Ganodermaovisporum sp. nov. (Polyporales, Polyporaceae) from Southwest China

Abstract Background Ganoderma is a white-rot fungus with a cosmopolitan distribution and includes several economically important species. This genus has been extensively researched due to its beneficial medicinal properties and chemical constituents with potential nutritional and therapeutic values. Traditionally, species of Ganoderma were identified solely based on morphology; however, recent molecular studies revealed that many morphology-based species are conspecific. Furthermore, some type species are in poor condition, which hinders us from re-examining their taxonomic characteristics and obtaining their molecular data. Therefore, new species and fresh collections with multigene sequences are needed to fill the loopholes and to understand the biological classification system of Ganoderma. New information In a survey of Ganoderma in Guizhou Province, southwest China, we found a new species growing on soil and, herein, it was identified by both morphology and phylogenetic evidence. Hence, we propose a new species, Ganodermaovisporum sp. nov. This species is characterised by an annual, stipitate, laccate basidiome, with a red–brown to brownish-black pileus surface and pale white pores, duplex context, clavate pileipellis terminal cells, trimitic hyphal system, ellipsoid basidiospores with dark brown eusporium bearing coarse echinulae and an obtuse turgid appendix. Phylogenetic analyses confirmed that the novel species sisters to G.sandunense with high bootstrap support. Furthermore, the RPB2 sequence of G.sandunense is supplied for the first time. Notably, we re-examined the type specimen of G.sandunense and provide a more precise description of the duplex context, pileipellis terminal cells and basidia. All species collected are described and illustrated with coloured photographs. Moreover, we present an updated phylogeny for Ganoderma, based on nLSU, ITS, RPB2 and TEF1-α DNA sequence data and species relationships and classification are discussed.


Introduction
Ganodermataceae Donk is a large family of Polyporales and Ganoderma P. Karst is the most speciose genus in the family (Hapuarachchi et al. 2016a, Hapuarachchi et al. 2017, He et al. 2019. Before the molecular era, Polyporales with double-walled basidiospores with a pigmented endosporium ornamented with columns or ridges and a smooth hyaline exosporium were usually placed in Ganodermataceae (Moncalvo and Ryvarden 1997). This family is comprised of ten genera: Amauroderma Murrill, Amaurodermellus Costa-Rezende, Drechsler-Santos & Góes-Neto, Foraminispora Robledo, Costa-Rezende & Drechsler-Santos, Furtadomyces Leonardo-Silva, Cotrim & Xavier-Santos, Ganoderma P. Karst, Haddowia Steyaert, Humphreya Steyaert, Sanguinoderma Y.F. Sun, D.H. Costa & B.K. Cu,Tomophagus Murrill and Trachyderma Imazeki ( Richter et al. 2014, Costa-Rezende et al. 2017, Costa-Rezende et al. 2020, Sun et al. 2020, Leonardo-Silva et al. 2022. However, Ganodermataceae has been treated as a synonym of Polyporaceae (Justo et al. 2017). There have been several discrepancies regarding the treatment of Justo et al. (2017); in particular, the studied collection of Ganodermatoid specimens was insufficient to establish a stable taxonomic and systematic placement in a phylogenetic context because some herbarium materials have been destroyed or cannot be found, lacking molecular and morphological data and the characterised double-walled basidiospores in Ganodermataceae are quite different from those in Polyporaceae , Costa-Rezende et al. 2020. In this study, we subsequently followed Justo et al. (2017) since the phylogenetic analyses are more convincing and objective than morphological results.
Despite their economic importance, the taxonomy of Ganoderma remains uncertain due to a slew of confusion and misconceptions. During the past several decades, many species of Ganoderma have been delimited, based on the presence of stipe, laccate or non-laccate, the context of pileus and the microscopic characteristics of basidiospores (Chang and Chen 1984, Seo and Kitamot 1998, Wu et al. 2004, Torres-Torres and Guzmán-Dávalos 2012, Tchotet-Tchoumi et al. 2019. In general, it is difficult and subjective to identify Ganoderma species solely based on morphological evidence, as their phenotypic traits are sensitive to extrinsic factors, such as illumination, ventilation and humidity (Szedlay et al. 1999, Demoulin 2010, Yang and Feng 2013, Hapuarachchi et al. 2019a). Therefore, morphology-based identification brought Ganoderma into a state of taxonomic chaos (Smith and Sivasithamparam 2003, Coetzee et al. 2015, López-Peña et al. 2019, Náplavová et al. 2020. Compared to morphology, molecular methods have turned out to be more effective in resolving intraspecific relationships with Ganoderma (Yamashita and Hirose 2016, Fryssouli et al. 2020, Gunnels et al. 2020, Shen et al. 2021. Phylogenetic markers, such as IGS, nrSSU, ITS, nrLSU, mtSSU, β-TUB, RPB1, RPB2 and TEF1-α sequences, were independently or conjointly used to infer intraspecific relationships within Ganoderma (Cao et al. 2012, Zhou et al. 2015, Xing et al. 2018, Hapuarachchi et al. 2019a, Ye et al. 2019. In particular, the multilocus phylogeny incorporating sequences from ITS, nrLSU, TEF1-α and RPB2 was applied to give a phylogenetic framework for species delimitation in this genus (Xing et al. 2018, Ye et al. 2019, Tchotet-Tchoumi et al. 2019, Wu et al. 2020. Furthermore, some researchers steered using a combination of morphological, chemotaxonomic and molecular strategies to elevate a steady taxonomy for Ganoderma and resolve taxonomic ambiguities (Richter et al. 2014, Welti et al. 2015. Ganoderma has a cosmopolitan distribution and most of the species are known from tropical and sub-tropical regions (He et al. 2019). This fungus grows as saprobes or parasites on deciduous and coniferous trees and some of them are considered as plant pathogens that cause basal stem butt rot and root rot (Pinruan et al. 2010, Ding et al. 2020, Mafia et al. 2020, Mohd et al. 2020). Species of Ganoderma play an important role in the nutrient mobilisation process of woody plants. They possess lignocellulose-decomposing enzymes with effective mechanisms for bioenergy production and bioremediation (Coetzee et al. 2015, Kües et al. 2015. In the natural environment, a basidiome has the ability to produce innumerable basidiospores that can be spread by air-or rain-driven and insect vectors (Tuno 1999, Kadowaki et al. 2011, Almaguer et al. 2014, Sadyś et al. 2014. The infection of a plant host by pathogenic Ganoderma species starts with the landing of the basidiospore on the wound trunk or root, followed by germination and colonisation (Rees et al. 2009, Rees et al. 2012, Hushiarian et al. 2013, Ayin et al. 2019. Basal stem rot caused by G. boninense is the main disease that leads to yield losses and death of oil palm, which account for 50% of substantial economic losses to Southeast Asia's palm oil industry (Hushiarian et al. 2013, Lee and Chang 2016, Midot et al. 2019. Red roots caused by G. philippii are a serious disease of commercial Acacia mangium in Malaysia and India (Glen et al. 2014). Since different Ganoderma species produce different characteristics and pathogenicity, species identification is difficult, which in turn, leads to significant difficulty in disease control (Wong et al. 2012).
Ganoderma was first reported from China by Teng (1934), with four species including G. lucidum and one variety. More than 80 species have been introduced so far and several extensive studies have been carried out to investigate Ganoderma diversity in China, with new species being introduced (Zhao and Zhang 2000, Wu et al. 2004, Dai et al. 2009, Cao et al. 2012, Hapuarachchi et al. 2015, Hapuarachchi et al. 2018c). However, the majority of Ganoderma species reported from China have not been subjected to systematic studies (Wang 2012, Hapuarachchi et al. 2016b, Hapuarachchi et al. 2018a. The objective of the present study is to introduce a novel Ganoderma species, from Guizhou Province, southwest China, with descriptions, colour photographs, illustrations and a multigene phylogeny.

Materials and methods
Ganoderma samples were collected from Sandong Township, Sandu Shuizu Autonomous County, Guizhou Province, China, during the rainy season of July 2020. They were dried and preserved as outlined in Hapuarachchi et al. (2019b). The materials used in this study were deposited at Guizhou University (GACP) and the Herbarium of Kunming Institute of Botany Academia Sinica (HKAS).

Morphological study
Macro-morphological characteristics were described, based on dried material and the photographs provided here. Colour codes (e.g. 8E8) are from Kornerup and Wanscher (1978). Pileus was sectioned with a razor blade and mounted in 5% potassium hydroxide (KOH) solution. Pileipellis, hyphal systems of pileus, basidia and basidiospores were observed and captured using a compound microscope (Leica DM2500) equipped with a camera. Images were measured with Leica Application Suite X (LAS X). In the description section, the number, length, width and length/width ratio of the measured basidiospores are denoted with symbols n, L, W and Q, respectively. The Faces of Fungi number was registered by following Jayasiri et al. (2015).

DNA Extraction, PCR and Sequencing
Genomic DNA was extracted from dried specimens using an HP Fungal DNA Kit (OMEGA, USA) following the protocol of the manufacturer. PCR amplification was performed in a final volume of 50 µl reaction mixture that contained 25 µl 2x BenchTopTM Taq Master Mix (Biomigas), 19 µl distilled water, 2 µl (10 µM) of each primer and 2 µl template DNA. The large subunit ribosomal RNA (LSU), the internal transcribed spacer (ITS), the translation elongation factor (TEF1-α) and the RNA polymerase II second largest subunit (RPB2) were amplified with primer pairs LROR/LR5 (Vilgalys and Hester 1990), ITS5/ITS4 (White et al. 1990), EF1-983F/EF1-1567R (Rehner and Buckley 2017) and RPB2-5f/RPB2-7cR (Liu et al. 1999). PCR amplification reactions were performed with a T100 Thermal Cycler (T100™, Bio-Rad, USA). The procedures used for amplification of ITS were as follows: initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, elongation at 72°C for 1 min and a final extension at 72°C for 5 min. The cycling conditions of LSU, TEF1-α and RPB2 consisted of initial denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, elongation at 72°C for 1.3 min and a final extension at 72°C for 10 min. PCR products were verified by 1% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai, China) for purification and sequencing.

Sequence Alignment and Phylogenetic Analysis
The raw sequences generated in this study were assembled with ChromasPro (2.1.8). Megablast analysis was conducted using the assembled ITS and RPB2 sequences as the query to check the closely-related taxa. The taxa used in our phylogenetic analysis were selected, based on megablast results and related publications (Table 1). Alignments were performed using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html, Katoh and Standley 2013). The resulting alignments were improved manually when necessary, using BioEdit v. 7.0.5.2 (Hall 1999). The introns in TEF and RPB2 were removed, based on the published CDS sequence in GenBank. The aligned ITS1, 5.8S, ITS2, LSU, TEF1-α and RPB2 sequences were concatenated with SequenceMatrix v. (ITS1 and ITS2) were selected as the best model. Two runs of four chains were run until the average standard deviation of split frequencies dropped below 0.01, which occurred after 2,360,000 generations. Tree was sampled every 1000th generation and the chain temperature was decreased to 0.05 to improve convergence. The convergence of the runs was checked using TRACER v.1.6 (Rambaut et al. 2013). The first 25% of the resulting samples were discarded as burn-in and posterior probabilities were calculated from the remaining sampled trees (Larget and Simon 1999). In both ML and BY analyses, Foraminispora concentrica (Cui 12644) and Foraminispora yinggelingensis (Cui 13618) were selected as the outgroup (Sun et al. 2020). ML bootstrap values and BY posterior probabilities greater than or equal to 70% and 0.95, respectively, were considered significant support. The phylogenetic tree was visualised with FigTree version 1.4.0 available at http://tree.bio.ed.ac.uk/software/figtree/ (Rambaut 2012

Etymology
Referring to the ovoid basidiospores.

Notes
Ganoderma ovisporum clusters with G. sandunense in the multigene phylogenetic tree (Fig. 3), the former is similar to the latter by having 98% and 97% homology in ITS and RPB2 sequence data, respectively. These two species are similar in having wide ovoid basidiospores and inhabiting deciduous coniferous mixed forests. However, G. ovisporum differs from G. sandunense in having inconspicuously concentric rings near the pileus margin, lateral stipe and shorter pileipellis terminal cells (18-29 × 6-11 μm), while conspicuously concentric zones and vertically-arranged ridges or grooves, central stipe and longer pileipellis terminal cells (50-95 × 8-13.5 μm) have been observed in the latter. By considering both phylogenetic evidence and morphological observations, we conclude our collection is a new species in Ganoderma.

Notes
Ganoderma sandunense was introduced by Hapuarachchi et al. (2019b) with ITS sequence. In addition, the description of its basidia is absent in their publication. In this study, the holotype of G. sandunense was loaned from Herbarium (GACP) and reexamined. We have refined this species with a more detailed illustration. Furthermore, we provided RPB2 sequence data of this species, which is an important phylogenetic marker used for intraspecific delimitation within Ganoderma.

Analysis Phylogenetic analyses
Eight sequences of ITS, LSU and RPB2 were successfully amplified, but we failed to obtain the TEF1-α sequence from the two specimens HKAS123193 and GACP20071602. The newly-generated sequences and sequences from GenBank represented 132 specimens from 66 species, of which 21 were the type. The combined alignment of sequences comprised 3028 characters of 606, 1020, 809, 593 belonging to TEF1-α, RPB2, ITS and LSU, respectively. The final ML optimisation log-likelihood was -17354.28. The Bayesian Inference stopped at 2915000 generations when the average standard deviation of split frequencies reached 0.009904. The tree topologies derived from ML and BY were identical. Therefore, only the ML tree is shown (Fig. 3). The new species G. ovisporum and G. sandunense formed an individual clade in the phylogenetic tree (Fig. 3).

Discussion
In this study, both phylogeny and morphology support G. ovisporum as a new species. Morphologically, it resembles other dark-coloured, laccate, stipitate Ganoderma species. However, it can be distinguished by having larger (12.5-15.5 × 9.0-11.5 μm), wide ovoid, dark brown-pigmented basidiospores. It is mostly similar to G. sandunense in having brownish-black pileus and similarly-sized basidiospores, as well distribution in Guizhou Province (Hapuarachchi et al. 2019b). The former species is distinct from the latter by having a lateral stipe and shorter pileipellis terminal cells (18-29 × 6-11 μm). Phylogenetically, G. ovisporum and G. sandunense are closely related, forming a distinct clade with basal position with strong support.
Ganoderma was extensively researched by the Chinese because it applied to medicine and food, together with the symbolic happiness and immortality culture, those being recognised as long as 2,000 years ago (Hapuarachchi et al. 2016b, Hapuarachchi et al. 2018b, Li et al. 2018, Du et al. 2021. Chinese taxonomists emphasised Phylogram for Ganoderma generated from Maximum Likelihood analysis of ITS, LSU, TEF1-α and RPB2 sequence data. Bootstrap support values for Maximum Likelihood and maximum parsimony greater than 70% and posterior probabilities of Bayesian Inference ≥ 0.95 are given above branches. Type specimens are marked with letter (T) and new species in this study are indicated in red.
the morphological characteristics, such as stipe, pileus, pores, context, pileipellis terminal cells and basidiospores as keys to identity (Zhao 1989, He and Yu 1989, Zhang 1997. Keeping this method, Zhao and Zhang (2000) recorded 76 Ganoderma species from China, providing detailed illustrations. Wu and Dai (2005) identified 77 Ganoderma species with full description and colour photographs. Studies have been implemented to revise the taxonomy of Ganoderma in China by using molecular and morphology methods in the recent decade. The results indicated at least 23 species names are synonyms and confirmed that 24 species are distributed in China, 16 of which possess laccate basidiomes (Wang 2012, Chao 2013. Since then, six species with laccate basidiomes have been described from China: G. bambusicola, G. dianzhongense, G. esculentum, G. sandunense, G. shanxiense and G. weixinense (Hapuarachchi et al. 2019b, Ye et al. 2019, Wu et al. 2020. Ganoderma taxonomy has undergone tremendous changes since both phenotypic features and phylogeny were used to delineate species (Gottlieb et al. 2000, Hapuarachchi et al. 2018c, Hapuarachchi et al. 2018a, Lin and Yang 2019, Tchotet-Tchoumi et al. 2019, He et al. 2022. Based on the aforementioned characteristics, we have provided a dichotomous key to 22 laccate species, including our new species from China.
Ganoderma could originate from Southeast Asia and later dispersal to the Northern Hemispheres, the Southern Hemispheres and the neotropics before 30 Mya years, during which species radiation and diversification events happened (Moncalvo and Buchanan 2008). Overviewing Ganoderma species worldwide, Imazeki (1939) concluded using subgenera Euganoderma and Elfvingia to accommodate species with laccate and nonlaccate characters, respectively. In this study, a phylogenetic analysis was carried out using combined LSU, ITS, TEF1-α and RPB2 sequences from 66 species that included species previously placed in the above two subgenera. The topology of our phylogenetic tree is consistent with the morphology that the laccate species and non-laccate species tend to form groups. It is worth mentioning that the new species G. ovisporum group with the laccate species of G. carnosum, G. dianzhongense, G. leucocontextum, G. lucidum, G. oregonense, G. sandunense, G. shandongense, G. shanxiense, G. tsugae and G. weixiensis had strong support in both ML and Bayesian analyses. Those species were found in only or few ecological niches, except the widely cultivated G. leucocontextum, G. lucidum and G. tsugae (Gottlieb et al. 2000, Moncalvo and Buchanan 2008, Hapuarachchi et al. 2018b, Lin and Yang 2019. Therefore, many Ganoderma species are geographically restricted (He et al. 2022). However, the phylogenetic tree in the case of the laccate species G. pfeifferi and G. mutabile grouped with the non-laccate species G. adspersum, G. australe, G. eickeri, G. ellipsoideum, G. gibbosum, G. knysnamense, G. lobatum, G. podocarpense and G. williamsianum, indicating Euganoderma and Elfvingia are polyphyletic (Gottlieb et al. 2000). However, in fact, they are similar in having a substipitate to sessile basidiome and living as saprobes or parasites ( Hapuarachchi et al. 2018c, Tchotet-Tchoumi et al. 2019. Consequently, biogeographic patterns and convergent evolution could explain the population structure and evolution of Ganoderma. Thus, a phylogeography study would help better understand the evolution of Ganoderma.