Phylogeny and taxonomy of Laetiporus (Basidiomycota, Polyporales) with descriptions of two new species from western China

Abstract Laetiporus is a cosmopolitan genus of brown rot fungi. In this study, L.medogensis and L.xinjiangensis are described as new species from western China, based on morphological and molecular evidence. L.medogensis has only been found on gymnosperms so far and is distinguished by pinkish-buff to clay-buff pileal surface and buff-yellow pore surface, azonate to faintly zonate pileus and ellipsoid to ovoid basidiospores (5–6.2 × 4.2–5.2 μm). L.xinjiangensis is found on angiosperms and is characterised by pale-buff to clay-pink pileal surface, cream to light yellow pore surface, azonate to faintly zonate pileus, large pores (2–3 per mm) and small basidiospores (4.5–5 × 3–4.2 μm). The phylogeny of Laetiporus is reconstructed with multi-gene sequences including the internal transcribed spacer regions (ITS), the large subunit (nrLSU) and small subunit (nrSSU) of the nuclear ribosomal RNA gene, the small subunit of the mitochondrial rRNA gene (mtSSU), the translation elongation factor 1-α gene (EF-1α) and the second subunit of RNA polymerase II (RPB2). The results show that L.medogensis and L.xinjiangensis formed two distinct lineages belonging to Laetiporus. Illustrated descriptions of the two new species are presented. An identification key to species of L.sulphureus complex is provided.


Introduction
Laetiporus Murrill (Fomitopsidaceae, Polyporales) is a cosmopolitan genus, causing brown rot on living hardwoods and conifers (Murrill 1904). Some species of the genus are known as forest pathogens and some are edible with medicinal functions (Dai et al. 2007(Dai et al. , 2009. According to previous studies, 15 species have been accepted in the genus worldwide and 11 species have been confirmed in the L. sulphureus complex by phylogenetic analyses, of which six have been reported from China: L. ailaoshanensis B.K. Cui & J. Song, L. cremeiporus Y. Ota & T. Hatt., L. montanus Černý ex Tomšovský & Jankovský, L. sulphureus (Bull.) Murrill, L. versisporus (Lloyd) Imazeki and L. zonatus B.K. Cui & J. Song (Tomšovský and Jankovský 2008, Ota et al. 2009, Banik et al. 2012, Song et al. 2014, Song and Cui 2017. The species in the L. sulphureus complex are characterised by annual basidiocarps, soft and fleshy context and a dimitic hyphal system composed of simple septate generative hyphae and binding hyphae (Burdsall and Banik 2001, Núñez and Ryvarden 2001, Ota et al. 2009).
A molecular phylogenetic study of Laetiporus in Japan identified three species, viz. L. cremeiporus, L. montanus and L. versisporus Hattori 2008, Ota et al. 2009). Recently, systematic studies have been carried out to define the species and explore the historical biogeography of the genus Laetiporus in China. Song et al. (2014) described two new Laetiporus species from south-western China based on morphological and molecular evidence. Further comprehensive study of Song and Cui (2017) indicated that there are two additional undescribed Laetiporus species.
In the present study, the two new Laetiporus species from western China (Clade P and Clade Q) are described based on morphological and phylogenetic analyses.

Morphological studies
Morphological studies followed Han et al. (2016). The studied specimens were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions were based on field notes. Colour terms followed Petersen (1996). Microscopic measurements and drawings were made from slide preparations of dried specimens stained with Cotton Blue and Melzer's reagent, following Han et al. (2016). Sections were studied at a magnification of 1000× using a Nikon Eclipse 80i microscope and phase contrast illumination. Drawings were made with the aid of a drawing tube. Spores were measured in tube sections. In presenting spore size variation, 5% of measurements were excluded from each end of the range and given in parentheses. The following abbreviations were used: KOH = 5% potassium hydroxide, CB = cotton blue, CB+ = cyanophilous, CB-= acyanophilous, IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, L = mean spore length (arithmetic average), W = mean spore width (arithmetic average), Q = variation in the L/W ratios between specimens studied, n (a/b) = number of spores (a) measured from a given number of specimens (b).
Bayesian Inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses were applied to the combined dataset. The best fit model of nucleotide evolution to each individual genetic marker and the combined dataset was selected with AIC (Akaike Information Criterion) using MrModeltest 2.3 (Posada andCrandall 1998, Nylander 2004). The best fit models were GTR for ITS, nrLSU, nrSSU, EF-1α, mtSSU, RPB2 and GTR+I+G for the combined dataset. The partitioned mixed model, which allows for model parameters estimated separately for each genetic marker, was used in the Bayesian analysis. BI was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) with 2 independent runs, each one beginning from random trees with 4 simultaneous independent chains, performing 4,000,000 replicates, sampling one tree every 100 generations. The first 25% of the sampled trees were discarded as burn-in and the remaining ones were used to reconstruct a majority rule consensus and calculate Bayesian posterior probabilities (BPP) of the clades.
ML searches were conducted with RAxML-HPC2 on Abe through the Cipres Science Gateway (www.phylo.org) and comprised 100 ML searches under the GTR-GAMMA model, with all model parameters estimated by the programme. Only the maximum likelihood best tree from all searches was kept. In addition, 100 rapid bootstrap replicates were run with the GTRCAT model to assess the reliability of the nodes. MP analysis was applied to the combined dataset as in Song and Cui (2017). Tree construction was performed in PAUP* version 4.0b10 (Swofford 2002) with the following settings. All characters were equally weighted and gaps were treated as missing data. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000, branches of zero length were collapsed and all most parsimonious trees were saved. Clade robustness was assessed using a bootstrap analysis with 1000 replicates (Felsenstein 1985). The descriptive statistics of tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for each most parsimonious tree generated.
Branches that received bootstrap support for maximum parsimony (MP), maximum likelihood (ML) and Bayesian posterior probabilities (BPP) greater than or equal to 75% (MP/ML) and 0.95 (BPP) were considered as significantly supported.

Phylogenetic analyses
The combined dataset (ITS+nrLSU+nrSSU+mtSSU+EF-1α+RPB2) included sequences from 55 samples representing 19 taxa. Antrodia serialis and Fomitopsis pinicola were used as outgroups. The dataset had a total aligned length of 3963 characters, of which 3137 (79.2%) were constant, 301 (7.6%) were variable and parsimony uninformative and 525 (13.2%) were parsimony informative. The parsimony analysis yielded 68 equally parsimonious trees (TL = 1173, CI = 0.812, RI = 0.865, RC = 0.702, HI = 0.188). The multiple sequence alignment and tree files were deposited at TreeBase (submission ID 21249; www.treebase.org). MP analysis and BI resulted in similar topologies as the ML analysis. The consensus tree inferred from the ML analysis with MP, ML and BPP values is shown in Figure 1.
Samples of Laetiporus clustered together with significant support (100% MP, 100% ML and 1.00 BPP; Figure 1). Sampled specimens of the two new species L. medogensis and L. xinjiangensis formed well-supported lineages (Figure 1).
Etymology. Medogensis (Lat.): referring to the locality (Medog County) of the type specimens. Basidiocarps. Annual, sessile to laterally substipitate, imbricate, fleshy when fresh, crumbly when dry, without odour or taste. Pileus flabelliform to dimidiate, applanate, projecting up to 9 cm, 12 cm wide and 1 cm thick. Pileal surface pinkish-buff to clay-buff when fresh, becoming pale yellow upon drying, glabrous, azonate to faintly zonate. Margin soft and slightly viscous, fawn when juvenile, fading to reddish-brown when dry. Pore surface buff-yellow when fresh, becoming pale yellow to cream when dry; sterile margin cream when fresh, up to 3 mm wide; pores angular, 2-4 per mm; dissepiments thin, entire to lacerate. Context white when fresh, becoming cream to pale yellow when dry, up to 8.5 mm thick. Tubes concolorous with pore surface, crumbly or chalky, up to 1.5 mm long.
Etymology. Xinjiangensis (Lat.): referring to the locality (Xinjiang Autonomous Region) of the type specimens.
Basidiocarps. Annual, sessile to laterally substipitate, imbricate, odour distinctive, taste with acid flavor, fleshy when fresh, crumbly when dry. Pilei flabelliform to dimidiate, applanate, projecting up to 15 cm, 20 cm wide and 3 cm thick. Pileal surface pale-buff to clay-pink when fresh, becoming pale-buff to cream upon drying, glabrous, azonate to faintly zonate when fresh. Margin blunt, clay-buff to greyish-  brown to brown when juvenile, fading to dark brown when dry. Pore surface cream to light yellow when fresh, becoming pale yellow when dry; sterile margin pale yellow when fresh, up to 2 mm wide; pores angular, 2-3 per mm; dissepiments thin, entire to lacerate. Context white when fresh, becoming cream to pale yellow when dry, up to 2.2 cm thick. Tubes concolorous with pore surface, crumbly or chalky, up to 8 mm long.
Additional specimens (

Discussion
Recent studies indicated that Laetiporus sulphureus in East Asia is a species complex, comprising several morphologically and ecologically distinct species (Ota et al. 2009, Song et al. 2014). The current study recognised two new species, namely, L. medogensis and L. xinjiangensis and, altogether, eight Laetiporus species have been found in China thus far. The multi-gene phylogenetic topology showed that the new species formed two separate lineages (Figure 1).
Laetiporus medogensis and L. ailaoshanensis group together with moderate to low MP and ML support (50% MP and 53% ML). Both L. medogensis and L. ailaoshanensis are found in Southwest China. Morphologically, L. ailaoshanensis is similar to L. medogensis by producing orange to yellow pileal surface, white context and ellipsoid to ovoid basidiospores. However, L. medogensis is found on conifers and the pore surface is yellow; L. ailaoshanensis grows on hardwoods and has a white pore surface (Song et al. 2014). L. sulphureus resembles L. medogensis by producing yellow to orange pileal surface and yellow pore surface; however, L. sulphureus usually grows on hardwoods and produces thicker basidiocarps and larger basidiospores (5-7 × 4-5 μm; Ota et al. 2009). L. versisporus and L. medogensis share similar characters including yellow pileal surface, yellowish pore surface and ovoid to ellipsoid basidiospores; however, L. versisporus differs from L. medogensis in having smaller pores (2-6 per mm) and larger basidiospores (4-6.8 × 3-5.5 μm). In addition, L. versisporus grows on hardwoods and is mainly distributed in subtropical to tropical areas (Ota et al. 2009, Song andCui 2017). Laetiporus xinjiangensis, L. sulphureus and L. montanus are all common in Northwest China. Both L. xinjiangensis and L. sulphureus grow on angiosperms and group together in the phylogenetic tree with moderate MP, ML and significant BI support (65% MP, 51% ML and 0.98 BPP). Morphologically, L. sulphureus is similar to L. xinjiangensis in having yellowish pore surface and ovoid to ellipsoid basidiospores; however, L. sulphureus produces larger basidiospores (5-7 × 4-5 μm) and has smaller pores (2-5 per mm; Burdsall and Banik 2001). L. montanus is similar to L. xinjiangensis by producing a burlywood pileal surface and a yellowish pore surface; however, L. montanus differs by producing pyriform basidiospores (6-8 × 4-5.5 μm) and by growing on gymnosperms (Tomšovský and Jankovský 2008).
Our research expanded the number of Laetiporus species to 17 around the world. However, studies in the Southern Hemisphere are still few and the relationships amongst Laetiporus species remain unresolved (Lindner and Banik 2008, Pires et al. 2016, Song and Cui 2017. More comprehensive studies on Laetiporus depend on more collections and data from poorly sampled areas. The main morphological characters, host trees and distribution areas of species in the L. sulphureus complex are provided in Table 2. An identification key to the known species of Laetiporus is provided.