﻿Taxonomy and molecular phylogeny of Trametopsis (Polyporales, Basidiomycota) with descriptions of two new species

﻿Abstract Trametopsis is a worldwide genus belonging to Irpicaceae in the phlebioid clade, which can cause a white decay of wood. Previously, only three species were ascribed to the genus. In this study, we performed a morphological and phylogenetic study of Trametopsis. Molecular phylogenetic analyses of multiple loci included the internal transcribed spacer (ITS) regions, the large subunit nuclear ribosomal RNA gene (nLSU), the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor 1-α gene (TEF1). Phylogenetic trees were inferred from the combined datasets of ITS+nLSU sequences and ITS+nLSU+RPB1+RPB2+TEF1 sequences by using maximum parsimony, maximum likelihood and Bayesian inference analyses. Combined with molecular data, morphological characters and ecological traits, two new species of Trametopsis are discovered. Trametopsisabieticola is characterised by its pileate, solitary or imbricate basidiomata, buff to buff-yellow pileal surface when fresh, becoming pinkish buff to clay-buff when dry, cream to buff pore surface when fresh, becoming pinkish buff to greyish brown upon drying, round to angular and large pores (0.5–1 per mm), cylindrical basidiospores (5.8–7.2 × 1.9–2.6 μm), distributed in the high altitude of mountains and grows on Abies sp. Trametopsistasmanica is characterised by its resupinate basidiomata, cream to pinkish-buff pore surface when fresh, becoming honey-yellow to snuff brown upon drying, cylindrical basidiospores (5.2–6.3 × 1.8–2.2 μm), and by growing on Eucalyptus sp. Detailed descriptions and illustrations of the two novel species are provided.

During our investigations of wood-decay fungi, some specimens of the phlebioid clade were collected. These specimens possess glabrous or velutinate to strigose pileal surface, round to angular, irregular, daedaleoid to irpicoid pores, saprophytic on dead wood and causing white rot. Preliminary morphological observations showed that these specimens may belong to Trametopsis. To determine the phylogenetic positions of these specimens, we performed phylogenetic analyses of Irpicaceae with emphasis on Trametopsis based on the combined sequences datasets of ITS+nLSU and ITS+nLSU+RPB1+RPB2+TEF1. Combining morphological and molecular evidence, two new species, viz., T. abieticola and T. tasmanica are described and illustrated.

Morphological studies
The examined specimens were deposited at the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Morphological descriptions and abbreviations used in this study follow Cui et al. (2019) and Song et al. (2021).

Molecular studies and phylogenetic analysis
The procedures for DNA extraction and polymerase chain reaction (PCR) used in this study were the same as described by Liu et al. (2021a) and Sun et al. (2022). The ITS regions were amplified with the primer pairs ITS4 and ITS5, the nLSU regions were amplified with the primer pairs LR0R and LR7, RPB1 was amplified with primer pairs RPB1-Af and RPB1-Cr, RPB2 gene was amplified with the primer pairs fRPB2-f5F and bRPB2-7.1R, and TEF1 gene was amplified with the primer pairs EF1-983F and EF1-1567R (White et al. 1990;Rehner 2001;Matheny et al. 2002;Matheny 2005).
The PCR cycling schedules for different DNA sequences of ITS, nLSU, RPB1, RPB2 and TEF1 genes used in this study followed those used in Liu et al. (2021bLiu et al. ( , 2022 with some modifications. The PCR products were purified and sequenced at Beijing Genomics Institute, China, with the same primers. All newly generated sequences were submitted to GenBank and were listed in Table 1. Sequences were aligned with additional sequences downloaded from GenBank (Table  1) using ClustalX (Thompson et al. 1997). Alignment was manually adjusted to allow maximum alignment and to minimise gaps in BioEdit (Hall 1999). Sequence alignment was deposited to TreeBase (https://treebase.org/treebase-web; submission ID 29580). In phylogenetic reconstructions, the sequences of Phanerochaete albida Sheng H. Wu and P. alnea (Fr.) P. Karst. obtained from GenBank were used as outgroups. The reason for choosing these two species as outgroup taxa is that they belong to Phanerochaete in Phanerochaetaceae, and are closely related to Irpicaceae (Chen et al. 2021), which conforms to the outgroup selection rules. Furthermore, species of Phanerochaete were also selected as outgroups in other phylogenetic studies of Irpicaceae, such as in El-Gharabawy et al (2021).

Present study
Newly generated sequences for this study are shown in bold.

Phylogeny
The combined 2-gene (ITS+nLSU) sequences dataset had an aligned length of 1893 characters, including gaps (619 characters for ITS, 1274 characters for nLSU), of which 1307 characters were constant, 105 were variable and parsimony-uninformative, and 481 were parsimony-informative. MP analysis yielded 26 equally parsimonious trees (TL = 2150, CI = 0.409, RI = 0.776, RC = 0.317, HI = 0.591). The best-fit evolutionary models applied in Bayesian analyses were selected by MrModeltest2 v. 2.3 for each region of the two genes, the model for ITS was GTR+I+G with equal frequency of nucleotides, while the model for nLSU was SYM+I+G with equal frequency of nucleotides. ML analysis resulted in a similar topology as MP and Bayesian analyses, and only the ML topology is shown in Fig. 1. The combined 5-gene (ITS+nLSU+RPB1+RPB2+TEF1) sequences dataset had an aligned length of 4609 characters, including gaps (619 characters for ITS, 1274 characters for nLSU, 1170 characters for RPB1, 1001 characters for RPB2, 545 characters for TEF1), of which 2675 characters were constant, 272 were variable and parsimonyuninformative, and 1662 were parsimony-informative. MP analysis yielded 36 equally parsimonious trees (TL = 9247, CI = 0.362, RI = 0.652, RC = 0.236, HI = 0.638). The best-fit evolutionary models applied in Bayesian analyses were selected by MrMod-eltest2 v. 2.3 for each region of the two genes, the model for ITS, RPB1, RPB2 and TEF1was GTR+I+G with equal frequency of nucleotides, while the model for nLSU was SYM+I+G with equal frequency of nucleotides. ML analysis resulted in a similar topology as MP and Bayesian analyses, and only the ML topology is shown in Fig. 2.
The phylogenetic trees inferred from ITS+nLSU and ITS+nLSU+RPB1+RPB2+TEF1 gene sequences were all obtained from 78 fungal samples representing 42 taxa of Irpicaceae and two taxa of Phanerochaetaceae within the phlebioid clade (Figs 1, 2). Phylogenetic analyses showed that Trametopsis abieticola, T. aborigena, T. brasiliensis, T. cervina and T. tasmanica    Etymology. Abieticola (Lat.): referring to the species grows on Abies sp. Fruiting body. Basidiomata annual, pileate, solitary or imbricate, soft corky to corky, without odour or taste when fresh, becoming corky and light in weight upon drying. Pilei applanate to flabelliform, projecting up to 9.5 cm long, 5.5 cm wide, and  2 cm thick at base. Pileal surface buff to buff-yellow when fresh, becoming pinkish buff to clay-buff when dry, strigose or glabrous; margin white to cream when fresh, becoming cream to buff-yellow when dry, obtuse to acute. Pore surface cream to buff when fresh, becoming pinkish buff to greyish brown upon drying; pores round to angular, 0.5-1 per mm; dissepiments slightly thick, entire to lacerate. Context corky, cream to buff yellow, up to 8 mm thick. Tubes concolorous with pore surface, corky, up to 7 mm long.
Etymology. Tasmanica (Lat.): referring to the species collected from Tasmania in Australia.
Fruiting body. Basidiomata annual, resupinate, not easily separated from the substrate, without odour or taste when fresh, becoming corky to fragile and light in weight upon drying; up to 5.5 cm long, 2 cm wide, and 7 mm thick at centre. Pore surface cream to pinkish-buff when fresh, becoming honey-yellow to snuff brown upon drying; pores round to angular, 2-4 per mm; dissepiments slightly thick, entire to lacerate. Context very thin, corky, cream to buff, up to 2 mm thick. Tubes concolorous with pore surface, corky, up to 4 mm long.

Discussion
In this study, the phylogenetic analyses of Trametopsis and related genera are inferred from the combined datasets of ITS+nLSU sequences (Fig. 1) and ITS+nLSU+RPB1+RPB2+TEF1 sequences (Fig. 2). The genera; Raduliporus Spirin & Zmitr., Resiniporus Zmitr. and Trametopsis grouped together and formed a highly supported lineage (Figs 1 and 2), which was called the Trametopsis lineage by Chen et al. (2021). Morphologically, Raduliporus and Resiniporus differ from Trametopsis by having a monomitic hyphal system and ellipsoid basidiospores (Chen et al. 2021). Phylogenetically, T. abieticola and T. tasmanica clustered with other Trametopsis species (Figs 1, 2) with high supports (100% MP, 100% ML, 1.00 BPP; Figs 1, 2). The main morphological characters and ecological habits of species in Trametopsis are provided in Table 2. The geographical locations of the Trametopsis species distributed in the world are indicated on the map (Fig. 7).
Trametopsis tasmanica is distributed in Tasmania, Australia and grows on Eucalyptus sp. Before that, there was no report of Trametopsis in Oceania. Morphologically, T. tasmanica and T. cervina share similar-sized pores, but T. cervina differs from T. tasmanica by its pileate to effused-reflexed basidiomata, larger basidiospores (6-9 × 2-3 μm; Tomšovský 2008). Trametopsis aborigena, T. brasiliensis and T. tasmanica are only distributed in the southern hemisphere and grow on angiosperm trees. However, T. aborigena differs from T. tasmanica by having pileate, effused-reflexed to occasionally resupinate basidiomata, larger basidia (19-22 × 5-6 μm) and basidiospores (5-7 × 1-2.5 μm), and being distributed in neotropical regions of Argentina In summary, we performed a taxonomic and phylogenetic study of Trametopsis. The concepts and species number of the Trametopsis are updated. So far, five species are accepted in the Trametopsis around the world. Currently, Trametopsis is characterised by an annual growth habit, effused-reflexed to pileate or resupinate, solitary or imbricate basidiomata, pinkish buff to cinnamon or clay-buff, zonate or azonate, glabrous or velutinate to strigose pileal surface, cream, pale yellow to greyish brown pore surface with round to angular, irregular, daedaleoid to irpicoid pores, a monomitic hyphal system in context, dimitic in trama, clamped generative hyphae, and allantoid to cylindrical basidiospores; it grows on different angiosperm and gymnosperm trees, causing white rot of wood (Tomšovský 2008;Gómez-Montoya et al. 2017).