et al. (2009)
Polyporus tuberaster, DAOM7997B
US
AY218420
AF261544
Moncalvo et al. (2002)
Polyporus s.l. Dichomitus campestris, O103769 Dichomitus gunnii, Ryvarden18829 Dichomitus sp., IFP14643 Polyporus hapalopus, Yuan 5809
NW AU CN CN
– – KX832053 KC297219
AJ487512 AJ487513 KX832062 KC297220
Krueger (2002) Krueger (2002)
Polyporus minutosquamosus, Runnel 690 Polyporus umbellatus, Pen13513 Polyporus umbellatus, TENN5079 Pseudofavolus
GF CN US
KU189803 KU189772 –
KU189803 KU189803 AJ418818
Runnel and Ryvarden (2016) Zhou et al. (2016) Krueger (2002)
Pseudofavolus cucullatus, WD2157 Pseudofavolus cucullatus, TENN11221 Trametes Trametes hirsuta, RLG5133T Trametes versicolor, FP135156sp Trametes villosa, FP71974R
JP AR
AB587637 AF516600
AB368114 AJ488124
Sotome et al. (2008) Krueger (2002)
US US US
JN164941 JN164919 JN164969
JN164801 JN164809 JN164810
Justo and Hibbett (2011) Justo and Hibbett (2011) Justo and Hibbett (2011)
run remotely at the CIPRES Science Gateway (Miller et al. 2010). A node was considered to be strongly supported if it showed a BPP ≥ 0.95 and/or BS ≥ 90%, while moderate support was considered when BPP < 0.95 and/or BS < 90%. In addition, a pairwise comparison of ITS variation was conducted for Bresadolia species and close related taxa, with available sequences (i.e., P. tuberaster, P. hapalopus, P. minutosquamosus, P. umbellatus, and Pi. badius), using the total extension of the aligned ITS region. Distance matrix was
Xue and Zhou (2014)
generated using an uncorrected p distance in MEGA 7.0 (Kumar et al. 2016).
Results and discussion Phylogenetic inference The combined dataset included 62 sequences of ITS with a total of 704 characters, 62 sequences of nLSU with 878
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characters, and 137 coded gaps, representing 68 terminals. The dataset had an aligned length of 1719 characters, of which 1039 characters were constant, 174 were uninformative variable, and 612 were parsimony-informative characters. The bests model for ITS and nLSU were TVM + I + G and GTR + I + G, respectively. Insertion and deletion events (i.e., indels) are transformational events that molecular sequences may exhibit during evolution. Therefore, as for any heritable transformation (e.g., transitions and transversions), indels can be used as evidence to test phylogenetic hypotheses (Giribet and Wheeler 1999; Simmons and Ochoterena 2000). Indel events are represented by gaps in multiple sequence alignments, and their quantity and distribution are greatly influenced by the alignment algorithm and by the cost of the matrices used. These properties can have strong topological impacts on the optimal tree (Ford and Wheeler 2016); for this reason, a standard discrete model was implemented for the gaps scored as absence/presence data. The topology of the BI and ML showed no inconsistency in any supported clades. Topology from BI analysis was presented along with ML-BS support values (Fig. 1). Phylogenetic studies have shown that Polyporus is polyphyletic, and, currently, several genera have been segregated and accepted, i.e., Atroporus Ryvarden, Cerioporus Quél., Lentinus Fr., Picipes Zmitr. & Kovalenko, Favolus Fr., Neofavolus Sotome & T. Hatt., Neodictyopus Palacio, Robledo, Reck & Dreschler-Santos, and Pseudofavolus Pat. (Krueger and Gargas 2004; Sotome et al. 2008, 2013; Binder et al. 2013; Dai et al. 2014; Seelan et al. 2015; Zmitrovich and Kovalenko 2016; Zhou et al. 2016; Palacio et al. 2017). The topology recovered in our phylogenetic analysis (Fig. 1) was overall consistent with the previous results and showed that all sampled species within the traditionally Polyporus infrageneric group Polyporus s.s. (i.e., P. craterellus, P. squamosus, P. tuberaster, and P. udus) are distributed into three distinct clades: 1. Polyporus craterellus and P. udus were recovered in the clade recognized as Bresadolia, which forms an independent lineage within Polyporus s.l. with high support (BPP = 1, BS = 99%), also encompassing B. cuticulata and B. paradoxa. Bresadolia is a sister taxon of the clade formed by P. tuberaster (Polyporus s.s.) and (P. umbellatus (P. minutosquamosus (P. hapalopus (D. gunnii (D. campestris, Dichomitus sp.))))) with high to moderate support (BPP = 0.95, BS = 78%). The morphological revision of new collections from the Atlantic Forest and type specimens, in addition to the phylogenetic evidence, showed that B. paradoxa and B. uda are not conspecific, as previously suggested (Rajchenberg and Wright 1987; Audet 2017), and therefore, B. paradoxa is recognized as the type species of the genus (see the BTaxonomy^ section). The ITS region, the most commonly used genetic
marker for fungal species discriminations and molecular identification (Nilsson et al. 2009; Ghobad-Nejhad et al. 2010), was used to compare the genetic distance (uncorrected p) within and among species of Bresadolia and closely related taxa (Table 2). Intraspecific distance in B. paradoxa species ranged between 0.3 and 0.8%, which was lower than the mean value for intraspecific variation in basidiomycetes (Nilsson et al. 2008). These values contrast with the high intraspecific variation between specimens of B. uda (i.e., B uda H6518 from India and B. uda FLASF6005 from USA) (uncorrected p ≈ 16.8%). This genetic distance is reflected in our topology, since specimens of B. uda did not form a monophyletic group; this suggests that, with the current data, B. uda is a species complex. The interspecific distance between Bresadolia species and P. tuberaster ranged between 17.9 and 20.1%, which was greater than the distance between P. tuberaster and the well-established genera Picipes (ranged between 13.9 and 17.7%). This interspecific distance observed between Bresadolia and taxa belonging to related genera is similar to distances reported in studies dealing with other genera of basidiomycetes (Paulus et al. 2000; Ghobad-Nejhad et al. 2010; Oliveira et al. 2014) and, as such, supports the separation of Bresadolia species from other genera in Polyporus s.l. 2. Polyporus tuberaster forms a sister clade with (P. umbellatus (P. minutosquamosus (P. hapalopus (D. gunnii (D. campestris))))), with high to moderate support (BPP = 1, BS = 81). Depending on the interpretation, this clade could be recognized overall as Polyporus s.s. (Fig. 1). Nonetheless, we recognize P. tuberaster as the only member of Polyporus s.s. (Polyporus infrageneric group Polyporus s.s., sensu Núñez and Ryvarden 1995), mainly because we only used GenBank sequences and no morphological examinations of types or new collections were conducted. Additionally, we are aware that many of these specimens have missing data in the molecular matrix (i.e., only one gene), which can sometimes lead to poorly resolved phylogenetic relationships (Jiang et al. 2014). We found that the heterogeneous nature of D. campestris, D. gunnii, P. hapalopus, P. minutosquamosus, P. tuberaster, and P. umbellatus group is reflected by morphological characteristics (see the BTaxonomy^ section) and also by genetic distance (Table 2). The interspecific distance between these species ranged between 17 and 22.4%, strongly contrasting with the lower distance observed between P. tuberaster and the well-established genera Picipes (ranged between 13.9 and 17.7%). New evidence and a more complete and comparative morphological analyses are needed to properly delimit Polyporus s.s. and to determine whether the closely related species are congeneric or not.
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Fig. 1 Combined phylogenetic analysis of ITS and nLSU region conducted by Bayesian inference, showing main lineages within Polyporus s.l. and allied genera. All sequences generated for this study are indicated in bold faces. Numbers at branches indicate Bayesian
posterior probability values/bootstrap frequency. Red stars indicate the type species of the genus and (T) = type specimens. The bar indicates number of expected substitutions per position
0
0.002
0.004
0.003
0.214 0.222
0.232
0.309
0.313
0.309
0.275
0.276
0.254
0.284
0.003
0
0.008
0.207 0.202
0.219
0.316
0.309
0.305
0.271
0.264
0.254
0.294
B. paradoxa, MV161
0.004
0
B. paradoxa, MV23
0.287
0.254
0.272
0.283
0.324
0.315
0.310
0.213
0.212 0.222
0.005
0.003
0
B. paradoxa, MC4402
0.282
0.254
0.261
0.254
0.315
0.309
0.298
0.239
0.207 0.221
0.008
0
B. paradoxa, Robledo 1958
0.284
0.252
0.273
0.255
0.322
0.312
0.306
0.223
0.215 0.222
0
0.236
0.135
0.187
0.201
0.255
0.261
0.290
0.147
0 0.168
0.170
0.144
0.179
0.156
0.226
0.280
0.297
0.148
0
B. para- B. uda, B. uda, H6518 FLASFdoxa, 60005 Robledo 1959
0.255
0.19
0.218
0.228
0.279
0.274
0.288
0
0.234
0.204
0.186
0.175
0.280
0.307
0
B. cuticu- Dichomitus lata sp. IFP14643
Evolutionary divergence matrix of ITS sequences (uncorrected Bp^) in Bresadolia and closely related genera
B. paradoxa, MV23 B. paradoxa, MV161 B. paradoxa, MC4402 B. paradoxa, Robledo1958 B. paradoxa, Robledo 1959 B. uda, H6518 B. uda, FLASF60005 B. cuticulata, Dai13101 Dichomitus sp., IFP14643 P. hapalopus, Yuan5809 P. minutosquamosus, Runnel690 P. tuberaster, DAOM7997 P. tuberaster, WD2382 P. umbellatus, Pen13513 Pi. badius, WD2341
Table 2
0.225
0.111
0.2
0.164
0.273
0
P. hapalopus
0.213
0.124
0.224
0.187
0
P. minutosquamosus
0.177
0.135
0.002
0
P. tuberaster, DAOM7997
0.139
0.126
0
P. tuberaster, WD2382
0.201
0
P. umbellatus, Pen13513
0
Pi. badius
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3. Polyporus squamosus is recovered in the clade recognized as Cerioporus with high support (BPP = 1, BS = 100%), according to results found by Zmitrovich and Kovalenko (2016) and Zhou et al. (2016). Polyporus squamosus was morphologically treated as a member of the Polypous infrageneric group Polyporus (sensu Núñez and Ryvarden 1995); it must be noted that it shares morphological characters with P. tuberaster and Bresadolia species, such as the presence of scales in the pilei and/or inflated skeleton-binding hyphae, respectively (see the BTaxonomy^ section); nevertheless, this has not been considered a difficulty for the recognition of Cerioporus as a good genus based on the phylogenetic evidence.
Taxonomy Bresadolia Speg., Anales de la Sociedad Científica Argentina 16(6): 277 (1883). Type species. Bresadolia paradoxa Speg. Basidiocarp annual, lateral, centrally to eccentrically stipitate, fleshy, sometimes watery when fresh, papery and brittle when dry, pale yellow to ochraceous. Pileus flabelliform to deeply infundibuliform; pilear surface azonate, with a wrinkled and papery cuticle, sometimes with darker radial fibers or pink to purple spots, but never with scales. Margin sterile, hyaline to white when fresh to dark brown upon drying. Pores round to angular, usually decurrent on the stipe; poroid surface whitish to cream when fresh to buff-yellow upon drying. Context fleshy and watery when fresh to leathery upon drying, homogeneous, white to cream. Stipe cylindrical, fleshy when fresh to woody and hard upon drying, expanded at the base, without formation of sclerotium. Hyphal system dimitic with generative and skeleton-binding hyphae; generative hyphae with clamp connections, dominant to almost monomitic in trama, characterized by two morphologies: (1) up to 7 μm wide, with abundant clamps and (2) inflated up to 15 μm wide, with sparse clamps; skeletonbinding hyphae from the context and stipe usually dominating, frequently branched, IKI−. Cystidia absent. Fusoid cystidioles present in the hymenium. Basidia clavate, foursterigmate. Basidiospores cylindrical to sub-ellipsoid, hyaline to slightly yellowish, thin-walled, smooth, large oil droplets present in most basidiospores, IKI−, CB+. Ecology and distribution. All the species grow on dead wood, causing white rot on the substrate. So far, the genus is distributed in tropical to warm-temperate areas. Comments. Bresadolia is distinguished macroscopically from other genera currently recognized in Polyporus s.l. by the combination of fleshy and sometimes watery basidiomata when fresh, papery, and brittle upon drying; with a papery and wrinkled cuticle on the pilear surface. Microscopically, the
inflated generative hyphae with clamp connections are characteristic, and, in general, the generative hyphae are dominant in trama, which is almost monomitic. Inflated generative hyphae are a convergent characteristic described in different species in Polyporus s.l. (Table 3) and in some species of Lentinus Fr., but is more typical in Agaricales (Grand et al. 2011). These hyphae are sometimes difficult to differentiate from the inflated skeleto-binding hyphae; however, the former is infrequently branched, strongly stained in aqueous phloxine solution, and restricted to the trama of the tubes. The hyphal system in Bresadolia is particular and rare in Polyporales since it is different from several other polypores that have a monomitic hyphal system in the context and dimitic hyphal system in the trama of the tubes. Additionally, this dimiticism is generally based on skeletal hyphae, not in skeleton-binding hyphae [e.g., Trulla duracina (Pat.) Miettinen (Miettinen and Ryvarden 2016), Flaviporus subhydrophilus (Speg.) Rajchenb. & J.E. Wright (Westphalen and Silveira 2013), Phellinotus Dreschler-Santos, Robledo, and Rajchenb. (Dreschler-Santos et al. 2016)]. Phylogenetically, Bresadolia is included in the core polyporoid clade (sensu Binder et al. 2013), Polyporaceae Corda family (sensu Justo et al. 2017). Bresadolia is recovered as a sister taxon of the clade formed by P. tuberaster (Polyporus s.s.) and (P. umbellatus (P. minutosquamosus (P. hapalopus (D. gunnii (D. campestris))))) with high to moderate support (BPP = 0.95, BS = 78%). Morphologically, Bresadolia species share with P. hapalopus, P. minutosquamosus, P. tuberaster, and P. umbellatus the fleshy when fresh and brittle upon drying basidiomata and the inflated generative hyphae, dominant in the trama, which is almost monomitic. However, Bresadolia species differ from P. tuberaster and P. umbellatus because the latter usually grow on the ground from a sclerotium close to stumps of hardwoods (Núñez and Ryvarden 1995; Dai 1999; Zhou et al. 2016). Additionally, P. hapalopus and P. minutosquamosus are differentiated by smaller basidiospores, which are generally larger in Bresadolia species [e.g., 8.0– 13.8 μm long in B. paradoxa (5.5–)6.1–6.9 (− 7.6) μm long in P. hapalopus (Xue and Zhou 2014), and (4.0–)4.5–5.5 μm long in P. minutosquamosus (Runnel and Ryvarden 2016)]. Morphologically, D. campestris differs from Bresadolia species by resupinate and coriaceous basidiomata that, according to Ryvarden (1991), could be derivatives of P. squamosus, presenting marginal remnants of black stipe, and D. gunni differs by coriaceous-woody basidiomata, laterally attached by a broad base (Cunningham 1965), in contrast to the fleshy and sometimes watery basidiomata of Bresadolia species. Based on morphological characteristics, Núñez (1993) suggested that D. campestris and tropical Dichomitus spp. might be closely related to Polyporus s.s. Phylogenetic analysis by Krueger (2002) showed that D. campestris and D. gunnii were positioned close to P. tuberaster. Nevertheless,
Circular
Pilear surface Context consistency
Radially striate Woody to corky to finely wrinkled Round to angular Flabellate to With a papery Fleshy and watery when pores infundibuliform and wrinkled fresh to leathery upon cuticle drying
Circular pores
Hymenophoral Pilei shape Cylindrical
Stipe
Morphological comparison of the genera currently segregated and accepted in Polyporus s.l.
With a black cuticle
Surface stipe
Generative hyphae
With clamp connections, non-inflated Bresadolia With clamp Cylindrical and With brown expanded at the fibrils connections; base inflated, dominating in trama Cerioporus Angular pores Reniform to With concentric Fleshy when fresh to brittle Very short to almost With a brownish With clamp flabelliform scales or hard upon drying missed to black felt connections; non-inflated Favolus Regular to Spathulate to With minute Fleshy-tough to leathery Cylindrical, Glabrous With clamp radially dimidiate hairs or when fresh to corky upon flattened, often connections or elongated scales drying reduced simple-septate; pores non-inflated Lentinus Lamellae Flabellate to With hairs or Fleshy-tough when fresh to Sub-cylindrical Squamous to With clamp infundibuliform scales to coriaceous upon drying glabrous connection; inflated, glabrous dominating in trama With clamp Neodictyopus Circular pores Reniform to Glabrous, Fleshy-tough to brittle or Cylindrical Reticulated to connections flabelliform radially hard upon drying longitudinally striate striate, with a black cuticle Cylindrical, often Glabrous With clamp Neofavolus Angular to Reniform to With flattened Fleshy-tough to leathery when fresh to brittle or reduced connections; diamond-semicircular scales or corky upon drying non-inflated shaped pores smooth Corky to coriaceous when Cylindrical, often With a brownish With clamp Picipes Round to angular Fan-shaped to With hard fresh and hard upon reduced to black connections or pores infundibuliform cuticle, drying cuticle simple-septate; without non-inflated scales Polyporus Angular to Circular to With Fleshy when fresh and Cylindrical, usually Glabrous With clamp s.s. (P. radially semi-circular agglutinated brittle upon drying originating from a connections; tuberaster) elongated scales to sclerotium inflated, dominating pores glabrous in trama Pseudofavolus Angular to Flabelliform to Glabrous to Coriaceous when fresh and Cylindrical, often Glabrous With clamp hexagonal spathulate sometimes hard upon drying reduced connections; pores striated non-inflated
Atroporus
Table 3
Basidiospores shape
Oblong-ellipsoid to navicular Cylindrical to navicular
Cylindrical to ellipsoid Subcylindrical to bacilliform
Cylindrical
Oblong to cylindrical or fusiform Cylindrical to oblong-ellipsoid Cylindrical
Arboriform dominating the context Arboriform dominating the trama
Arboriform
Arboriform, dominating the basidiocarp
Arboriform, dominating the basidiocarp Strongly arboriform, dominating the trama
Arboriform, dominating the basidiocarp
Arboriform
Usually arboriform, Narrowly dominating in context and ellipsoid to stipe, dextrinoid in trama subcylindrical Arboriform dominating in Cylindrical to context and stipe sub-ellipsoid
Skeleton-binding hyphae
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since then, the phylogenetic position of these species has not been exhaustively tested. Although they are shown to be closely related to P. tuberaster in our phylogenetic analysis (Fig. 1), the results are not conclusive since they are based solely on the available LSU sequences. A morphological comparison of the genus Bresadolia with the currently accepted genera in Polyporus s.l. is presented in Table 3. Bresadolia craterella (Berk. & M.A. Curtis) Audet, Index Fungorum 311: 1 (2016) [MB# 552685]. Basionym: Polyporus craterellus Berk. & M.A. Curtis, Journal of the Linnean Society 10: 305 (1869) [MB#227289] (Fig. 2). Complete description in Núñez and Ryvarden (1995). Comments. Polyporus craterellus was described from Cuba and recorded in subtropical and tropical America. We
Fig. 2 Basidiospores of Bresadolia species. a Basidiospores of B. craterella (TENN59383). b Basidiospores of B. paradoxa (SP445677). c Basidiospores of B. uda (HLB 63263, holotype). Scale bar = 5 μm. Illustrations by V. MotatoVásquez
could not have access to the type specimen, but we reviewed the only specimen with an available sequence in GenBank (TENN59383). Following the protologue (Berkeley and Curtis 1868) and the available morphological descriptions (Núñez and Ryvarden 1995; Silveira and Wright 2005, as P. cyathiformis Lév.), we conclude that the sequenced specimen is a clear representative of B. craterella. It shares with other species of the genus the fleshy basidiomata and the papery pilear surface. However, it differs by the smaller and paler basidiomata without watery consistency when fresh, usually deeply infundibuliform pileus and smaller pores (5–7 per mm). Our phylogenetic analyses suggest that B. craterella forms a sister clade with B. paradoxa with high support (BPP = 1, BS = 100; Fig. 1). Specimens examined: Brazil, Paraná, Foz de Iguaçu, Iguaçu National Park, in seasonal semi-deciduous sub-montane forest, on decayed trunk, 150 m a.s.l., 31.XII.1992, leg.
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A.A.R. Meijer 2396 (TENN59382)—Ecuador, Napo, Anangu, Rio Napo, Tropical Rainforest, on old fallen trunks, 78° 35′N, 0° 10′S; 10.VII.1983, T. Læssøe 44971 (TENN59383). Bresadolia cuticulata (Y.C. Dai, Jin Si & Schigel) Audet Index Fungorum 311: 1 (2016) [MB# 822674]. Basionym: Polyporus cuticulatus Y.C. Dai, Jin Si & Schigel, Mycosystema 35(3): 276 (2016) [MB#811143]. Complete description in Si and Dai (2016). Comments. Bresadolia cuticulata, recently described from China, is morphologically related to B. uda. However, both species differ in the color and structure of the pilear surface, being pale gray to grayish brown, with buff-yellow to cinnamon radial stripes in the former, and brown to dark brown, with pink to purple spots in the latter. Macroscopically, B. cuticulata also resembles B. paradoxa (cf. Fig. 2a; Si and Dai 2016, Fig. 2), but these are geographically distant and morphologically differ in the pore sizes (3–5 per mm in the former and 2–3 per mm in the latter). Although our results are not conclusive regarding the phylogenetic position of B. cuticulata, it is clear that it does not nest within the South American species of Bresadolia (i.e., B. paradoxa and B. craterella). Bresadolia cuticulata was also considered similar to P. varius (Pers.) Fr. due to the radial stripes on its upper surface (Si and Dai 2016). However, P. varius differs by the presence of a black stipe and smaller basidiospores (6.7–9.2 × 3.2–3.8 μm) (Niemelä 2005). In addition, recent phylogenetic studies showed that P. varius is related to Cerioporus clade (Zhou et al. 2016; Palacio et al. 2017). Bresadolia paradoxa Speg., Anales de la Sociedad Científica Argentina 16(6): 277 (1883) [MB#75377] (LPS!) (Figs. 2, 3, and 4). = Polyporus aquosus Henn., Hedwigia 43: 199 (1904) [MB#176974] (S!) Basidiomata annual, pileate, lateral to eccentrically stipitate, solitary, up to 7–12 cm long; pileus sub-reniform to flabellate, up to 5–10 cm-wide and 0.7–4 cm thick, pilear surface azonate, with a papery and wrinkled cuticle upon drying, sometimes with darker radial fibers, brown (N60 A90 M60), some specimens becoming dark brown (N50 A99 M99) to reddish dark brown (N60 A60 M90); stipe cylindrical, fleshy when fresh to woody and hard when dry, up to 5–8 cm long and 0.9–3.0 cm thick, expanded at the base, with abundant brown fibrils; pileus margin entire, hyaline when fresh, thin, glabrous, in dried specimens mostly involute, sterile, up to 0.3 cm thick; context fleshy and watery when fresh, leathery when dry, white to cream (N00 A10 M00), homogeneous, up to 3.5 cm thick; pore surface cream when fresh to pale-brown when dry, pores large, regular, round, or more commonly angular, 2–3 pores per mm, decurrent on the stipe; dissepiments thin, partially dilacerate upon drying, up to 35–90 μm
thick; tube layer not stratified, slightly darker than the context in dry specimens, up to 0.5 cm deep. Hyphal system dimitic with generative and skeletonbinding hyphae; generative hyphae with clamp connections, dominant in trama of tubes, which may seem almost monomitic, and are characterized by two morphologies: (1) thin-walled and non-inflated, (2.5–)3.0–6.0 (− 7.0) μm diameter, and (2) inflated, with sparse clamps, slightly branched, thin-walled, slightly tortuous, (9.0–)10–12.5 μm diameter, context hyphae mostly running horizontally, generative hyphae infrequent, skeleton-binding hyphae abundant, frequently branched to arboriform, straight to sinuous, hyaline to yellowish, with thick walls, 5.0–9.0 μm wide. Stipe with hyphal structure similar to the context. Fusoid cystidioles present in the hymenium. Cystidia absent. Basidia clavate, foursterigmate, (20.5–) 22–30.5 (− 31.5) × (5.5–) 6.0–8.0 (− 8.5) μm; basidioles clavate, thin walled, 17.5–22.5 μm long. Basidiospores cylindrical to sub-ellipsoid, 8.0–13.8 × (2.5–) 3.5–4.0 (− 4.5) μm, L = 11.2 μm, W = 4.0 μm, Q′ = 1.8–3.75 (− 5.5) μm, Q = 2.5 μm (n = 325/16), hyaline to slightly yellowish, thin-walled, smooth, large oil droplets present in most basidiospores, weakly CB+, IKI−, uninucleate. Ecology and distribution. Growing on hardwood logs in areas of the Atlantic and Amazonia Forests. Basidiomata appearing mainly in the rainy season from February to April. Comments. The above description incorporates data of type specimens that were previously not mentioned and characters from fresh specimens that were collected and sequenced. Polyporus aquosus Henn. is a rare species only known from its type locality at Serra da Cantareira, São Paulo, Brazil (Hennings 1904; Fidalgo and Fidalgo 1957; Fidalgo 1965); the holotype was kept in Berlin Museum (B) and was considered lost after the Second World War. However, it was recently found at the Swedish Museum of Natural History (S) and, based on the interpretation of the hyphal system as monomitic, the species was transferred to Tyromyces P. Karst (Ryvarden 2012). Recent collections of poroid fungi in Serra da Cantareira, Brazil (type locality of P. aquosus), around the type locality of B. paradoxa in East Paraguay (unfortunately the exact point of the type locality of B. paradoxa is unknown) and additional collections in the Atlantic Forest of NE Argentina, allowed us to conduct a morphological analysis and comparison with type specimens. We could not find any morphological or DNA differences between B. paradoxa and P. aquosus; therefore, we recognized these as conspecific and the synonymy was proposed, with B. paradoxa being the oldest name available for the taxon. Previously, B. paradoxa was morphologically compared with the Paleotropical species B. cuticulata and B. uda ( R a j c h e n b e r g a n d Wr i g h t 1 9 8 7 ; A u d e t 2 0 1 7 ) . Morphologically, B. cuticulata differs by the smaller pores
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Fig. 3 Macromorphological characteristics of B. paradoxa. Bresadolia paradoxa (Robledo1958) (a–d). a, b Basidiocarp. c Pore surface detail. d Context detail. Bresadolia paradoxa (LPS15714, holotype). e
Basidiocarp. Polyporus aquosus (S-F15335, holotype). f Basidiocarp. Pictures by G.L. Robledo (a, c–e) and V. Motato–Vásquez (b, f). Scale bar: a, b, e, f = 1 cm, c, d = 1 mm
(3–5 per mm), pale gray to grayish brown pilear surface, with radial buff-yellow to cinnamon grayish stripes, absence of cystidioles and slightly wider basidiospores [(3.0–) 3.3–4.4 (− 5) μm] (Si and Dai 2016). On the other hand, B. uda differs macroscopically by the bigger pores (1–3 per mm) and the presence of pink to purple spots on the pilear surface and, microscopically, by the wider basidiospores (4.5–6.0 μm). Phylogenetically, B. paradoxa forms a separated clade from
B. cuticulata and B. uda, as sister taxon of B. craterella (Fig. 1). Thus, these species cannot be considered conspecific, and accordingly, B. paradoxa is recognized as the type species of the genus. Bresadolia paradoxa has also been compared macroscopically to C. squamosus (Huds.) Quél., with both species sharing the large and fleshy lateral to eccentrically stipitate basidiomata, inflated skeleton-binding hyphae, and large
Mycol Progress Fig. 4 Bresadolia paradoxa (SP445677). a Basidia, basidioles, and cystidioles. b Hyphal ends from the dissepiments. c Inflated generative hyphae. d Generative and skeleton-binding hyphae from the context. Scale bar = 5 μm. Illustrations by V. MotatoVásquez
basidiospores, but differing by the scaly pilear surface present in the latter (Núñez and Ryvarden 1995). Also, both species have different ecological requirements, as B. paradoxa has been found only on dead wood, whereas C. squamosus has frequently been recorded on living hardwood trees and causing stem rot (Schwarze et al. 2000). Our phylogenetic analysis agrees with previous studies showing that C. squamosus forms a separate lineage from Bresadolia and Polyporus s.s. clade (Zmitrovich and Kovalenko 2016; Zhou et al. 2016). In the literature, there are other species names described from the Neotropics that have been related to or treated as synonyms of B. uda, viz., P. cornucopiae Lloyd from Brazil, P. discoideus Berk. & M.A. Curtis. from Cuba, and P. marbleae Murrill from Puerto Rico (Núñez and Ryvarden 1995). In this study, we could only study the holotype of P. discoideus. The specimen was in poor condition and the pilear surface was quite destroyed. We could not find and measure more than two basidiospores, and although the hyphal structure is similar to Bresadolia species, we could not attribute this specimen to any of the known Bresadolia species. Further studies of Bresadolia, especially in the Neotropics, should
include a re-examination of type specimens, further collections in type localities, and the addition of coding genes with the purpose of clarifying the evolutionary history of these species. Specimens examined: Argentina, Chaco, Resistencia, 20.XI.1975, leg. J.R. Deschamps & Del Busto (BAFC 2764, BAFC 50842); Misiones, San Pedro, Tobuna, 26° 31′S, 54° 00′W, 600 m a.s.l., leg. G.L. Robledo 1958 & K. Cockle (FCOS 44); Ibid., leg. G.L. Robledo 1959 (FCOS 45); Iguazú, 26.IX.1984, leg. D.M. Job 3825 (BAFC 30410)— Brazil, São Paulo, Serra da Cantareira, III.1913, leg. A. Puttemans 796 (S-F15335) (holotype of P. aquosus); Ibid., Parque Estadual da Cantareira, 20.III.2007, leg. F. Karstedt, N. Menolli Jr. & M. Capelari FK887 (SP 392852); Ibid., leg. FK886 (SP 392854); Ibid., 3.X.2008, leg. M. Capelari, & L.A. Ramos 4402 (SP 445678); Ibid., 21.X.2011, leg. V. MotatoVásquez 23 & A.M. Gugliotta (SP 445677); Ibid., leg. V. Motato-Vásquez 27 (SP 445681); Ibid., 07.III.2012, leg. V. Motato-Vásquez 157 (SP 445680); Ibid., leg. V. MotatoVásquez 161 (SP 445679); Ibid., 10.XI.2016, leg. R.M. Pires 345 (SP 466617); Ibid., XI.2016, leg. G. Jerônimo (SP
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466618); Ibid., 21.II.2017 leg. V. Motato-Vásquez 909 & A.M. Gugliotta (SP 466616); Ibid., Parque Estadual das Fontes do Ipiranga, 11.IX.2001, leg. M. Capelari (SP 381515); Ibid., 11.X.2001, leg. U.C. Peixoto (SP 381514); Ibid., 23.IV.2002, leg. M. Capelari (SP 381516); Rio Grande do Sul, São Leopoldo, 1909, leg. J. Rick (SP 22841)— Paraguay, 3913 sur le tronc des arbres, III.1883 (LPS 15714) (holotype of B. paradoxa); Ibid., III.2015, Alto Paraná, Reserva Itaipu, on dead fallen trunk, leg. G. Robledo & E. grassi 2955 (FCOS 43). Additional specimens examined: Cuba, leg. C. Wright 379 (K 57288) holotype of P. discoideus Berk. & M.A. Curtis. Bresadolia uda (Jungh.) Audet Index Fungorum 311: 1 (2016) [MB#552687]. Basionym: Polyporus udus Jungh., Tijdskr. Nat. Gesch. Physiol. 7: 289 (1940) [MB#201065] (HLB!) = Polyporus fuscomaculatus Bres. & Pat. Mycological Writings 1(6): 49 (1901) [MB#171411] (S!). Complete description in Núñez and Ryvarden 1995. Comments: Bresadolia uda was described from Indonesia (Java; Junghuhn 1840) and subsequent collections were recorded from Africa (Kenya, Malawi, Tanzania; Ryvarden and Johansen 1980), Asia (India, Japan; Núñez and Ryvarden 1995) and the Neotropics (Argentina, Bolivia, Brazil, Cuba, Paraguay and Peru; Corner 1984; Silveira and Wright 2005). Some Neotropical specimens to which we had access were re-identified as B. paradoxa (see specimens examined); however, many others require re-examination. The holotype of P. fuscomaculatus Bres. & Pat., described from Samoa (Polynesia), was revised; the specimen is preserved although very contaminated and we could not find morphological differences with B. uda; therefore, we treated it as a synonym. Previously, no focus had been placed on B. uda and its phylogenetic relationships within Polyporus s.l. No recent collections of specimens from the type locality are available and the few studies that dealt with other genera showed that B. uda was placed in an uncertain position, unrelated to any of the major phylogenetic clades recognized in Polyporus s.l. (Sotome et al. 2008). The only specimen collected close to the type locality and with ITS sequence available in GenBank is from India (DAOM 72382). This specimen was sequenced by Krueger (2002), and we obtained the morphological annotations and digitalized images of the author, which fits with the type and descriptions of B. uda (Ryvarden and Johansen 1980; Núñez and Ryvarden 1995; Silveira and Wright 2005). In our phylogenetic analyses, specimens of B. uda group within the genus Bresadolia (Fig. 1). Nevertheless, specimens of B. uda were not recovered as a monophyletic group; which suggests that it is a species complex, as already suggested by Corner (1984) and Silveira and Wright (2005) based on morphological evidence. The empirical consequences of limited
taxonomic sampling and scarce molecular evidence in phylogenetic analyses are widely discussed in the literature (Rosenberg and Kumar 2001, 2003). Despite not having a general consensus, it is widely recognized that the inclusion of as many evidence and as much diversity as possible will make the test of a hypothesis more accurate. For this reason, collections in the type locality (Indonesia) and more molecular evidence will allow that the identity of B. uda, in relation to similar specimens recorded from Africa, North America, Polynesia, and the Caribbean area, to be revealed. For the time being, we leave the resolution of this species complex as an open question. Specimens examined: India, Kulu division Panjab, 23.VI.1955 (DAOM 72382, examined through photographs of the basidiocarp identified and annotated by Dr. D. Krueger). Indonesia, Java, and Pangarango (HLB 63263, holotype of P. udus). Samoa, I.1900, leg. C.G. Lloyd 5004 (S 14792) (holotype of P. fuscomaculatus Bres. & Pat.).
Excluded and doubtful taxa Bresadolia caucasica Schestunov, Hedwigia 101 (1910) [MB#217195]. Comments. Bresadolia caucasica was described from the Caucasus region (Magnus 1910). In the description, Magnus and later Saccardo and Trotter (1912) stressed the possibility of this species being synonymous of P. squamosus, mainly by its geographic distribution and by the pilear surface with scales, a feature that is absent in other species of Bresadolia. As far as we known, there is no record in the literature of new collections of this species since the original description. We were unable to locate the type specimen. Bresadolia hapalopus (H.J. Xue & L.W. Zhou) Audet, Index Fungorum 311: 1 (2016) [MB#552686]. Basyonim: Polyporus hapalopus H.J. Xue & L.W. Zhou, Mycological Progress 13: 814 (2014) [MB#802658]. Comments. Polyporus hapalopus is only known from one specimen collected in China (Xue and Zhou 2014). It has been considered closely related to B. uda since both are distributed in Pantropical to warm-temperate areas. They present a cuticle on the pilear surface and generative hyphae that is dominant in trama, which is almost monomitic. Audet (2016) propose the combination of P. hapalopus in Bresadolia; nevertheless, the evidence used for this nomenclatural decision was not presented. Based on the protologue of P. hapalopus (Xue and Zhou 2014) and collected data of Bresadolia species, we found that morphologically, P. hapalopus differs from Bresadolia species by the imbricate basidiomata, soft when fresh but tough upon drying context, and the smaller basidiospores, which are generally larger in Bresadolia species [e.g., 8.0–13.8-μm length in B. paradoxa and (5.5–) 6.1–6.9 (− 7.6)-μm length in P. hapalopus].
Mycol Progress
Additionally, our phylogenetic analyses do not support the combination of P. hapalopus into Bresadolia, showing that P. hapalopus clusters in a clade closely related to P. tuberaster, in accordance with results presented by Xue and Zhou (2014) and Zhou et al. (2016). Delimitation of genera based only on morphological characteristics has become an increasingly difficult task in Polyporus s.l. For this reason, although we agree that phylogenetic hypothesis and nomenclatural changes are always plausible to be tested, in order to maintain nomenclatural stability in an era of fast changes in the systematics of the kingdom Fungi, we emphasize the importance of supporting hypotheses combining all of the empirical evidence possible (e.g., morphological, biological, molecular) before implementing new taxonomic decisions regarding the species. Bresadolia mangifera Pat., Flore Cryptogamique des Antilles Françaises 36 (1903) [MB#217051]. Comments. The specimen was collected from Guadeloupe Is., French Antilles, and described by Patouillard (Duss 1903). In the original description, as well as in Saccardo (1905), the type species was described as sterile. In addition, the type specimen was destroyed, making a morphological study impossible (Ryvarden 1983). New specimens collected in the type locality are needed to define the status of this name. Acknowledgements The authors thank the curators of BAFC, DAOM, FCOS, LPS, HLB, K, S, TENN, and SP herbaria for the loan of type or original collections. VMValso gratefully acknowledges the PhD financial support received from Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Brazil; International Association for Plant Taxonomy (IAPT); and Rufford Small Grant Foundation. Authors also acknowledge the assistance of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Universidad Nacional de Córdoba, both of which support facilities used in this project. Financial support was also provided by (1) International Cooperation Project BR10RED01, Argentina–Brasil MINCyT–CAPES BDiversidad y Ecología De Hongos Degradadores (Aphyllophorales, Basidiomycota) Del Sur De Brazil y Argentina^; (2) FONCYT (PICT 1676) and Fondo IBOL CONICET; and by (3) FONCYT (PICT-2015-0830). Authorities that give permits to collect in Paraguay, Argentina, and Brazil (Instituto Florestal) are kindly acknowledged. Technical equipment support was also provided by Idea Wild. Authors express sincere thanks to the two anonymous reviewers for their valuable and constructive comments and suggestions to improve the quality of the paper.
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