Introduction

Phylloporia Murrill is a widely distributed polypore genus in Hymenochaetaceae (Hymenochaetales, Agaricomycetes, Basidiomycota) typified by P. parasitica Murrill1. Species of Phylloporia occur mainly in the tropics but are also known from higher latitude ecosystems well2,3,4,5. Phylloporia sensu Murrill was characterized by resupinate and annual basidiomata with the ability to grow on the underside of living leaves, monomitic hyphal system, and absence of the setae which characterize most members of the Hymenochaetaceae1. Although the genus was erected in 1904, it remained undocumented until Ryvarden6 re-examined the type material and recognized four additional species: P. bibulosa (Lloyd) Ryvarden, P. chrysites (Berk.) Ryvarden, P. fruticum (Berk. & M. A. Curtis) Ryvarden, and P. weberiana (Bres. & Henn. ex Sacc.) Ryvarden, each sharing the microscopic features characterizing the type species. Subsequently, with the advent of DNA barcoding, Phylloporia taxonomy was revisited7. Based on phylogenetic analysis inferred from nuc 28S rDNA sequences as well as morphological and anatomical features, Wagner and Ryvarden7 demonstrated that Phylloporia is monophyletic, with Fulvifomes Murrill as a sister genus. From this pioneering molecular work, Phylloporia received much more attention, resulting in the current recognition of 61 species worldwide8,9,10,11,12,13. The current morphological concept for Phylloporia species includes annual to perennial basidiomata with resupinate, pileate-sessile, or pileate-stipitate habits, homogenous to two-layered context, monomitic to dimitic hyphal system, presence or absence of cystidioles, and subglobose to ellipsoid basidiospores8, 10, 13, 14. The presence of setae in Phylloporia was not noted until reported by Wu et al.10. Ecologically, some Phylloporia species are putatively host-specific parasites on living leaves, bushes, branches, and trees7, 13, 15,16,17, others are saprotrophs on wood18,19,20, with the trophic status of others still unknown10.

Despite the reasonably well-defined generic diagnostic features, Phylloporia remains heterogeneous and the species can be difficult to separate morphologically from those of related hymenochaetoid genera. As an example, Douanla-Meli et al.21 described a new species from Cameroon as P. resupinata Douanla-Meli and Ryvarden, but subsequent molecular phylogenetic analysis placed P. resupinata within the hymenochaetoid Fomitiporella clade and the species was transferred to that genus22.

Currently 14 species have been reported from tropical Africa with eight of these described from type material collected in the region2, 7, 9, 12, 18, 23. These species are mainly Central or East African and to our knowledge, only P. weberiana was known from West Africa7 until Olou et al.24 reported two species identified as Phylloporia sp. However, since species of Phylloporia can be host specific, we have since re-examined both of the species of Olou et al.24. Utilizing morphological and molecular phylogenetic analyses, we found that one of these species is P. littoralis Decock & Yombiyeni, previously known only from Gabon, while the second is new to science. Here we describe the new species and provide a key to the known tropical African Phylloporia species.

Material and methods

DNA extraction, amplifications, and sequencing

We extracted DNA from dried specimens using the microwave method25. Although previous studies involving Phylloporia species have used primarily the nuclear ribosomal large subunit, here we amplified two nuclear ribosomal DNA regions (nrDNA), the internal transcribed spacer (ITS) and the D1–D4 domain of large subunit (LSU). The primer pairs ITS-1F/ITS426, 27 and LR0R/LR528 were used to amplify both target DNA regions. For the polymerase chain reaction (PCR) procedure, the PCR products purification, and Sanger sequencing, we followed Olou et al.24. A total of six sequences, composed of two ITS and four LSU, were generated in this study and deposited in GenBank. Table 1 gives the accession numbers for all taxa included in this study.

Table 1 Taxa names with collection details and GenBank accession numbers of all sequences of Phylloporia spp. used in this study.
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Sequence alignment and species delimitation

To place our newly generated sequences accurately in the phylogenetic tree, we aligned them in addition to 126 LSU sequences retrieved from GenBank and used by previous studies on Phylloporia29. Sequences were aligned using the online mode of MAFFT version 730, with the algorithm FFT-NS-i as the most suitable. The resulting multiple sequences alignment was checked in Geneious 5.6.7 (https://www.geneious.com)31, where the ends rich in gaps were manually trimmed. Further, the multiple sequences alignment was inspected and some bases were manually adjusted using AliView32. Two model-based methods for species delimitation namely the Automated Barcode Gap Discovery (ABGD)33 and the Poisson Tree Process (PTP)34 were performed. The ABGD analysis detect potential barcode gap and use the identified barcode gap to sort the datasets into a hypothetical species. This analysis was performed on ABGD web interface using the Jukes-Cantor (JC69) and Kimura two-parameter (K2P). The relative gap width was set to 1.0 because if the gap is too large, the model will sort the dataset into a single species. We kept all other parameters as default. Like the ABGD method, the PTP is another species delimitation method that inferred putative species boundaries on a given phylogenetic input tree. To run the PTP analysis, we first built a single phylogenetic tree using IQ-tree 1.6.12 (http://www.iqtree.org/) in command line mode. The resulted tree without annotations in Newick format was used as the input tree to run the PTP analysis on a web server (http://species.h-its.org/ptp/) for 500,000 generations and 25% were discarded as burn-in. To compare both species delimitation models to the phylogenetic analysis, Maximum likelihood (ML) analysis under the Ultrafast Bootstrap with 5000 replicates was performed on the dataset using IQ-tree 1.6.12 (http://www.iqtree.org/) in command line mode with TM3 + F + I + G4 as the best substitution model selected using the command TESTONLY.

Phylogenetic analyses

For phylogenetic analyses, 73 sequences from the LSU region out of the 126 sequences previously used to inform species delineation in Phylloporia were selected and aligned with the 4 newly generated sequences in this study. In addition, 34 sequences from the ITS region including the type material of the genus were downloaded from GenBank and aligned together with the sequences newly generated in this study. Inonotus andersonii (Ellis & Everh.) Nikol. and I. hispidus (Bull.) P. Karst. were chosen as outgroup for both regions. Each region was aligned separately using the online mode of MAFFT version 730, with the algorithm L-INS-i. The multiple sequences alignments were checked and concatenated in Geneious 5.6.7 (https://www.geneious.com)31.

Given the gap in terms of number of sequences between the ITS and LSU regions (36 vs. 77), the concatenated alignment was considered as a single region and the best-fit evolutionary model was estimated as GTR + I + G using IQ-tree 1.6.12 (http://www.iqtree.org/) and the command TESTONLY. Following this substitution model, two phylogenetic tree inference methods, ML and Bayesian inference (BI) were performed. The ML was run using RAxML 8.2.1035 under standard bootstrap at the Cipres Science Gateway V.3.336. The BI was executed using MrBayes 3.2.7 in command line mode (https://github.com/NBISweden/MrBayes)37 for five million generations until the standard deviation of split frequencies reached 0.01. Chain convergence was determined using Tracer.v1.7.1 (http://tree.bio.ed.ac.uk/software/tracer/) and the first 25% (5000) trees was discarded as burn-in. The remaining trees were used to build the consensus tree using the Phylogenetic Tree Summarization (SumTrees) program within DendroPy 4.3.0. (https://github.com/jeetsukumaran/DendroPy)38. The topology of the ML tree was better resolved than that of BI, so the ML tree was targeted. To add the posterior probabilities (PP) of BI on the ML tree, the Phylogenetic Tree Summarization (SumTrees) program within DendroPy 4.3.0. (https://github.com/jeetsukumaran/DendroPy)38 was used. Then, the bootstrap values were added to the ML best tree already having the posterior probabilities using IQ-tree39. The resulting tree with (PP/BS) is presented in Fig. 3 and the support values ≥ 80% of PP and ≥ 70% of BS are indicated on each node. Alignment and phylogenetic tree generated in the study are deposited in TreeBASE: http://purl.org/phylo/treebase/phylows/study/TB2:S27303.

Morphological examination

Morphological descriptions were based on dried herbarium specimens. Macro-morphological characters were described with the aid of a stereomicroscope Leica EZ4 while microstructures were described using a Leica DM500 light microscope. For the microstructures, fine sections through the basidiomata were prepared for observation using a razor blade under a stereomicroscope and mounted in distilled water and 5% aqueous solution of potassium hydroxide (KOH) mixed with 1% aqueous solution of phloxine. Melzer’s reagent (to test for dextrinoid or amyloid reactions) and cotton blue (to test for cyanophilic reaction) were used and then examined at a magnification of 1000×. Leica Application Suite EZ V.3.4 software (Leica Microsystems Ltd., Switzerland) was used to capture images from the microscope. Measurements from captured images were done with the software “Makroaufmaßprogramm” from Jens Rüdigs (https://ruedig.de/tmp/messprogramm.htm) and analyzed with the software “Smaff” version 3.240.

Results

Species delimitation

The ABGD method with parameters JC69 and K2P gave identical results and partitioned the LSU dataset into 6 partitions. The first five partitions with interspecific priority divergence ranging from P = 0.001 to P = 0.0077 contained 83 groups each while the sixth partition with interspecific priority divergence P = 0.0129 contained only one group (Fig. 1). Each group within each partition represented a hypothetical species with one or several sequences (Supplementary Table S1). Given the congruence between the first five partitions (83 groups in each), we have chosen one of them as the one that reflects well our dataset. Thus, all 130 sequences contained in our dataset represent 83 hypothetical species (Supplementary Table S1). The four newly generated LSU sequences in this study were sorted into two groups. The new sequence named Phylloporia sp. OAB0204 clustered together with other sequences of P. littoralis retrieved from GenBank. The other sequences (Phylloporia sp. OAB0107, Phylloporia sp. OAB0142, and Phylloporia sp. OAB0511) grouped together to form a distinct group (Supplementary Table S1).

Figure 1
figure 1

LSU data partition from Automatic Barcode Gap Detection (ABGD).

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The PTP species delimitation estimated that the number of species in LSU dataset was between 82 and 109, with the Mean of 97 species. The PTP species delimitation was supported by the maximum likelihood solution (PTP_Mls) and the Bayesian solution (PTP_Bs). Both solutions gave two different results in terms of the number of estimated species. The PTP_Mls yielded into 82 putative species (Supplementary Table S2) while PTP_Bs gave 100 putative species (Supplementary Table S3). Although the PTP_Mls and PTP_Bs yielded different results, the newly generated sequences formed two distinct species and are grouped identically in both outcomes (Supplementary Table S2, 3). Since species delimitation with PTP_Mls and PTP_Bs gave same results for our newly generated sequences with good support values, and considering the ML tree and ABGD results, we chose the results from PTP_Mls as the most suitable for our dataset. The Fig. 2 presents the ML tree with the putative species as found with ABGD and PTP_Mls.

Figure 2
figure 2

Maximum likelihood tree of the LSU dataset of Phylloporia with rapid bootstrap values and species delimitation as recovered in ABGD and PTP analyses. The sequence names are followed by voucher or strain number and country of origin.

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Phylogenetic analyses

The combined ITS-LSU alignment contained 78 sequences with 2397 characters, of which 711 were parsimony-informative, 277 singleton sites, and 1409 constant sites. Four well supported major clades namely Fomitiporella (PP = 1.00/BS = 99), Fulvifomes (PP = 1.00/BS = 98), Inonotus (PP = 1.00/BS = 100), and Phylloporia (PP = 1.00/BS = 88) were recovered from the phylogenetic analyses inferred from the ITS-LSU (Fig. 3). Phylloporia appeared as a well-supported monophyletic clade, which split into two well-supported groups, here named A and B (Fig. 3). Group A (PP = 1.00/BS = 76) contained the sequences of the most Phylloporia species, including the generic type (P. parasitica), while group B (PP = 1.00/BS = 96) consisted of just three species of Phylloporia. The newly generated sequences nested within group A. The sequence OAB0204 clustered together with P. littoralis as it was found in the species delimitation analyses with high support (PP = 1.00/BS = 95). Sequences OAB0107, OAB 0142, and OAB0511 formed a distinct well-supported lineage (PP = 0.97/BS = 93) and had as a sister lineage an unidentified Phylloporia species from Kenya with high support (PP = 1.00/BS = 95). Since the sequences OAB0107, OAB 0142, and OAB0511 grouped together and had always formed a distinct lineage in all analyses (Figs. 2, 3; Supplementary Table S1–3), we proposed here as a new species and performed a detailed anatomical–morphological description on these specimens.

Figure 3
figure 3

Bayesian analysis (BY) and maximum likelihood (ML) analyses of the combined ITS-LSU dataset of Phylloporia. Branch support values given as PP/BS. Newly generated sequences are highlighted in red. The sequence names are followed by voucher or strain number and country of origin.

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Taxonomy

Phylloporia beninensis Olou & Langer, sp. nov.

MycoBank No. MB839326

Figures 4, 5, 6

Figure 4
figure 4

Macromorphology of Phylloporia beninensis. (A) Basidiomata on dead wood stump, (B) Basidiomata showing effused-reflexed attachment, (C) Basidiomata on dead part of living tree showing the margin of actively growing specimens, (D) poroid hymenophore, (E) Context showing the black line separating the tomentum and the lower context.

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Figure 5
figure 5

Line drawing of the hymenium of a pore of the type specimen of Phylloporia beninensis (OAB0511) showing the basidiospores, hyphae, basidia, basidioles, and cystidioles. Most elements with one or several guttulae. On the top left corner, we have the location where the microscopic preparation was taken. Scale bar = 5 μm.

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Figure 6
figure 6

Microstructures of the type specimen of Phylloporia beninensis. (A) Hyphae from tomentum, (B) generative hyphae from trama in KOH mixed with 1% phloxine, (C) Section through the hymenium in KOH mixed with 1% phloxine showing basidia, basidioles, and basidospores, (D) section through the hymenium in KOH showing hyaline basidia and basidioles, some with several guttulae, (E) Basidiospores in KOH mixed with 1% phloxine, (F) Basidospores with one or two guttulae in KOH.

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Diagnosis

Phylloporia beninensis differs from other known species of Phylloporia by the combination of the following characteristics: basidiomata imbricate; pileus projecting up to 3 cm, 5 cm wide, and 1 cm thick at base, surface concentrically sulcate and zonate; cystidioles present, variable in size and shape; basidia 9–12 × 4–5 μm; basidiospores ellipsoid to subglobose, 3–4.6 × 2.1–3.6 μm.

Holotype

BENIN. Borgou province, Woodlands of Okpara/Parakou, 9° 15′ 36.62″ N, 2° 43′ 28.40″ E, altitude 331 m.a.s.l., on dead stump of an unidentified angiosperm tree., leg. Boris A. Olou, sampling date: 11.09.2019, OAB0511 (dried specimen, holotype in UNIPAR and isotype in KAS). Holotype sequences: LSU, accession number: MW244096.

Etymology

beninensis (lat.): referring to the country of the type locality.

Description

Basidiomata annual, pileate, sessile, imbricate with overlapping pilei, broadly attached or effused-reflexed (Fig. 4a–c), hard when dried, without odour or taste, projecting up to 3 cm, 5 cm wide, and 1 cm thick at the base. Pileus applanate to slightly convex, surface mustard and ferruginous brown in young or actively growing specimens and almost blackish in old specimens, velvety under stereomicroscope; surface concentrically sulcate and zonate; margin undulate, obtuse, yellowish when young or in actively growing specimens (Fig. 4c), concolorous with the pileus at maturity. Pore surface buff-yellow to honey, not shining or at least in the dried specimens, pore very small, 7–9 per mm, isodiametric to angular (Fig. 4d). Context two-layered, with a black line separating the upper context (tomentum) from the lower context, mustard brown, tomentum softer and lighter coloured than the lower context, tomentum up to 5 mm thick at the base and in the middle and thinner toward the margin, lower context up to 2 mm thick at the base and thinner at the margin (Fig. 4e). Tube layer concolorous with pore surface, up to 2 mm long.

Hyphal system dimitic (Fig. 5), skeletal hyphae of tomentum golden yellow in water, darker in KOH, thick-walled, unbranched, simple septate, interwoven, 3–6 μm in diam. (Fig. 6a). Skeletal hyphae in the lower context golden yellow in water, darker in KOH, thick-walled, unbranched, septate, 3–4 μm in diam., slightly interwoven. Trama with generative hyphae (Figs. 5, 6c); these hyaline, thin to thick-walled, occasionally branched, frequently simple septate, without clamp, 2–3 μm in diam. (Fig. 6b); skeletal hyphae abundant, dominating the trama, unbranched, septate, 3–4.5 μm in diam., thick-walled, wall thickness up to 1 μm, slightly interwoven to partially arranged.

Basidiospores normally abundant, smooth, with one or two guttulae, ellipsoid to subglobose, thin- to thick-walled, yellow–brown, hilar appendix nearly inevident (Figs. 5, 6e,f), inamyloid, acyanophilous, (3–)3.3–4.3(–4.6) × (2.1–)2.4–3.3(–3.6) µm, L = 3.8 μm, W = 2.8 μm, Q = 1.08–1.6 (n = 1088/1). Basidia tetrasterigmate; sterigmata up to 2.3 μm long, hyaline, clavate, 9–12 × 4–5 μm, with several guttulae; basidioles abundant, similar in shape to basidia, 9–11 × 4–6 μm, with several guttulae (Figs. 5, 6c,d). Cystidioles frequent, variable in size and shape.

Ecology and distribution

On deadwood or dead parts of living trees of woody angiosperms, including Trichilia emetica Vahl. Currently known from the type locality and other localities of Benin.

Additional materials examined

BENIN. Collines province, woodlands of Kilibo/Ouèssè, leg. Boris A. Olou, on dead wood stump of T. emetica, 17.08.2017, 8° 32′ 36.39″ N, 2° 41′ 12.80″ E, altitude 312 m.a.s.l., OAB0107 (UNIPAR); Borgou province, Ouémé Supérieur reserve forest, on dead part of an unidentified angiosperm living tree, 9° 45′ 29.09″ N, 2° 19′ 58.78″ E, altitude 334 m.a.s.l., 24.08.2017, OAB0142 (KAS).

Discussion

Phylogenetic analyses inferred from the LSU and ITS-LSU datasets, coupled with macro- and microscopic examinations and ecological analyses, support the recognition of P. beninensis as a new species. Phylloporia beninensis is morphologically distinguished from other Phylloporia species by its annual, sessile, pileate, and imbricate basidiomata, two-layered context with the layers separated by a black line, dimitic hyphal system, and presence of cystidioles that vary in size and shape.

Phylloporia beninensis is macroscopically most similar to P. rattanicola F. Wu, G.J. Ren & Y.C. Dai; the two species share the pileate and imbricate basidiomata, velutinous pileus surface, two-layered context separated by a black line, presence of cystidioles, and dimitic hyphal system8. Phylloporia rattanicola differs from P. beninensis in its perennial basidiomata; smaller pores (9–11 per mm), and cyanophilic basidiospores8. Phylloporia minutipora L.W. Zhou is also similar in its annual, sessile basidioma with velutinate pileus surface, duplex context, and a dimitic hyphal system41. However, P. minutipora can be easily differentiated from P. beninensis by its much smaller pore size (12–15 per mm), larger basidiomata that project up to 10 cm from the substratum, absence of cystidioles, and smaller basidiospores 2.5–3 × 1.5–2.5 μm41. In addition to these morphological differences, P. beninensis clustered in a strongly supported and distinct lineage within Phylloporia clade in our molecular phylogenetic analyses (Figs. 2, 3). In these analyses P. beninensis has a strong phylogenetic relationship (PP = 1.00, BS = 95%) with an unidentified species of Phylloporia from Kenya (MUCLKE 16107, GenBank KY349147)17 and is phylogenetically distant from P. rattanicola and P. minutipora.

We cannot yet confirm whether or not P. beninensis is saprotrophic even though it was mainly found on dead wood (Fig. 4a,b), because it is well evidenced, that the habit of a fungus to produce fruit body on dead wood does not necessarily indicate a saprotrophic lifestyle42. However, although the lifestyle of P. beninensis is not yet well known, the fact that it was mainly found on dead wood we can reasonably say that the latter is saprotroph. As saprotroph, P. beninensis is therefore ecologically different from P. minutipora and Phylloporia sp., which are mainly collected from living trees17, 41. Like P. beninensis, P. rattanicola is also saprotrophic because it was collected from dead rattan8. However, knowing that Phylloporia species display a high level of host specificity7, 10, 15, 43, and that P. rattanicola is only collected on rattan while P. beninensis is collected on hardwood, we can safely say that P. beninensis and P. rattanicola do not belong to the same morpho- ecological group as stated above.

We also reported here P. littoralis Decock & Yombiyeni on the basis of molecular and morphological analyses, constituting the first record of the species from Benin (Figs. 2, 3, 7). The Benin P. littoralis specimen fits well morphologically and genetically to the Central African type specimen (see Fig. 2, in Yombiyeni and Decock 2017). To our knowledge, this is the first time P. littoralis has been reported outside its type locality Gabon, and suggests that the species may be more widely distributed in sub-Saharan Africa.

Figure 7
figure 7

Field photos of Phylloporia littoralis (OAB0204). (A) Basidioma attached to a branch of…, (B) Pileus surface and hymenophore.

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The recognition of P. beninensis brings the number of described Phylloporia species to 62 worldwide. Among these 62 species, nine were described from tropical Africa9, 12, 17, 18, 23. Phylloporia are more diverse in tropical Africa in comparison with Europe, where only P. ribis (Schumach.) Ryvarden has been reported4 to date. Considering that tropical Africa remains poorly explored for wood-decay fungi, it is likely that many more Phylloporia species remain to be found. We are also confident that new investigations of new still unexplored habitats and re-examination of herbarium specimens initially assigned to the genus Phellinus will reveal more new species of Phylloporia from tropical Africa. Aside the nine species described with type specimens, six other Phylloporia species have been reported from tropical Africa2, 7, which brings the number of regional Phylloporia species to 15. To facilitate future taxonomic studies in the genus, we provide a dichotomous key for identification of tropical African Phylloporia species.

Identification key to African Phylloporia species

  1. 1.

    Basidiomata resupinate on the underside of living leaves…P. parasitica

  2. 1.

    Basidiomata sessile to stipitate…2

  3. 2.

    Basidiomata stipitate…3

  4. 2.

    Basidiomata sessile…5

  5. 3.

    Context homogenous, black line lacking…P. minutispora

  6. 3.

    Context duplex, black line present…4

  7. 4.

    Pores 7–9 per mm…P. spathulata

  8. 4.

    Pores 10–11 per mm…P. afrospathulata

  9. 5.

    Perennial, pore surface glancing…P. pectinata

  10. 5.

    Annual, pore surface not glancing…6

  11. 6.

    Basidiomata gregarious…7

  12. 6.

    Basidiomata solitary to imbricate…9

  13. 7.

    Clustered in more than 100 individuals, pileus shiny…P. flabelliformis

  14. 7.

    Clustered in a small groups of less than 100 individuals, pileus dull…8

  15. 8.

    Hyphal system monomitic, pores 5–6 per mm…P. gabonensis

  16. 8.

    Hyphal system dimitic, pores 9–11 per mm…P. fulva

  17. 9.

    Cystidioles present…10

  18. 9.

    Cystidioles absent…11

  19. 10.

    Cystidioles fusoid, pores sinuous to subdaedaleoid, (1.5–) 2–3 per mm…P. inonotoides

  20. 10.

    Cystidioles variable in shape and size and up to 30 μm long, pores round to angular, 7–9 per mm…P. beninensis

  21. 11.

    On living trees and bushes…12

  22. 11.

    On dead and Q3living trees…14

  23. 12.

    Host specific, found on species of Rinorea (Violaceae)…P. rinoreae

  24. 12.

    Not host specific…13

  25. 13.

    Basidioma 0.5–3 cm in diam., 0.5–1 cm thick, basidia 8.5 × 5 µm…P. littoralis

  26. 13.

    Basidioma 1–5 cm in diam., up to 2 cm thick, basidia 8–10 × 3–4 µm…P. fruticum

  27. 14.

    Basidiospores 3–4.5 × 2.5–3.5 µm…P. weberiana

  28. 14.

    Basidiospores 2.5–3.5 × 2–2.5 μm…P. pulla