Decrypting the Polyporus dictyopus complex: Recovery of Atroporus Ryvarden and segregation of Neodictyopus gen. nov. (Polyporales, Basidiomyocta)

Polyporus dictyopus, with a large number of heterotypic synonyms, has been traditionally considered a species complex, characterized by wide morphological variation and geographic distribution. Thus, neotropical specimens previously identified as P. dictyopus from Amazonia, Cerrado and Atlantic Forest biomes were studied based on detailed macro- and micromorphological examination and phylogenetic analyses, using distinct ribosomal and protein-coding genomic regions: the nuclear ribosomal internal transcribed spacer (nrITS), nuclear ribosomal large subunit (nrLSU), and RNA polymerase II second subunit (RPB2). Two unrelated generic lineages, each one represented by different species, are reported: Atroporus is recovered and re-circumscribed to include A. diabolicus and A. rufoatratus comb. nov.; Neodictyopus gen. nov. is proposed to accommodate N. dictyopus comb. nov. and two new species, N. atlanticae and N. gugliottae. Our study showed that at least five distinct species were hidden under the name P. dictyopus. Detailed descriptions, pictures, illustrations, and a key are provided for Atroporus and Neodictyopus species.


Phylogenetic analyses
Two distinct datasets were constructed: the first based on three molecular markers (nrITS, nrLSU, and RPB2), and the second based on two (nrITS and nrLSU). The generated sequences, including related sequences downloaded from GenBank (Table 1), were aligned using Mafft v.7 [33], under the Q-INS-I strategy for nrITS and G-INS-i strategy for nrLSU and RPB2 for both datasets. The alignments were manually examined and adjusted with MEGA 6 [34]. We coded the nrITS and nrLSU indels present in the datasets as binary characters following the simple indel coding method [35], performed in the SeqState software [36]. An intron in RPB2 was separated and analyzed as a distinct partition. The first dataset was subdivided into nine partitions: ITS1, 5.8S, ITS2, nrLSU, RPB2 -1st, -2nd, -3rd codon positions, RPB2 intron, and ITS/LSU Indels; the second was subdivided into five partitions, excluding the partitions related to RPB2. The best-fit evolutionary model for every partition was selected using jMo-delTest v. 1.6 [37,38] following the Bayesian Information Criterion (BIC). The final alignments were deposited at TreeBASE (submission ID: S20479). To test the congruence of the distinct nucleotide partitions, we applied the Partition Homogeneity Test (PHT), as implemented in PAUP Ã [39]. Since this test does not show any incongruence among the partitions, we proceeded with the concatenated analyses. Two distinct analyses were performed for each dataset: Bayesian Inference (BI) and Maximum Likelihood (ML). Bayesian Inferences were conducted using MrBayes 3.2.6 as available in CIPRES Science Gateway 3.1 [40], and implemented with two independent runs, each one with four chains and starting from random trees. The runs performed 20.000.000 generations and trees were sampled every 1000 th generation. Twenty five percent of sampled trees were discarded as burn-in, while the remaining ones were used for calculating a 50% majority consensus tree and Bayesian Posterior Probabilities (BPP). To check the convergence and stability of the runs, the average standard deviation of split of frequencies (<0.01) was evaluated in Tracer v.1.6 [41], as well as the potential scale reduction factor (PSRF). ML trees were obtained using RAxML v.8.1.4 [42], in CIPRES science gateway [40]. The analysis first involved 100 ML searches, each one starting from one randomized stepwise addition parsimony tree, under a GTRGAMMA model, with no proportion of invariant sites and all other parameters estimated by the software. We provided a partition file to force RAxML software to search for a separate evolution model for each dataset. Bootstrap support values (BS) were obtained with multi-parametric bootstrapping replicates under the same model, allowing the program halts bootstrapping automatically by the autoMRE option. 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%. Trametes hirsuta (Wulfen) Lloyd and Trametes versicolor (L.) Lloyd were used as the outgroup based on previous studies [10,12].

Nomenclature acts
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publi-cation of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www. mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Phylogenetic analysis
A total of thirty one sequences were newly generated in this study (12 nrITS Table 2. The topology of the BI and ML of the first and second dataset analyses showed no inconsistency in any supported clades, as is shown in the BI tree (Fig 1). For the second dataset, the topology of the ML analyses has no inconsistency with the BI, and they recovered the same clades of the first dataset. The bootstrapping criteria of RAxML indicated 360 pseudo replicates as sufficient to access the internal branch support for the first dataset, and 204 for the second dataset.
All phylogenetic analysis performed showed that specimens of P. dictyopus complex were grouped into two distinct, strongly supported clades, hereafter named "Atroporus clade" and "Neodictyopus clade".
Type species. Atroporus diabolicus (Berk.) Ryvarden. Remarks: Basidiospores descriptions and Melzer reagent reaction of the skeletal-binding hyphae are new diagnostic information for the genus. Atroporus resembles morphologically Polyporus sensu lato and Echinochaete Reid, however, the combination of narrowly ellipsoid to subcylindrical basidiospores, strongly dextrinoid skeletal-binding hyphae with a differentiated apex, and the black cuticle on the pileus are unique to the group Atroporus. All the species grow on dead wood, typically dead fallen branches of relatively thin diameter (up to 10 cm diam.) and cause white rot on the substrate. So far the genus is only known for the neotropics (Fig 5A and 5B). Basidiomata annual to biannual, central to eccentrically stipitate, solitary; pileus circular, up to 3.2 cm in diameter and 4 mm thick; pilear surface reddish black (10R2.5/1) to very dark red (2.5YR2.5/2), glabrous, radially striate to finely wrinkled; margin rounded/truncate, sterile, with a black cuticle. Pore surface light brown (7.5YR6/4) to dark brown (7.5YR3/2), in some specimens a black cuticle covering the surface; pores circular, regular, 5-8 per mm, 90-140(-150) μm (ave = 111.5 μm, n = 80/2); dissepiments entire, 30-100.5(-120) μm thick, (ave = 51.1 μm, n = 80/2). Tubes concolorous with pore surface, not stratified to stratified into 3 layers, up to 7 mm long each one. Context homogeneous, light brown (7.5YR6/4), 1.5 mm thick. Stipe cylindrical, solid, glabrous, longitudinally striate, bearing a black cuticle up to 3.2 cm long, up to 5 mm diam., with a robust appearance. Hyphal system dimitic with generative hyphae and skeletal-binding hyphae. Generative hyphae with clamps, hyaline, thin-walled, 2-3 μm thick, difficult to observe. Skeletal-binding hyphae of two types; arboriform type, present in stipe and context, up to 230 μm long, 2.5-4 μm wide, thick-walled, with a short unbranched stalk (17.5-48 μm), 4-6 branches with an alternating arrangement, and shortened as getting closer to the trama of the tubes, hyaline to yellowish in KOH and water, IKI-(Figs 3A and 4A). In the trama of the tubes are present the second type of hyphae (Figs 3C and 4B), skeletal-binding hyphae short (41-75 μm long) and "prickly" always with acute apex that is projected above hymenium, golden yellow in KOH and water, strongly dextrinoid changing to dark brown in Melzer reagent, thick-walled, just after the septa (3-5 μm wide) developing a stalk, that is considerably enlarged at the central portion (7-11 μm wide), between the middle portion and apical portion arise 2 to 6 branches (1-3 μm wide), stalk ending in an acute apex, as small spines,   Remarks: Atroporus diabolicus is characterized by the presence of strongly dextrinoid skeletal-binding "prickly" hyphae with a pointed apex in the trama of the tubes that arises above the hymenium, the rounded/truncate and sterile margin and the robust appearance of the basidiomata. Atroporus dibolicus is microscopically similar to A. rufoatratus and A. infernalis, however A. rufoatratus has tramal skeletal-binding hyphae developing a stalk that tend to be slightly longer and narrower (49−93 × 2−8 μm), with a rounded and projected apex (lacking spine-like short branches), similar to those of A. infernalis. Macrocopically, A. infernalis is hitherto known by a short and lateral stipe with pileus flat and flabelliform, A. diabolicus is central to eccentrically stipitate with pileus flat and circular, and P. rufoatratus centrally stipitate with pileus circular, depressed to slightly infundibuliform.  tubes, hyaline to yellowish in KOH, water, and lactofenol, IKI-. In the trama of the tubes they differ in the second type of hyphae (Fig 7A), skeletal-binding hyphae with a wider main stalk (49−93 μm long) developed just after the clamp scar (2−3 μm wide) that is enlarged especially in the central portion (4−8 μm wide), between the middle and apical portion 2 to 5 branches arise (1−3 μm wide), up to 76 μm long, with dichotomous branches, thin to thick-walled, the hyphal Polyporus dictyopus complex from Neotropics apex is round and projected above hymenium, hyaline to yellowish in KOH, water, and lactofenol, strongly dextrinoid changing to dark brown. Pileipellis as an anamorph matrix, 20−28 μm thick, pale yellow to dark orange. Cystidioles subulate, 13−20 × 5−7 μm, with a basal clamp; basidia clavate, 4-sterigmate, with a basal clamp, 17−21 × 6−8 μm. Basidiospores narrowly Remarks: This species is well characterized by the narrowly ellipsoid to rarely subcylindrical basidiospores and the skeletal-binding hyphae of the trama, strongly dextrinoid, with a widened main stalk and a round apex projected above the hymenium; macroscopically, it is characterized by its centrally stipitate basidiomata, infundibuliform dark reddish brown pilei, and slender stipe. Atroporus diabolicus is a related species but it has tramal skeletal-binding hyphae with a slightly shorter and wider stalk (41-75 × 3-11 μm) and a pointed and "prickly" apex, besides a more robust appearance of the basidiomata.
Remarks: the type specimen is damaged, only a pilear fragment of 2 cm remaining in the voucher specimen. Berkley [43] described P. infernalis based on a collection from Minas Gerais (Brazil) as an allied species of P. varius (Pers.) Fr. and P. dictyopus, but as a "very distinct species". Polyporus infernalis was later transferred to Atroporus [16] based on the dextrinoid and modified skeletal-binding hyphae in the trama of the tubes. After our revision of the type we confirm the presence of this feature, endorsing that this species belongs to Atroporus; we also observed the sterile margin as mentioned in the protologue. Atroporus infernalis is related to A. rufoatratus but it differs in having a short and lateral stipe, and flabelliform pileus (protologue information [43]). Unfortunately, basidiospores were not able to be observed and the poor condition of the type did not allow us to compare it to the other specimens. Basidiomata annual, lateral to eccentrical, rarely centrally stipitate; pileus reniform to flabelliform; pilear surface glabrous, radially striate, dark reddish brown; margin irregular, wavy, and lobed to decurved and entire. Pores circular. Context homogenous, yellow to light brown. Stipe cylindrical, solid, reticulated to longitudinally striate, bearing a black cuticle (Fig 2A, 2B, 2C and 2D). Hyphal system dimitic; generative hyphae clamped, hyaline, thin-walled, branched skeletal-binding hyphae dominating, arboriform, hyaline, IKI− to slightly dextrinoid (only in mass) in the trama of the tubes. Basidia clavate, 4-sterigmate. Basidiospores subcylindrical to bacilliform, thin-walled, smooth, hyaline, IKI- (Figs 3B, 3D, 3G, 3H, 4D, 4E, 4F1, 4F2, 4F3 and 4F4).
Type species. Neodictyopus atlanticae Palacio, Robledo & Drechsler-Santos. Remarks: Neodictyopus is characterized by its subcylindrical to bacilliform basidiospores, reniform to spatulate pileus, and skeletal-biding hyphae of arboriform type, slightly dextrinoid (when in mass) in the trama of the tubes. So far, the genus is neotropical (Fig 5C, 5D and 5E), but probably pantropical, since some specimens from the Paleotropics clustered together with the Neodictyopus clade. All the species grow on dead wood, typically dead fallen branches of relatively thin diameter (up to 10 cm diam) and produce white rot on the substrate. Neodictyopus is morpholically similar to other Polyporus species; however, P. tuberaster, the type species of Polyporus, has fleshy basidiomata when fresh, and pileus upper surface whitish to ochraceous covered with scales. Macroscopically, Neodictyopus is more similar to Atroporus, but the ellipsoid to subcylindrical basidiospores and strongly dextrinoid skeletal-biding hyphae from the trama of the tubes are unique to latter.
Remarks: Neodictyopus atlanticae is well characterized by lateral to eccentrically stipitate basidiomata, well developed and slender stipe, reniform pileus with irregular, wavy and lobed margin, and the gregarious habit.
Substrate: on fine woody debris. Distribution: This species was originally described from temperate forest of the Juan Fernández archipelago (Chile), being currently and also found in the Cerrado province of Mato Grosso state (Brazil) (Fig 5D). Remarks: Neodictyopus dictyopus is characterized by having basidioma laterally stipitate, with short, robust, black, and reticulated stipe, margin irregular, wavy and lobed, variable pilear surface color, and subcylindrical to rarely narrowly cylindrical basidiospores. The Brazilian specimens examined for this study are linked to the type specimen by morphological comparison, despite the disjunct distribution. In order to better define the circumscription and distribution of N. dictyopus, more collections from the type locality are needed. Neodictyopus dictyopus can be differentiated from N. atlanticae by the short, robust, and lateral stipe, and the smaller basidiospores. Etymology: in honor of Dr. Adriana Gugliotta, a Brazilian expert in polypores, for its contributions to our knowledge of polypore fungi diversity.
Basidiomata annual, laterally stipitate, solitary; pileus flabelliform to slightly spathulate, up to 1.5 cm in diameter and 2 mm thick; pilear surface reddish brown (2.5YR4/4) to dark reddish  Context homogeneous, yellow (10YR8/8), up to 1 mm thick. Stipe cylindrical, solid, slender, longitudinally striate, glabrous, bearing a black cuticle, up to 2.3 cm long and 2 mm in diam. Hyphal system dimitic with generative hyphae and skeletal-binding hyphae. Generative hyphae with clamps, hyaline, thin-walled, 2−3 μm thick, IKI-, CB-, more easily observed in the tubes. Skeletal-binding hyphae hyaline to yellowish in KOH and water, IKI-, CB-. Stipe, context and trama of the tubes composed mainly of skeletal-binding hyphae with a loose arboriform branching pattern, up to 310 μm long, 3−5 μm wide, thick-walled, geniculated, with a short unbranched stalk (92−155 μm) and then with 2−4 branches (up to 190 μm long) with an alternating arrangement. Skeletal-binding hyphae from the tubes are shorter (up to 120 μm) than in stipe and context, and become shorter (up to 90 μm) approaching the dissepiments where there are more (3−6) and shorter ramifications (Fig 10A). Cystidiole subulate, 12 Remarks: Neodictyopus gugliotae is characterized by subcylindrical to bacilliform basidiospores, the eccentrically stipitate basidiomata with a circular pileus. Neodictyopus atlanticae has a similar slender and developed stipe but it differs in its irregular, wavy and lobed pileus margin and shorter basidiospores. Remarks: Type specimen damaged, only a pilear fragment remaining in the voucher specimen. Based on the cylindrical basidiospores and skeletal-binding hyphae IKI-, it is possible to recognize P. blanchetianus as a Neodictyopus member; however, given the poor condition of the holotype, we prefer to consider P. blanchetianus as a dubious species. Additional specimens from the type locality, Salvador (Bahia) according to Góes-Neto [44], are needed to confirm its correct placement.

Discussion
Inferences from previous phylogenetic studies including specimens identified as P. dictyopus have been limited by the small number of sequences and did not link the results with morphological studies [7,8,10]. Our reconstructions revealed that P. dictyopus, as it is currently understood [1,2,15], is polyphyletic. Moreover, it includes species belonging to two well defined lineages, both presenting distinct morphological features, supporting them as distinct genera, Atroporus and Neodictyopus. Atroporus conforms a strongly supported clade in both BI and MP analyses (Fig 1) and includes A. diabolicus, the generic type species, and A. rufoatratus. Within Atroporus, two highly supported lineages (Fig 1) represent 2 species: A. diabolicus represented by one specimen from the Imerí province (Amazonas, Brazil), and A. rufoatratus represented by three specimens from the Atlantic province (Santa Catarina, Brazil). Both species have ellipsoid to rarely cylindrical basidiospores (Q = 1−2), strongly dextrinoid skeletal-binding hyphae from the trama of tubes, protruding the hymenium with an acute apex, and centrally to eccentrically stipitate basidiomata.
Neodictyopus is strongly supported by both BI and MP analyses (Fig 1) and encompasses four lineages. N. atlanticae is represented by a strongly supported clade with three specimens from the Atlantic province (Santa Catarina, Brazil), the type species of the genera. The clade of N. dictyopus is composed by three specimens from the Cerrado province (Mato Grosso, Brazil). Neodictyopus gugliottae is represented by a strongly supported clade formed by two specimens from Araucaria and Paraná Forest provinces (São Paulo, Brazil and Misiones, Argentina), respectively. Finally, there is a paleotropical clade (BPP = 1.00, BS = 100%) with three samples from subtropical Asia, which are not taxonomically treated in this work. Neodictyopus atlanticae, N. dictyopus, and N. gugliottae share cylindrical basidiospores, reniform pileus, and a lateral to occasionally eccentrical stipe.
The study of the type specimen of P. dictyopus showed morphological similarities with the Cerrado specimens. The reticulated stipe surface (Fig 2B1), the short (up to 1.5 cm) and wide (up to 8 mm) stipe, and the flabeliform pileus are macromorphologically similar features. Micromorphologically, basidiospore shape and size (Fig 9B and 9B1) and skeletal-binding hyphae with a loose arboriform branching pattern and weakly dextrinoid reaction in the dissepiments are identical. Molecular data from the N. dictyopus type or from other type locality specimens were not possible to be used in our study. However, based on the morphological similarities, our specimens (GAS60; GAS272; GAS281, VFL18) were assumed as conspecific.
Atroporus and Neodictyopus share similar hyphal system in the context of pileus and stipe, with generative hyphae with clamps and dominant skeletal-binding hyphae. Both genera have basidiomata with a dark reddish brown cuticle on the pilear surface, becoming even blackish in A. diabolicus, and have a substrate preference for fine woody debris (diameter 5-9 cm). Atroporus species can be differentiated by its ellipsoid basidiospores, strongly dextrinoid skeletal-binding hyphae in the trama of tubes with projected apex, and centrally to eccentrically stipitate basidiomata. Differently, Neodictyopus species have cylindrical basidiospores, nondextrinoid to weakly dextrinoid (only in mass) skeletal-binding hyphae, and lateral to eccentric stipitate basidiomata.
The distinct skeletal-binding hyphae of the trama, here treated as typical for Atroporus, were once considered as cystidia [16] and/or as modified skeletal-binding hyphae for the P. dictyopus s.l. [1,[15][16][17]. Meticulous examinations of the hyphal system according Decock et al. [24] allow us to observe and describe whole hyphae, and then reinterpret as a unique type of skeletal-binding hyphae characteristic of Atroporus.
In this study, Neodictyopus was recovered as a sister group of Picipes. Our results also bring new phylogenetic information about Atroporus, which appears as a sister clade of the remaining Neodictyopus and Picipes. These three genera formed a strongly supported clade (BPP = 1.00, BS = 98% , Fig 1), in which all the species share the black cuticle in the stipe, the principal character that defines Melanopus sensu Patouillard and Melanopus group sensu Núñez & Ryvarden [1]. However, other species (e.g. P. leprieurii, P. guianensis, and P. varius), that present the same cuticle, are not related to those clades, reinforcing Melanopus group is an artificial group as previously pointed out [10].
Atroporus can be easily differentiated from Picipes and Neodictyopus by its mainly ellipsoid basidiospores and strongly dextrinoid skeletal-binding hyphae from the tubes with protruding apex. Polyporus austroandinus (Pers.) Fr. is another related species that also has basidiomata with a stipe bearing a black cuticle, similar to Neodictyopus species. Nevertheless, the species has larger pores (4-5 per mm) and basidiospores [(-8)9-11.5 × 3-3.8(-4)], and grows in the temperate zones of the southern Andes forest [10].
The reexamination of morphological groups and species complexes within Polyporus is required in order to classify the genus in a less artificial way. Independent inspection of the hyphal system from the trama of the tubes, context, and pileus, the dextrinoidity of the structures, as well as basidiospore shape comparison (from the Q value), could assist the detection of morphological patterns within clades already recognized as the Melanopus clade sensu Dai et al. [10].
Key to the species of Atroporus and Neodictyopus 1. Basidiospores usually ellipsoid, skeletal-binding hyphae from the tubes strongly dextrinoid with a well differentiated and protruding apex protruding into the hymenium Atroporus 2 1. Basidiospores subcylindrical to bacilliform, skeletal-binding hyphae from the tubes IKI− to occasionally weakly dextrinoid, without differentiated apex Neodictyopus 3 2. Skeletal-binding hyphae from the tubes with a sharply spinose pointed apex, basidiomata robust, generally with 2−3 tube layers, sometimes in old specimens with a black cuticle covering the hymenophore, stipe robust (up to 3.2 cm long × 0.5 cm diam.)