Neotropical Studies on Hymenochaetaceae: Unveiling the Diversity and Endemicity of Phellinotus

Phellinotus, a neotropical genus of wood-decay fungi commonly found on living members of the Fabaceae family, was initially described as containing two species, P. neoaridus and P. piptadeniae. The members of this genus, along with six other well-established genera and some unresolved lineages, are the current representatives of the ‘phellinotus clade’. On the other hand, based on a two-loci phylogenetic analysis, some entities/lineages of the ‘phellinotus clade’ have been found in Fomitiporella s.l. In this work, we performed four-loci phylogenetic analyses and based on our results the genera of the ‘phellinotus clade’ are shown to be monophyletic groups. In addition to the natural groups confirmed as different genera, morphological revisions, phylogenetic relationships, and host distribution of different specimens resembling P. neoaridus and P. piptadeniae revealed three new species in the Phellinotus genus, referred to here as P. magnoporatus, P. teixeirae and P. xerophyticus. Furthermore, for P. piptadeniae a narrower species concept was adopted with redefined morphological characters and a more limited distribution range. Both P. neoaridus and P. teixeirae have a distribution range restricted to seasonally dry tropical forests in South America. Additionally, based on detailed morphological revisions Phellinus badius, Phellinus resinaceus, and Phellinus scaber are transferred to the Phellinotus genus. The geographic distribution and host range of the genus are then discussed.


Introduction
Phellinotus Drechsler-Santos, Robledo & Rajchenb. is a genus of wood-decay fungi characterized by perennial basidiomata with dark lines and a mycelial core, as well as a monomitic hyphal system in the context, a dimitic hyphal system in the tubes, and yellowish basidiospores with a flattened side that become chestnut brown in KOH solution [1].

Molecular Phylogenetic Analyses
Two combined datasets were developed concatenating the newly generated nITS, nLSU, TEF1-α, and RPB2 sequences of Phellinotus specimens and sequences of specimens/species of the 'phellinotus clade' retrieved from GenBank (NCBI) ( Table 1). The sequences used in this study represented species and genera closely related to Phellinotus such as Drechsler-Santos et al.
Two distinct combined datasets were constructed: one with taxa the of the 'phellinotus clade' (with 36 specimens), and the other with species of Phellnotus (with 35 specimens). The two four-loci combined datasets (i.e., nITS, nLSU, TEF1-α and RPB2) were subdivided into 13 data partitions (ITS1, 5.8S, ITS2, LSU, TEF1-α-1st, -2nd, -3rd codon positions, TEF1-α introns, RPB2-1st, -2nd. and -3rd codon positions). The best-fit model of nucleotide evolution for the datasets was selected using AIC (Akaike Information Criterion) as implemented in jModelTest2 v.1.6 [37,38]. For phylogenetic inferences based on the combined dataset of the 'phellinotus clade' taxa, we selected Sanghuangporus vaninii (Ljub.) L.W. Zhou & Y.C. Dai, Tropicoporus drechsleri Salvador-Montoya & Popoff, and Inonotus griseus L.W. Zhou as outgroup species. For phylogenetic inferences from the combined dataset, species of Phellinotus, T. drechsleri, and I. griseus were selected as the outgroup species. Maximum likelihood (ML) and Bayesian inference (BI) analyses were applied to the combined datasets. ML searches were conducted with RaxMLv.8.1 [49], and the GTRGAMMA model was selected to search for the best-scored trees, with all other parameters estimated by the software. The analyses first involved 1000 ML independent searches, each one starting from one randomized stepwise addition parsimony tree. Only the best-scored ML tree was kept, and the reliability of the nodes was accessed through nonparametric Bootstrap (BS) pseudoreplicates under the same model, allowing the program to halt bootstrapping automatically with the autoMRE option. An additional alignment partition file to force RAxML software to search for a separate evolutionary model for each partition was used. BI analyses were carried out in MrBayes 3.2.6 [50] and implemented with two independent runs, each beginning from random trees with four simultaneous independent chains. A total of 2 × 10 7 generations was carried out, sampling one tree every 1000th generation. The first 25% of the sampled trees were discarded as burn-in and checked by the convergence criterion (frequencies of average standard deviation of split <0.01) in Tracer v.1.6 [51], while the remaining ones were used to reconstruct a 50% majority-rule consensus tree and to estimate the Bayesian posterior probabilities (BPP) of the branches. jModelTest2 v.1.6, MrBayes 3.2.6, and RaxMLv.8.1 were used in CIPRES science gateway [52]. A node was strongly supported if it showed a BPP ≥ 0.9 and/or BS ≥ 90% while moderate support was considered if BPP ≥ 0.8 and/or BS ≥ 60%. For phylogenetic inferences of taxon based on the molecular data of the nITS region, we followed the recommendation of Jeewon and Hyde [53]. The topology from the ML tree is depicted, and the statistical values of ML and BI are indicated for each node. The alignments were deposited in TreeBASE under the accession number 28692 (http://purl.org/phylo/treebase/phylows/study/TB2:S28692, accessed on 2 January 2022).

Taxonomy
Habitat and distribution: The substrata and distribution data of species are still unknown [54]. According to Larsen [55], this is from North America; however, Cunningham [56] believes that this taxon belongs to the West Indies.  Notes: This species, described by Berkeley [57], is considered a species of Phellinus by its perennial basidioma and dimitic hyphal system (Phellinus s.l.) [54][55][56]58,59]. However, we revised the lectotype of species (K-M 199720) and observed the slightly cracked pileal surface, the hymenophore with 4-5 pores/mm, the context with a dark line, a monomitic hyphal system in the context and dimitic in the tubes, absence of setae, and broadly ellipsoid to ellipsoid basidiospores (7-8 × 5-6 µm) with a flattened side that turns darker in KOH solution ( Table 2, Figures 3D-K and 4A-D). In this case, this taxon is transferred to Phellinotus because it fits the morphological concept of the genus [1]. Phellinotus badius resembles P. neoaridus and P. piptadeniae, but these last two have smaller basidiospores (5.5-6.5 × 4-5 µm in P. neaoridus; 5-5.5 × 3.5-4.5 µm in P. piptadeniae) ( Table 2). Additionally, P. neoaridus has basidiomata with a rimose pileal surface and a mycelial core in the context [1], whereas P. piptadeniae has basidiomata with a lobulate pileal surface in well-developed specimens (Table 2, Figure 5D Diagnosis: Basidioma perennial, ungulate, pileal surface slightly cracked at the base of basidioma, margin round, hymenophore poroid (1-2/mm). Context with a mycelial core. Hyphal system monomitic in the context and dimitic in the tubes. Basidiospores broadly ellipsoid to ellipsoid with a flattened side, 4.5-5.5 × 4-4.4 µm, thick-walled, yellowish, chestnut to ferruginous in KOH, on living tree of O. aurantiodora.
Habitat and distribution: In the Central Andes Coast floristic group of SDTFs in South America, basidiomata of P. magnoporatus are found on living trees of Ocotea aurantiodora.
[1], and Elias et al. [7], P. piptadeniae has applanate to triquetrous basidiomata with cracked to rimose reddish yellow to grayish black pileal surface, a hymenophore with 4-6 pores/mm, a context with a dark line, tubes distinctly stratified, skeletal hyphae restricted to the trama of the tubes, and broadly ellipsoid to ellipsoid with a flattened side basidiospores that turn darker in KOH solution. Additionally, this is a SDTFs generalist species that is found predominately on living trees of P. gonoacantha (recurrent host). However, based on a phylogenetic and morphological reassessment of various specimens for this study, we revealed two entities within this taxon (P. piptadeniae s.l.). One entity possesses a rimose and dark grayish brown pileal surface in mature basidiomata and grows on living trees of Fabaceae in different FG of SDTFs in South America (described as P. teixierae sp. nov. below) ( Table 2, Figures 1 and 2). The second entity has a lobulate, cracked, olive gray pileal surface with deep concentric furrows delimiting wide lobes in mature specimens and grows predominately on living trees of P. gonoacantha in different types of vegetation (Table 2, Figure 5D,G,H). In this context, this last species is considered as P. piptadeniae s.str. in this work. Phellinotus piptadeniae resembles P. neoaridus. However, P. neoaridus has a rimose pileal surface, a mycelial core in the context, tubes indistinctly stratified and slightly larger basidiospores (5.5-6.5 × 4-5) (Table 2, Figure 6A Description: Basidioma perennial, pileate, sessile, broadly attached, solitary, ungulate, up to 41 mm long, 60 mm wide, and 53 mm thick, woody hard, pileal surface glabrous, brown (5E4-5F4), with some resinous, dark, and glossy substance, cracked, concentrically, and radially sulcate, mostly fine sulci but some coarse and deep, margin entire, round, thick, pubescent, and golden brown (5D7), pore surface flat to slightly convex, dark blond (5D4) to brown (5E4), pores rounded, regular, (2-)3-4(-5)/mm, (220-)230-400(-420) µm diam; dissepiments entire, (40-)50-120(-130) µm thick, context up to 13 mm thick, with a mycelial core at the base of the basidioma, with some resinous substance in the form of granules and thin layers, non-zonate and brown (6D8-6E8), tubes indistinctly stratified, up to 40 mm long, brown (6D8-6E8), with whitish mycelia strands usually filling the old tubes.
Habitat and distribution: This polypore is distributed in Papua New Guinea and Australia, growing on living tree of E. papuana.
Habitat and distribution: The perennial basidiomata are found on living trees of Acacia Notes: This taxon, described by Berkeley [61] as Polyporus igniarius var. scaber, was considered a synonym of Phellinus rimosus (Berk.) Pilát (current name = Fulvifomes rimosus (Berk.) Fiasson & Niemelä) on account of its perennial basidiomata with rimose pileal surface and dimitic hyphal system (Phellinus s.l.) [55,59,62]. We revised the type specimen of Polyporus igniarius var. scaber, and other material from Australia previously determined as P. rimosus. All material showed triquetrous to ungulate basidiomata with rimose and dark gray to black pileal surface, flat to concave, yellowish to olive, hymenophore with 3-4 pores/mm, the context with dark lines and a monomitic hyphal system, a dimitic hyphal system in the tubes, without setae, and broadly ellipsoid to oblong with a flattened side basidiospores (5.5-8 × 4-6 µm) that turn darker in KOH solution ( Table 2, Figures 9 and 10A-C). Accordingly, this species is transferred to Phellinotus as the best option because it fits into the morphological concept of the genus [1] as well. Phellinotus scaber resembles P. neoaridus. However, P. neoaridus has a reddish yellow to dark brown and flat to convex hymenophore (Table 2, Figure 6A Diagnosis: Basidioma perennial, triquetrous, pileal surface rimose, margin round, hymenophore poroid (4-6/mm). Context with dark lines. Hyphal system monomitic in the context and dimitic in the tubes. Basidiospores broadly ellipsoid to ellipsoid with a flattened side (4.5-6.5 × 3.5-5.5 µm), thick-walled, yellowish, chestnut to ferruginous in KOH, on living tree of Fabaceae in SDTFs.
Etymology: teixeirae, in honor of Dr. Alcides Ribeiro Teixeira for his valuable contribution to mycology in Brazil.
Habit and distribution: In the Chaquean province of South America, basidiomata are found on living trees of Prosopis sp.
Given these facts, we revised the type specimens of P. badius, P. resinaceus, and P. scaber in this work. Based on our observations, the types of these three species have characters that are present in taxa of the 'phellinotus clade', such as a dark line, a mycelial core, a monomitic hyphal system in the context, a dimitic hyphal system in the tubes, basidiomata without setae, and colored basidiospores with a flattened side that turn darker in KOH solution [1]. Based on the revision of the literature, Kotlaba and Pouzar [59] and Larsen [54] observed yellowish basidiospores that turn chestnut brown in KOH solution in the type specimens of P. badius and P. scaber. Therefore, in accordance with the results of this work and our reinterpretation of the literature, we transferred these three species to Phellinotus. However, molecular analyses are suggested to confirm their phylogenetic position in this genus.

Morphological, Host and Geographic Distribution Patterns in Species of Phellinotus
The species of Phellinotus are presented here with a narrower morphological concept and a redefined, more limited, distribution range. In Hymenochaetaceae, Phellinotus is considered a genus with a neotropical distribution [1]. Based on our results, the geographic distribution of Phellinotus is expanded due to the addition of P. badius, P. resinaceus, and P. scaber to this genus. Phellinotus resinaceus and P. scaber have a geographic distribution in the Australasian region. Phellinotus resinaceus grows on Eucalyptus papuana (Myrtaceae) and is characterized by the presence of resinous substances in the basidiomata and a mycelial core in the context (Table 2, Figure 8H-M). On the other hand, P. scaber is found on living members of the family Myrtaceae, Casuarinaceae R. Br. and Fabaceae, and is characterized by a rimose pileal surface and the presence of dark lines in the context (Table 2, Figure 9A-K). In the case of P. badius, a species characterized by a cracked pileal surface and a dark line in the context (Table 2, Figure 3D-H), its type locality and substrata remain unknown. However, based on our interpretation of the literature, this taxon is distributed in North and Central America, as well as the Caribbean region [54,56]. Considering these facts, Phellinotus has a geographic distribution in the tropical and subtropical climatic zones of America and Australia, growing on living members of different genera of angiosperms.
In South America, the geographic distribution pattern of P. piptadeniae has been studied [1, 6,7]. This species was described by Teixeira [71] as a parasitic polypore on P communis (current name = P. gonoacantha), from the Atlantic Forest of the Brazilian state of São Paulo. Sixty years later, Drechsler-Santos et al.
[1] extended its distribution to the Caatinga domain in northern Brazil, where it was found on living trees of the species Piptadenia Benth. Then, Salvador-Montoya et al. [6] revealed that the populations of this species showed variable morphology and had a disjunct distribution in different patches of SDTF in South America, within the limits of the Atlantic Forests domain in Brazil and northwestern Peru, growing on living tree species belonging to different genera of Fabaceae such as Libidibia Schltdl., Mimosa, Pithecellobium Mart., Pityrocarpa, and Senegalia. Recently, Elias et al. [7] showed that P. piptadeniae is a SDTF generalist species with a wide geographic distribution in South America that decays several hosts (with P. gonoacantha as a recurrent host).
Given this evidence, the wide taxonomic concept of P. piptadeniae has been questioned. Based on the results of this study, two taxa are revealed: P. piptadeniae s.str. and P. teixeirae (Figures 1 and 2). Morphologically, P. piptadeniae s.str. is characterized by a cracked lobulate with concentric deep furrows delimiting wide lobes, olive gray pileal surface in well-developed specimens (Table 2, Figure 5A-I). Furthermore, this parasitic polypore is distributed both in dry and wet forest in eastern South America, within the limits of the Caatinga, Cerrado, Atlantic, Parana Forest, and Pampean provinces and SDTFs ( Figure 11B), growing on living tree species of Fabaceae (ex. C. tweediei and P. gonoacantha (a recurrent host)) and Myrtaceae (ex. E. uruguayensis). Therefore, the evidence supports that this taxon is not a specialist, as suggested by Elias et al. [7].
Phellinotus teixeirae differs from P. piptadeniae s.str. mainly in terms of its rimose and dark grayish brown pileal surface (Table 2, Figure 6D-I). The morphological patterns of the pileal surface have been documented to distinguish species in the Hymenochaetaceae; for example, Fulvifomes squamosus Salvador-Montoya & Drechsler-Santos and F. cedrelae (Murrill) Murrill with F. robiniae (Murrill) Murrill, and the latter with F. rimosus [31,59]. Furthermore, P. teixeirae is distributed within the limits of SDTF in South America ( Figure 11C), growing on living tree species of Fabaceae such as Pithecellobium excelsum, Libidibia glabrata, and Pityrocarpa moniliformis. These three tree species are distributed within the limits of SDTF, which makes them related to some degree [14,[72][73][74]. Libidibia glabrata is a common species in the inter-andean seasonally dry tropical forest and in the lowland of the equatorial seasonally dry tropical forest [73], P. excelsum is found in the seasonally dry forests both in the Andean valleys in northern Peru and southern Ecuador and on the pacific coast of Ecuador and northern Peru [72], and P. moniliformis grows in the Caatinga, as well as the Atlantic Forests domain, in southeastern and northeastern Brazil [75][76][77]. Additionally, Särkinen et al. [14] mentioned that P. moniliformis is a SDTF habitat specialist. Therefore, unlike P. piptadeniae s.str., P. teixeirae is considered a SDTF host specialist in this work. The SDTF specialist species are entities that restrictedly grow in SDTF habitats [14,78].
Phellinotus neoaridus is a parasitic polypore that morphologically resembles P. piptadeniae s.str. and P. teixeirae. However, P. neoaridus differs from them by having a black rimose pileal surface and a mycelial core in the context (Table 2, Figure 6A-C). Furthermore, P. neoaridus is widely distributed in the Brazilian semiarid region (Caatinga dry woodlands), growing on living trees of Caesalpinia spp. and Cenostigma pyramidale [1]. Cenostigma pyramidale is an endemic species in the Caatinga domain and is widely distributed in northeastern Brazil [79][80][81][82]. Based on our results and interpretation of the literature, all records of P. neoaridus are found within the limits of SDTF in northeastern Brazil ( Figure 11A). Therefore, we consider P. neoaridus a SDTF host specialist, as suggested for P. teixeirae. Accordingly, both P. neoaridus and P. teixeirae are considered endemic polypore species from SDTF in this study. In the Hymenochaetaceae, some taxa are treated as endemic species. For example, Coltricia africana Masuka & Ryvarden is endemic to Africa [83].  [8,84].
With respect to P. magnoporatus and P. xerophyticus, both species are only registered from their type localities in South America ( Figure 11A). Both species have large pores (1-2 pores/mm in P. magnoporatus, 3-4 pores/mm in P. xerophyticus). However, P. magnoporatus is characterized by a cracked pileal surface and a context with a mycelial core (Table 2, Figure 3A-C) and grows on O. aurantiodora in the Central Andes Coast floristic group of SDTFs, while P. xerophyticus has a rimose pileal surface and a dark line in the context (Table 2, Figure 8A-E) and grows on species of Prosopis L. in the Chaquean province. Further surveys and studies would help shed light on their distributional ranges.

Classification System Status in the 'Phellinotus Clade'
Previous studies, based on multilocus molecular data, have been performed to delimit the lineages/entities in the Hymenochaetaceae family [85][86][87][88][89][90][91]. Pildain et al. [2] mentioned that the incorporation of more loci, as well as taxa, into phylogenetic analyses of the 'phellinotus clade' could help us to better understand the taxonomic inferences in this group. At that time, three taxonomic scenarios were proposed based on phylogenetic inferences of a two-loci combined dataset (nITS and nLSU). The first taxonomic scenario assumes that different lineages in the group of the 'phellinotus clade' are different genera (i.e., Arambarria, Inocutis, Fomitiporella sensu Pildain et al. [2], Fulvifomes, and Phylloporia), while the second scenario treats the taxa of the 'phellinotus clade' as a unique genus, Fomitiporella. The third taxonomic scenario keeps Phellinotus and Inocutis as independent genera and groups Arambarria under Fomitiporella [2]. The first taxonomic scenario was accepted as the best option despite the morphological and phylogenetic variability.  As suggested by Pildain et al. [2], in this work we conducted a phylogenetic analysis with a four-loci combined dataset (nITS, nLSU, TEF1-α, and RPB2) and based on our results we agree with the first scenario of classification proposed by Pildain et al. [2]. Despite our phylogenetic inferences suggesting that the 'phellinotus clade' genera (i.e., Arambarria, Inocutis, Fomitiporella s.str., Fulvifomes, Phylloporia, Rajchenbergia and other nontaxonomically treated lineages in Fomitiporella s.l.) is a monophyletic group with high support (Figure 1), we agree that it is a classification hypothesis. In the future, following further research on natural groups at the genera level on Hymenochaetaceae, this will become more evident. We would also like to reinforce the need to conduct more sampling with the inclusion of specimens from different ecosystems around the world, as well as analyses based on more molecular markers.
Key to the known species of Phellinotus