The genus Trametes (Polyporales, Basidiomycota) consists of wood-decay Fungi with a distribution covering all continents and all major climatic zones. Species of Trametes are characterised by a combination of a pileate basidioma, a poroid hymenophore, a trimitic hyphal system, and non-amyloid, thin-walled basidiospores. They are saprotrophs causing white rot during the decay of woody substrates. Species of the genus Trametes have a long ethnomycological history as medicinal Fungi in many cultures and some species are studied in the context of cancer research. Despite the global-scale distribution, importance for wood decomposition, and medicinal properties, the taxonomic and phylogenetic knowledge of Trametes spp. worldwide is still incomplete.
Since the first formal description of the genus Trametes by Elias Fries in 1835, based on the type species Trametes suaveolens, the concept of this genus was interpreted in different ways, resulting in different numbers of species attributed to the genus. Recently, based on phylogenetic analyses, the concept of Trametes was re-delimited and circumscribed. This concept includes in addition to species of Trametes sensu stricto, species of Artolenzites, Coriolopsis, Lenzites, and Pycnoporus.
Previous studies on Trametes spp. have mainly concentrated on specimens from temperate and boreal regions, and thus most Trametes spp. have been described from these regions. By contrast, little is known on Trametes spp. in tropical Africa, and most known specimens of Trametes spp. from this area are missing in most phylogenetic analyses.
For Benin, seven species of Trametes, namely Trametes cingulata, Trametes elegans, Trametes flavida, Trametes polyzona, Trametes sanguinea, and Trametes socotrana were reported by a 2019 study. Taking a closer look at these species, it was noticed that sequence data are lacking for specimens from tropical Africa and that the knowledge on taxonomical and phylogenetic placements is incomplete.
Additional to these known species in Benin, a putative new species of Trametes was recently discovered, although morphological and phylogenetic analyses are outstanding. In the same study, the occurrence of Trametes elegans was reported in Benin.
Trametes elegans has beenfound to be a species complex and has therefore recently been split into three distinct species, namely Trametes aesculi, Trametes elegans senso stricto, and Trametes repanda. However, this study did not include tropical African specimens although Trametes elegans exists in this area.
In a paper published in the journal MycoKeys on 10 March 2020, Boris Armel Olou of the Department of Ecology at Universität Kassel, the Research Unit Tropical Mycology and Plant-Soil Fungi Interactions at the University of Parakou, and the Laboratory of Applied Ecology at the University of Abomey-Calavi, Franz-Sebastian Krah of the Department of Ecology at Philipps-Universität Marburg, and the Bavarian Forest National Park, Meike Piepenbring of the Department of Mycology at Goethe Universität, Nourou Soulemane Yorou, also of the Research Unit Tropical Mycology and Plant-Soil Fungi Interactions at the University of Parakou, and Ewald Langer, also of the Department of Ecology at Universität Kassel, report on the diversity of Trametes species in Benin and their phylogenetic positions, with a focus on a new species of Trametes and the Trametes elegans species complex.
A total of 37 specimens of Trametes were collected in three different macroclimatic zones and different forests of Benin from July to September in 2017 and in 2018 (another series of surveys). Small pieces of fresh fruit bodies were placed in plastic bags half-filled with silica gel for DNA extraction. The rest of fruit bodies were air- or oven-dried at 45–50°C for 1–2 days depending on the consistency of the fruit body. The dried fruit bodies were then preserved in plastic bags for morphological investigation. Specimens are deposited at the mycological herbaria of the University of Parakou and the University of Kassel.
Genomic DNA of all specimens classified into nine morphotypes was extracted using the microwave DNA extraction method or the NucleoSpin Plant II DNA extraction kit.
The extracted genomic DNA was amplified targeting two nuclear ribosomal DNA regions, internal transcribed spacer and ribosomal large subunit-coding DNA (28S ribosomal RNA) for all specimens. Additionally, three protein-coding genes, RNA polymerase II largest subunit, RNA polymerase II second largest subunit, and translation elongation factor 1-alpha were amplified for specimens forming part of the Trametes elegans species complex and specimens of Trametes sp. The amplification of the 5.8S ribosomal RNA gene region, including internal transcribed spacer were performed in Mastercycler nexus gradient using the primer pair internal transcribed spacer-1F/internal transcribed spacer4. The Polymerase Chain Reaction procedure for the internal transcribed spacer region, was as follows: initial denaturation at 95°C for 3 minutes, followed by 35 cycles at 95°C for 30 seconds, 52°C for 30 seconds and 68°C for 1 minute, and a final extension at 68°C for 3 minutes. Amplifications of Large Subunit and three protein-coding genes were performed in a 96-well TGradient Thermocycler. Polymerase Chain Reaction procedure for amplifying partial Large Subunit ribosomal DNA using the primer pair LR0R/LR5 approximately 964 base pairs differed to the internal transcribed spacer only by the annealing temperature (55°C instead of 52°C) and increased cycle extension time (90 seconds per cycle). The primer pairs elongation factor1-983F/elongation factor1-1567R, RNA polymerase II largest subunit-Af/RNA polymerase II largest subunit-Cr, and RNA polymerase II second largest subunit-b6F/RPB2-b7.1R were used to amplify approximately 500 base pairs of translation elongation factor 1, 1000 base pairs of RNA polymerase II largest subunit, and 800 base pairs of RNA polymerase II second largest subunit. To amplify the protein-coding genes RNA polymerase II largest subunit and RNA polymerase II second largest subunit, the touchdown Polymerase Chain Reaction protocol was used. Polymerase Chain Reaction products were checked on 1% agarose gel stained with GelRed fluorescence dye in the Transilluminator Biometra Ti5 equipped with BioDocAnalyze software. They were further cleaned up with QIAquick PCR Purification Kit according to manufacturer’s instructions. Thereafter, Sanger sequenced at GATC Biotech in Germany.
At least one sequence per specimen was generated for each morphotype except for the morphotype named Trametes aff. versicolor. All newly generated sequences composed of 25 internal transcribed spacer, 20 Large Subunit, two RNA polymerase II largest subunit, four RNA polymerase II second largest subunit, and three translation elongation factor 1 were deposited in GenBank.
To place all the 25 generated internal transcribed spacer sequences of specimens of Trametes spp. in a phylogenetic context, Olou et al. aligned them in addition to 66 internal transcribed spacer sequences retrieved from GenBank. Further, 48 Large Subunit sequences were aligned with 20 Large Subunit sequences generated here. For the Trametes elegans species complex, seven newly generated sequences of protein-coding genes were aligned in addition to sequences used by Alexis Carlson, Alfredo Justo, and David Hibbett to establish that Trametes elegans was a species complex rather than a single species in 2014. Each marker was aligned separately using MAFFT version 7, with the algorithm L-INS-i and standard settings as default. The resulting multiple species alignments were slightly adjusted and trimmed at both ends a bit from incomplete sequences in Geneious 5.6.7. Eight different datasets were assembled for the phylogenetic analyses: (i) An internal transcribed spacer dataset with 91 sequences of Trametes spp., (ii) a combined internal transcribed spacer/Large Subunit dataset with 91 sequences Trametes spp., (iii) a combined RNA polymerase II largest subunit-RNA polymerase II second largest subunit dataset with 23 sequences of Trametes spp., (iv) an internal transcribed spacer dataset with 17 sequences of the Trametes elegans species complex, (v) a RNA polymerase II largest subunit dataset with ten sequences of the Trametes elegans species complex, (vi) a RNA polymerase II second largest subunit dataset with 12 sequences of the Trametes elegans species complex, (vii) a translation elongation factor 1-alpha dataset with 14 sequences of the Trametes elegans species complex, and (viii) combined dataset of four genes (internal transcribed spacer, RNA polymerase II largest subunit, RNA polymerase II second largest subunit, translation elongation factor 1-alpha) of the Trametes elegans species complex. The combined datasets were concatenated using Geneious 5.6.7. For the phylogenetic analyses, the partitioning of the combined datasets of Trametes spp. was considered. Lopharia cinerascens and Dentocorticium sulphurellum were chosen as the outgroup in all datasets. Two phylogenetic tree inference methods, Maximum Likelihood and Bayesian analyses were performed in each dataset. The Maximum Likelihood of all datasets were performed using RAxML 8.2.10 and the Bayesian analysis of all individual genes and combined dataset of Trametes elegans species complex were performed using MrBayes 3.2.6 at the Cipres Science Gateway V.3.3. The Bayesian analysis of the partitioned datasets of Trametes spp. were run independently using MrBayes 3.2.7. Two independent Markov Chain Monte Carlo processes were run, each in 4 chains, for 5 million generations, and 0.2 fraction were discarded as burn-in. The Phylogenetic Tree Summarization program within DendroPy 4.3.0. was used to build the consensus tree with branch supports (posterior probabilities). Further, by using IQ-Tree, Olou et al. assigned the bootstrap values of the Maximum Likelihood analysis to the consensus tree of the Bayesian analysis. The resulting phylogenetic trees were inspected in FigTree v. 1.4.2. All sequence alignments and phylogenetic trees generated in the study were deposited in TreeBASE.
Macro-morphological descriptions were based on fresh and dried herbarium specimens. Microstructures are described using dried herbarium specimens. Fine sections through the basidiomata were prepared for observation using a razor blade under a stereomicroscope Leica EZ4 and mounted in 5% aqueous solution of potassium hydroxide mixed with 1% aqueous solution of Phloxine. Melzer’s reagent (to test for dextrinoid or amyloid reactions), Cotton Blue (to test for cyanophilic reaction) were used and then examined at a magnification of 1000× using a Leica DM500 light microscope. Measurements were done with the software 'Makroaufmaßprogramm' from Jens Rüdigs and analysed with the software 'Smaff' version 3.2. In total, 135 basidiospores were measured from the sequenced specimen OAB0022 and additional examined specimen OAB0268.
The 25 internal transcribed spacer sequences obtained from Trametes spp. from Benin clustered in eight distinct clades. All sequences of Trametes spp. from Benin fell into the monophyletic corresponding clades except the clade of Trametes lactinea, which, besides sequences of Trametes lactinea, accommodated also sequences of Trametes cubensis with a very high support. Sequences of specimens of Trametes sp. (OAB0022 and OAB0023) from Benin formed a separated and well-supported clade within the Trametes clade.
Results of Maximum Likelihood and Bayesian analyses show higher congruency, higher support values, and a higher number of resolved nodes than the results obtained with internal transcribed spacer sequence data only. As evident by the internal transcribed spacer sequence dataset, the sequence of Trametes lactinea from Benin clustered in addition to other sequences of Trametes lactinea retrieved from GenBank with sequences of Trametes cubensis with high support. Like in the analysis of the internal transcribed spacer sequence dataset, sequences of Trametes sp. from Benin formed a distinct clade. The two sequences of the new species of Trametes from Benin clustered in a distinct lineage within the Trametes clade.
The phylogenetic trees generated from individual gene regions internal transcribed spacer, RNA polymerase II largest subunit, RNA polymerase II second largest subunit, translation elongation factor 1-alpha and the combined datasets show similar results for phylogenetic relationships within the Trametes elegans species complex. Four distinct and well-supported clades were evident in all datasets. The clade highlighted in grey is distinct from all other clades within Trametes elegans species complex and highly supported in all individual gene and combined dataset. This clade contains only sequences of Trametes elegans from Benin and Cameroon.
The new species identified by Olou et al. is named Trametes parvispora, where 'parvispora' refers to the small size of the spores. The species is described from two specimens, both collected from the dead part of a living Velvet Tamarind tree, Dialium guineense, in the dry dense forest of Pahou in Ouidah, in Atlantic Province, Benin.
Trametes parvispora differs from known species of Trametes in the combination of the following characteristics: daedaleoid hymenophore, context whitish, thin 1–1.5 mm, homogeneous, without black lines, small spores 3.2–4.6 × 2.1–2.8 μm, regular hyphal pegs 25–30 μm long, cystidia absent, abundance of basidioles, and basidia 12–15 × 3–5 μm.
Basidiomata probably perennial, sessile, pileate, applanate, semicircular, up to 13 cm long and 8 cm wide, up to 2.5 cm thick at the base, coriaceous to woody and hard when dry, without odour or taste when fresh. Pileus surface dull, glabrous, and whitish, zonate, margin thick, obtuse. Pore surface whitish, daedaleoid. Context whitish, thin (1–1.5 mm), homogeneous, without black lines.
Hyphal system trimitic, generative hyphae hyaline branched with clamp connections, thin-walled, 1.5–2.0 μm in diameter, acyanophilous; skeletal hyphae solid to thick-walled, hyaline, non-septate, 3–4 μm in diameter, totally dominating in the context, acyanophilous, tissues unchanged in potassium hydroxide, unbranched; binding hyphae very common in both the context and trama, hyaline, thick-walled, acyanophilous, and much branched.
Cystidia absent, but the branches of the binding hyphae may easily be mistaken for thick-walled cystidia in the hymenium unless a careful examination is undertaken. Hyphal pegs present, especially at the base of pores, and regular, 25–30 μm long.
Basidia 12–15 × 3–5 μm, clavate, tetrasterigmatic, sterigmata 3 μm long; Basidioles numerous, similar in shape to basidia but slightly smaller than basidia, up to 4 μm in diameter.
Basidiospores broadly ellipsoid, hyaline, thin-walled, smooth, usually with one guttule each, negative in Melzer’s reagent, acyanophilous.
In Benin, seven species of Trametes were previously reported up until 2019. in 2019 two additional species, namely Trametes lactinea and Trametes aff. versicolor, were recorded in addition to previous species. Thus, to Olou et al.'s knowledge, nine species of Trametes are currently known for Benin. Of these nine species, only two species, Trametes elegans and Trametes sanguinea, were reported in Benin prior to 2002. The remaining seven species, namely Trametes cingulata, Trametes flavida, Trametes lactinea, Trametes parvispora, Trametes polyzona, Trametes socotrana, and Trametes aff. versicolor, were recorded between 2017 and 2018. Given this history, it is most likely that more species will be found. Nonetheless, this number is significant when compared to the total diversity of 9–14 species of Trametes reported for Europe. Further studies are needed to document the overall diversity of species of Trametes in Benin.
To place specimens of Trametes spp. from Benin in a larger phylogenetic context, Olou et al. generated sequences of several genes. Generated sequences were placed into the phylogeny of the genus Trametes as established by Alfredo Justo and David Hibbett. Eight distinct clades corresponding to eight different species were obtained from these sequences.
Olou et al.'s phylogenetic analyses from internal transcribed spacer sequences and combined internal transcribed spacer sequence-Large Subunit datasets reveal sequence similarities and taxonomic misplacement within the clades of Trametes flavida and Trametes lactinea. The clade of Trametes flavida accommodated, in addition to sequences of Trametes flavida, sequences of Trametes sp. from French Guiana which is known as Leiotrametes sp.. This species was proposed as a new species by in 2012. In Olou et al.'s study Trametes sp. clustered together with Trametes flavida with high support in the internal transcribed spacer sequence dataset and combined internal transcribed spacer sequence-Large Subunit datasets. Both species share also high morphological similarity and a tropical distribution. Olou et al. therefore suggest that Trametes sp. from French Guiana should not be considered as a new species but should be referred to as Trametes flavida. In addition to the Trametes flavida clade, Olou et al.'s phylogenetic analyses showed that the Trametes lactinea clade contains not only sequences of Trametes lactinea, but also sequences of Trametes cubensis with high support in the internal transcribed spacer sequences and combined internal transcribed spacer sequence-Large Subunit datasets. This result is similar to previous phylogenetic analyses on Trametes using the internal transcribed spacer sequence marker. Trametes lactinea and Trametes cubensis are still valid names and both species share quite similar morphological characters. They are characterised by an applanate, broadly attached to dimidiate, white to cream basidiomata and a white to cream pore surface. Nevertheless, although both species are sharing quite similar morphological characters, they also differ in some characters. Trametes cubensis is characterized by an annual basidioma, small pores, almost invisible to the naked eye, 5–7 per mm, and cylindrical basidiospores 7–9 × 3–3.5 μm, while Trametes lactinea has an annual to perennial basidioma and large pores, which are visible to the naked eye, mostly 1.5–2 per mm, but can reach up to 3–4 (5) per mm in some specimens with cylindrical-ellipsoid basidiospores 4–7.5 × 2.2–3 μm. Olou et al.'s specimen of Trametes lactinea matches the morphological description of Trametes lactinea with 3–4 pores per mm, but Olou et al. did not observe any spore despite numerous attempts. Thus, considering the result of their phylogenetic analyses, absence of spores in our Trametes lactinea specimen, and the high morphological similarity between species within Trametes, Olou et al. cannot reasonably distinguish Trametes lactinea from Trametes cubensis. Further morphological, chemotaxonomic, and molecular studies integrating proteins coding genes (e.g. RNA polymerase II largest subunit, RNA polymerase II second largest subunit, and translation elongation factor 1-alpha) are therefore needed to confirm whether Trametes lactinea from Trametes cubensis refer to the same species.
Previously the phylogenetic resolution of Trametes cingulata was problematic due to low sequence availability. Olou et al. generated a total of 17 de novo sequences and show that Trametes cingulata appears as a monophyletic group within Trametes with high support in internal transcribed spacer sequence and combined internal transcribed spacer sequence-Large Subunit datasets. Thus, contrary to the uncertain position of Trametes cingulata within the genus Tramete, Olou et al.'s results revealed that the latter does not belong to Trametes sensu stricto in the sense of previous studies, but rather to Trametes sensu lato.
The specimens from Benin identified as members of the Trametes elegans species complex correspond to the morphological descriptions of Trametes elegans in previous studies. The clades evident in all datasets within the Trametes elegans complex represent three clades previously attributed to three different species by Alexis Carlson, Alfredo Justo, and David Hibbett, and a new clade represents specimens of Trametes elegans from Benin and Cameroon (Tropical Africa). This new clade contains only sequences of Trametes elegans from Benin and Cameroon due to the non-publication of most Trametes elegans sequences from tropical Africa. Thus, prior to this study, only sequences of Trametes elegans from Cameroon and Gabon are available in GenBank for Africa. However, the sequences of Trametes elegans from Gabon (GenBank accession number: KY449397, and KY449398) were not considered because they fell outside the Trametes elegans species complex and were instead closely related to Trametes lactinea. Olou et al., therefore, excluded these sequences from their analyses. All in all, since the sequences of Trametes elegans from tropical Africa investigated in Olou et al.'s study are demarcated from sequences of Trametes elegans sensu stricto, the adoption of another correct name for specimens of Trametes elegans from this area is necessary.
Specimens belonging to the Trametes elegans species complex have been reported in the past for tropical African countries, with the first name applied to such specimens being Daedalea amanitoides, which was based on a specimen from Nigeria (cited as kingdom of Oware by Ambroise-Marie-François-Joseph Palisot de Beauvois in 1804). The morphological characteristics evident in the very short description and illustration of a fruiting body of Daedalea amanitoides match the characteristics of the specimens examined in this study. However, for reasons that Olou et al. ignore, Elias Fries replaced this name (Daedalea amanitoides) with the name Daedalea palisotii in 1821, which is sanctioned and therefore must be used. The combination Trametes palisotii erected by Rokuya Imazeki in 1952 is available and must be used for African specimens known previously as Trametes elegans.
The sequences belonging to the new species named Trametes parvispora form a distinct and well-supported clade in the internal transcribed spacer sequence and the combined internal transcribed spacer sequence-Large Subunit datasets. This species forms a sister clade with the still formally undescribed Trametes sp. (KT896651) from Finland. However, unlike Trametes parvispora where fruiting bodies were available for morphological characterisation, the Finnish specimen was isolated as mycelium from the Bark Beetle Ips typographus. Thus, anatomical and morphological comparisons are currently not possible. Furthermore, both sequences of Trametes parvispora share a clade with Trametes meyenii. This clade was confirmed by phylogenetic analyses including two additional markers RNA polymerase II largest subunit, and RNA polymerase II second largest subunit. Trametes meyenii has hispid and cream-yellow pilei, irpicoid and white to ochraceous hymenophore, pores 1–3 per mm, 4.5–6 × 2–2.5 μm basidiospores, whereas Trametes parvispora has glabrous and whitish pilei, a daedaleoid and white hymenophore, 3.2–4.6 × 2.1–2.8 μm basidiospores, and the presence of regular hyphal pegs. These morphological differences confirm that Trametes parvispora and Trametes meyenii are distinct species as shown by the phylogenetic analyses. However, some species lacking DNA sequences, namely Trametes barbulata, Trametes daedaleoides, and Trametes rugosituba, share with Trametes parvispora a quite similar spore size range. But the latter species differs from each other species by the combination of macro- and microscopic characteristics outlined above. Thus, the rare anatomic features of the regular hyphal pegs and the small size of the basidiospores together with the phylogenetic placement within the Trametes clade, provide enough evidence for Trametes parvispora as a distinct new species.
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