Diversity of Trametes (Polyporales, Basidiomycota) in tropical Benin and description of new species Trametes parvispora

Abstract Trametes is a globally distributed genus of white-rot polypores and well sampled in temperate and boreal areas. However, the diversity, taxonomy, and phylogenetic positions of Trametes spp. are poorly known in tropical Africa. This study aims at documenting the diversity of Trametes species in Benin (tropical Africa) and their phylogenetic positions with a focus on the T. elegans species complex. Therefore, we collected specimens of Trametes from different forest types across Benin. To infer phylogenetic relationships between Trametes species, we investigated sequences of five gene regions and added available sequences from GenBank. Using Maximum likelihood and Bayesian phylogeny inference methods, we found eight supported species clades. For the T. elegans species complex, we re-establish the name Trametes palisotii for species previously known as T. elegans in tropical Africa. Furthermore, we propose Trametes parvispora as a species new to science and provide the description of this species. Our molecular phylogeny of Trametes with a focus on tropical Benin contributes to taxonomic clarity of an important wood-decay fungal genus, which is the basis for biodiversity assessments of Trametes in the tropics.


Introduction
The genus Trametes Fr. (Polyporales, Basidiomycota) consists of wood-decay fungi with a distribution covering all continents and all major climatic zones (Gilbertson and Ryvarden 1987;Ryvarden 1991). Species of Trametes are characterized by a combination of a pileate basidioma, a poroid hymenophore, a trimitic hyphal system, and non-amyloid, thin-walled basidiospores (Gilbertson and Ryvarden 1987). They are saprotrophs causing white rot during the decay of woody substrates (Wong and Wilkes 1988). Species of the genus Trametes have a long ethnomycological history as medicinal fungi in many cultures (Cui et al. 2011;Ss and Pandey 2012;Ueitele et al. 2017) and some species are studied in the context of cancer research (Zmitrovich et al. 2012;Cruz et al. 2016;Blagodatski et al. 2018). Despite the global-scale distribution, importance for wood decomposition, and medicinal properties, the taxonomic and phylogenetic knowledge of Trametes spp. worldwide is still incomplete (Carlson et al. 2014).
Since the first formal description of the genus Trametes by Fries (1835), based on the type species Trametes suaveolens (L.) Fr., the concept of this genus was interpreted in different ways, resulting in different numbers of species attributed to the genus (Karsten 1881;Murrill 1905;Kavina and Pilát 1936;Kotlaba and Pouzar 1957;Gilbertson and Ryvarden 1987;Corner 1989). Recently, based on phylogenetic analyses, the concept of Trametes was re-delimited and circumscribed (Justo and Hibbett 2011). Here, we apply the broad concept of Trametes as proposed by Justo and Hibbett (2011). This concept includes in addition to species of Trametes sensu stricto, species of Artolenzites Falck, Coriolopsis Murrill, Lenzites Fr., and Pycnoporus P. Karst.
Previous studies on Trametes spp. mainly concentrated on specimens from temperate and boreal regions (David 1967;Gilbertson and Ryvarden 1987;Hattori 2005;Tomšovský et al. 2006;Pieri and Rivoire 2007;Ryvarden et al. 2009;Gomes-Silva et al. 2010;Hattori and Sotome 2013), and thus most Trametes spp. have been described from these regions. By contrast, little is known on Trametes spp. in tropical Africa (Fig. 1A), and most known specimens of Trametes spp. from this area are missing in most phylogenetic analyses.
Additional to these known species in Benin, we recently found a putatively new species of Trametes (Olou et al. 2019), but morphological and phylogenetic analyses were outstanding. In the same study, we reported the occurrence of T. elegans in Benin.
Trametes elegans was found to be a species complex and has therefore recently been split into three distinct species, namely T. aesculi (Fr.) Justo, T. elegans s.str., and T. repanda (Pers.) Justo (Carlson et al. 2014). However, this study did not include tropical African specimens although T. elegans exists in this area.  Our study thus aims to report the diversity of Trametes species in Benin and their phylogenetic positions, with a focus on a new species of Trametes and the T. elegans species complex.

Specimens sampling and preservation
A total of 37 specimens of Trametes were collected in three different macroclimatic zones and different forests of Benin (Fig. 1A, C) from July to September in 2017 (Olou et al. 2019) 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 (UNIPAR; Thiers 2019) and the University of Kassel (KAS). The rows referring to sequences generated in this study are written in bold.
T. elegans species complex, seven newly generated sequences of protein-coding genes were aligned in addition to sequences used by Carlson et al. (2014). Each marker was aligned separately using MAFFT version 7, with the algorithm L-INS-i (Katoh et al. 2017) 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 (Kearse et al. 2012). Eight different datasets were assembled for the phylogenetic analyses: (i) ITS dataset with 91 sequences of Trametes spp., (ii) combined ITS-LSU dataset with 91 sequences Trametes spp., (iii) combined RPB1-RPB2 dataset with 23 sequences of Trametes spp., (iv) ITS dataset with 17 sequences of T. elegans species complex, (v) RPB1 dataset with ten sequences of the T. elegans species complex, (vi) RPB2 dataset with 12 sequences of T. elegans species complex, (vii) TEF1 dataset with 14 sequences of T. elegans species complex, and (viii) combined dataset of four genes (ITS, RPB1, RPB2, TEF1) of T. elegans species complex. The combined datasets were concatenated using Geneious 5.6.7 (Kearse et al. 2012). For the phylogenetic analyses, the partitioning of the combined datasets of Trametes spp. was considered. Lopharia cinerascens (Schwein.) G. Cunn., and Dentocorticium sulphurellum (Peck) M.J. Larsen & Gilb., were chosen as the outgroup in all datasets (Justo and Hibbett 2011). Two phylogenetic tree inference methods, Maximum likelihood (ML) and Bayesian (BY) were performed in each dataset. The ML of all datasets were performed using RAxML 8.2.10 (Stamatakis 2014) and the BY of all individual genes and combined dataset of T. elegans species complex were performed using MrBayes 3.2.6 (Ronquist et al. 2012) at the Cipres Science Gateway V.3.3. (Miller et al. 2010). The BY of the partitioned datasets of Trametes spp. were run independently using MrBayes 3.2.7 (Ronquist et al. 2012). The parameters in BY inference were set as follows: lset applyto = (all), nst = 6, rates = invgamma, ngammacat = 4, sampling frequency = 1000, and the command "unlink" was used to unlink parameters across characters on partitioned datasets. Two independent Markov Chain Monte Carlo (MCMC) processes were run, each in 4 chains, for 5 million generations, and 0.2 fraction were discarded as burn-in. The Phylogenetic Tree Summarization (SumTrees) program within DendroPy 4.3.0. (Sukumaran and Holder 2010) was used to build the consensus tree with branch supports (posterior probabilities). Further, by using IQ-Tree (Trifinopoulos et al. 2016), we assigned the bootstrap values (BS) of ML to the consensus tree of BY. The resulting phyloge-

Microscopic analyses of specimens of the new species of Trametes
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 (KOH) 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 (https:// ruedig.de/tmp/messprogramm.htm) and analysed with the software "Smaff" version 3.2 (Wilk 2012). In total, 135 basidiospores were measured from the sequenced specimen OAB0022 and additional examined specimen OAB0268. The basidiospore size is given as length and width of the spore. As measurements we present the mean with standard deviation and minimum and maximum values in parentheses (see below). The length (L), arithmetic average of all spore lengths, and the width (W), arithmetic average of all spore widths, were calculated. In addition, the ratio of length/width (Q) was calculated.

Availability of data and materials
All alignments and phylogenetic trees generated in this study are available in TreeBASE under this link: http://purl.org/phylo/treebase/phylows/study/TB2:S24354. Newly generated sequences are available in GenBank, and the accession numbers are given in Table 1. Alignments, phylogenetic trees, and accession numbers of newly generated sequences will be public after the paper is published. Collected specimens are available at the mycological herbarium of the University of Parakou (UNIPAR). The new species was registered in mycoBank, and the registration number is given in the taxonomy section of this paper. Translation elongation factor 1-alpha UNIPAR Mycological herbarium of the University of Parakou

Results
Phylogenetic analyses of sequences of Trametes species from Benin ITS dataset. The 25 ITS sequences obtained from Trametes spp. from Benin clustered in eight distinct clades (Suppl. material 2). All sequences of Trametes spp. from Benin fell into the monophyletic corresponding clades except the clade of Trametes lactinea (Berk.) Sacc., which, besides sequences of T. lactinea, accommodated also sequences of Trametes cubensis (Mont.) Sacc. with a very high support (BP = 1.00/BS = 100). Sequences of specimens of Trametes sp. (OAB0022 and OAB0023) from Benin formed a separated and well-supported clade within the Trametes clade (BP = 0.73/BS = 66).
ITS-LSU dataset. Results of ML and of BY show higher congruency, higher support values, and a higher number of resolved nodes than the results obtained with ITS data only. As evident by the ITS dataset, the sequence of T. lactinea from Benin clustered in addition to other sequences of T. lactinea retrieved from GenBank with sequences of T. cubensis with high support (BP = 1.00/BS = 92). Like in the analysis of the ITS dataset, sequences of Trametes sp. from Benin formed a distinct clade (Fig.  3). The two sequences of the new species of Trametes from Benin clustered in a distinct lineage within the Trametes clade (Figs 2I, J; 4). The clade of the T. elegans species complex is presented in the section below.
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.
Ecology and distribution. Saprotrophic, on dead part of living tree Dialium guineense and only known from dry dense forest of Pahou in southern Benin.

Trametes spp. diversity in Benin
In Benin, seven species of Trametes were previously reported (Olou et al. 2019). By the present, study two additional species, namely T. lactinea and T. aff. versicolor (Fig. 2E, F,  N), were recorded in addition to previous species. Thus, to our knowledge, nine species of Trametes are currently known for Benin. Of these nine species, only two species, T. elegans and T. sanguinea, were reported in Benin until 2002 (Yorou and De Kesel 2002). The remaining seven species, namely T. cingulata, T. flavida, T. lactinea, T. parvispora, T. polyzona, T. socotrana, and T. 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 (Ryvarden and Gilbertson 1994;Ryvarden and Melo 2014). Further studies are needed to document the overall diversity of species of Trametes in Benin.

Phylogenetic positions of Trametes species of Benin
To place specimens of Trametes spp. from Benin in a larger phylogenetic context, we generated sequences of several genes. Generated sequences were placed into the phylogeny of the genus Trametes as established by Justo and Hibbett (2011). Eight distinct clades corresponding to eight different species were obtained from these sequences.
Our phylogenetic analyses from ITS and combined ITS-LSU datasets reveal sequence similarities and taxonomic misplacement within the clades of T. flavida and T. lactinea (Fig. 3; Suppl. material 2). The clade of T. flavida accommodated, in addition to sequences of T. flavida, sequences of Trametes sp. from French Guiana which is known as Leiotrametes sp. (Welti et al. 2012). This species was proposed as a new species by Welti et al. (2012). Here, Trametes sp. clustered together with T. flavida with high support in the ITS dataset (PP = 0.84/BS = 89) and the combined ITS-LSU datasets (PP = 0.98/BS = 99). Both species share also high morphological similarity (Welti et al. 2012; Fig. 2C, D) and a tropical distribution. We therefore suggest that Trametes sp. from French Guiana should not be considered as a new species but should be referred to as T. flavida. In addition to the T. flavida clade, our phylogenetic analyses showed that the T. lactinea clade contains not only sequences of T. lactinea, but also sequences of T. cubensis with high support in the ITS and ITS-LSU datasets ( Fig. 3; Suppl. material 2). This result is similar to previous phylogenetic analyses on Trametes using the ITS marker (Justo and Hibbett 2011;Carlson et al. 2014). Trametes lactinea and T. cubensis are still valid names and both species share quite similar morphological characters. They are characterized by an applanate, broadly attached to dimidiate, white to cream basidiomata and a white to cream pore surface (Ryvarden and Johansen 1980;Gilbertson and Ryvarden 1987). 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 (Gilbertson and Ryvarden 1987), while T. 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 (Ryvarden and Johansen 1980). Our specimen of T. lactinea (Fig. 2E, F) matches the morphological description of T. lactinea with 3-4 pores per mm, but we did not observe any spore despite numerous attempts. Thus, considering the result of our phylogenetic analyses, absence of spores in our T. lactinea specimen, and the high morphological similarity between species within Trametes (Gilbertson and Ryvarden 1987), we cannot reasonably distinguish T. lactinea from T. cubensis. Further morphological, chemotaxonomic, and molecular studies integrating proteins coding genes (e.g. RPB1, RPB2, and TEF1) are therefore needed to confirm whether T. lactinea and T. cubensis refer to the same species.
Previously the phylogenetic resolution of T. cingulata was problematic due to low sequence availability. Here we generated a total of 17 de novo sequences and show that T. cingulata appears as a monophyletic group within Trametes with high support in ITS and combined ITS-LSU datasets respectively (PP = 1.00/BS = 97) and (PP = 1.00/BS = 100) ( Fig. 3; Suppl. material 2). Thus, contrary to the uncertain position of T. cingulata within the genus Trametes (Welti et al. 2012), our results revealed that the latter does not belong to Trametes sensu stricto in the sense of Justo and Hibbett (2011) and Welti et al. (2012) (Fig. 3; Suppl. material 2) but rather to Trametes sensu lato.

Species diversity in the Trametes elegans species complex
The specimens from Benin identified as members of the T. elegans species complex correspond to the morphological descriptions of T. elegans by Gilbertson and Ryvarden (1987) and Ryvarden and Johansen (1980). The clades evident in all datasets within the T. elegans complex (Figs 3, 5; Suppl. material 2, 3) represent three clades previously attributed to three different species by Carlson et al. (2014), and a new clade highlighted in grey ( Fig. 5; Suppl. material 3) represents specimens of T. elegans from Benin and Cameroon (Tropical Africa). This new clade contains only sequences of T. elegans from Benin and Cameroon due to the non-publication of most T. elegans sequences from tropical Africa (Olusegun 2015;Awala and Oyetayo 2016;Ueitele et al. 2018). Thus, prior to this study, only sequences of T. elegans from Cameroon and Gabon are available in GenBank for Africa. However, the sequences of T. elegans from Gabon (GenBank accession number: KY449397, KY449398) were not considered because they fell outside the T. elegans species complex and were instead closely related to T. lactinea. We, therefore, excluded these sequences from our analyses. All in all, since the sequences of T. elegans from tropical Africa investigated in this study are demarcated from sequences of T. elegans s. str., the adoption of another correct name for specimens of T. elegans from this area is necessary.
Specimens belonging to the T. elegans species complex have been reported in the past for tropical African countries (Ryvarden and Johansen 1980), with the first name applied to such specimens being Daedalea amanitoides P. Beauv., which was based on a specimen from Nigeria (cited as kingdom of Oware) (Palisot-Beauvois 1804). The morphological characteristics evident in the very short description and illustration of a fruiting body of D. amanitoides match the characteristics of the specimens examined in this study. However, for reasons that we ignore, Fries (1821) replaced this name (D. amanitoides) by the name Daedalea palisotii Fr., which is sanctioned and therefore must be used. The combination Trametes palisotii (Fr.) Imazeki (Imazeki 1952) is available and must be used for African specimens known previously as T. elegans (Fig. 5).

Phylogenetic position and taxonomy of the new species Trametes parvispora
The sequences belonging to the new species named T. parvispora form a distinct and well-supported clade in the ITS and the combined ITS-LSU datasets ( Fig. 3; Suppl. material 2). This species forms a sister clade with the still formally undescribed Tram-etes sp. (KT896651) from Finland. However, unlike T. parvispora where fruiting bodies were available for morphological characterization (Fig. 2I, J), the Finnish specimen was isolated as mycelium from the bark beetle Ips typographus L. (Linnakoski et al. 2016). Thus, anatomical and morphological comparisons are currently not possible. Furthermore, both sequences of T. parvispora share a clade with Trametes meyenii (Klotzsch) Lloyd. This clade was confirmed by phylogenetic analyses including two additional markers RPB1 and RPB2 (Suppl. material 4). Trametes meyenii has hispid and creamyellow pilei, irpicoid and white to ochraceous hymenophore, pores 1-3 per mm, 4.5-6 × 2-2.5 μm basidiospores (Zmitrovich et al. 2012), whereas T. 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 (Figs 2I, J, 4). These morphological differences confirm that T. parvispora and T. meyenii are distinct species as shown by the phylogenetic analyses ( Fig. 3; Suppl. material 2, 4). However, some species lacking DNA sequences, namely Trametes barbulata Corner, Trametes daedaleoides Corner, and Trametes rugosituba Corner (Corner 1989;Hattori 2005;Hattori and Sotome 2013), share with T. 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 T. parvispora as a distinct new species.