Morphological and molecular identification of two new Ganoderma species on Casuarina equisetifolia from China

Abstract Ganoderma is a cosmopolitan white rot fungal genus, famous for its medicinal properties. In the present study, two new Ganoderma species were collected from south-eastern China and described on the basis of morphological characters and phylogenetic analyses of sequences of the internal transcribed spacer (ITS) region, the translation elongation factor 1-α gene (EF1-α) and the second subunit of RNA polymerase II (RPB2). Specimens of both species were found on living trees of Casuarina equisetifolia. Ganoderma angustisporum sp. nov. is characterised by its sessile basidiomata and almond-shaped, slightly truncate, narrow basidiospores (9–11.3 × 4–5.2 µm). Ganoderma casuarinicola sp. nov. is characterised by its strongly laccate reddish-brown pileal surface, luminous yellow to yellowish-brown cutis and ellipsoid, truncate basidiospores (9–10.2 × 5–6 µm). The two new species are compared with their related taxa. Phylogenetic analyses confirmed that G. angustisporum and G. casuarinicola are distinct species within Ganoderma.


Introduction
Ganoderma P. Karst. is easily recognised by its characteristic appearance, double-walled and truncate basidiospores (Karsten 1881;Moncalvo and Ryvarden 1997). According to Christian (2015), there are 428 names in the Index Fungorum (http://www.indexfungorum.org/) and 456 records of taxa (420 with status legitimate) in MycoBank (http://www.mycobank. org/). Ganoderma products as dietary supplement are very popular in Asia, especially in China and China is very rich in Ganoderma species (Zhao and Zhang 2000;Wang et al. 2009;Cao et al. 2012;Cao and Yuan 2013;Li et al. 2014b). The great variability in macroscopic characters of basidiomes has resulted in a large number of synonyms and confusions in the taxonomy of this genus. Using DNA sequence data for the identification of Ganoderma species is of greatest importance.
Some Ganoderma species are well known for causing wood decay in a wide range of tree species around the world. For example, G. boninense Pat. is a causal agent of oil palm basal stem rot and is responsible for considerable yield losses in southeast Asian oil palm plantations (Pilotti 2005). Especially in Indonesia and Malaysia, G. boninense and G. philippii (Bres. & Henn. ex Sacc.) Bres. cause great economic loss of palm oil, tea and rubber (Zakaria et al. 2009).
Casuarina equisetifolia Forst. is used as an industrial raw material and wood fuel, as well as for conservation of coastal ecosystems and for agricultural land protection against salinity intrusion (Hossain et al. 1998;Chowdhury et al. 2009). In China, Casuarina equisetifolia is widely planted in the coastal areas of Guangxi, Guangdong, Fujian, Hainan and Taiwan provinces. During collections of wood-rotting fungi in South China in recent years, two Ganoderma species growing on Casuarina equisetifolia, which could not be identified to any known species, were collected. Those two species are here described based on morphological characters and phylogenetic analyses.

Morphological studies
The examined specimens were deposited in the herbarium of the Institute of Microbiology, Beijing Forestry University (BJFC). Macro-morphological descriptions were based on field notes. Special colour terms followed Petersen (1996). Micro-morphological data were obtained from the dried specimens and observed under a light microscope following Li et al. (2014a) and Han et al. (2016). Sections were studied at a magnification of up to 1000× using a Nikon E 80i microscope and phase contrast illumination. Drawings were made with the aid of a drawing tube. Microscopic features, measurements and drawings were made from slide preparations stained with Cotton Blue and Melzer's reagent. Spores were measured from sections cut from the tubes. To represent variation in the size of basidiospores, 5% of measurements were excluded from each end of the range and extreme values are given in parentheses.
The following abbreviations are used: IKI = Melzer's reagent, IKI-= neither amyloid nor dextrinoid, KOH = 5% potassium hydroxide, CB = Cotton Blue, CB+ = cyanophilous, Q is an average computed by dividing the length by the width of each spore separately, n (a,b) = a spores measured from b specimens.

Molecular study and phylogenetic analysis
The CTAB rapid plant genome extraction kit-DN14 (Aidlab Biotechnologies Co. Ltd., Beijing, China) was used to extract total genomic DNA from dried specimens according to the manufacturer's instructions with some modifications (Chen et al. , 2017. The genes ITS, EF1-α and RPB2 were amplified by polymerase chain reaction (PCR) technique. The ITS region was amplified with primer pair ITS5 (GGA AGT AAA AGT CGT AAC AAG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990). Part of the EF1-α gene was amplified with primer pair EF1-983F (GCY CCY GGH CAY CGT GAY TTY AT) and EF1-1567R (ACH GTR CCR ATA CCA CCR ATC TT) (Rehner and Buckley 2005). Part of the RPB2 gene was amplified with primer pairs 5F (GAY GAY MGW GAT CAY TTY GG) and 7CR (CCC ATR GCT TGY TTR CCC AT) (Liu et al. 1999). The PCR cycling for ITS was as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles at 94 °C for 40 s, 54 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min. The PCR cycling for EF1-α was as follows: initial denaturation at 95 °C for 3 min, followed by 34 cycles at 94 °C for 40 s, 56 °C for 45 s and 72 °C for 1 min and a final extension of 72 °C for 10 min. The PCR cycling for RPB2 was as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 1 min, 58 °C for 2 min and 72 °C for 1.5 min and a final extension of 72 °C for 10 min. The PCR products were purified and sequenced at Beijing Genomics Institute (China), using forward and reverse PCR primers. All newly generated sequences were deposited in GenBank (Table 1).
Phylogenetic analyses in this study followed the approach of Song et al. (2016a) and Song and Cui (2017). The maximum likelihood (ML) and Bayesian inference (BI) methods were used to analyse the combined dataset of ITS, EF1-α and RPB2 sequences. ML analysis was conducted with RAxML-HPC252 on the Cipres Science Gateway (Miller and Pfeiffer 2011) involved 100 ML searches; all model parameters were estimated by the programme. The ML bootstrap values (ML-BS) were obtained with 1000 rapid bootstrapping replicates. BI was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), with a mixed model partition. A suitable substitution model for each partition of the dataset was determined using the Akaike Information Criterion implemented in MrMODELTEST 2.3. Four Markov chains were run from the random starting tree for 1 million generations to make the average standard deviation of split deviation frequencies lower than 0.01. Trees were sampled every 100 generations. The burn-in was set to discard the first 25% of the trees. A majority rule consensus tree of all the remaining trees was used to calculate Bayesian posterior probabilities (BPP). The ML and BI algorithms generated congruent topologies in main lineages; thus, only the topology from the ML algorithm was presented along with BS and BPP greater than 75% and 0.95, respectively, at the nodes.

Phylogenetic analysis
The combined ITS, EF1-α and RPB2 dataset included sequences from 66 fungal samples representing 27 taxa. The selected models were K80 for 5.8S, K80 + G for ITS1, HKY + G for ITS2, GTR + I + G for ITS1+ ITS2 + 5.8S. The best model selected and applied in the BI analysis for the combined ITS, EF1-α and RPB2 partition was a GTR+I+G model. BI analysis and ML analysis resulted in the same topology with an average standard deviation of split frequencies = 0.006025 (BI). Diagnosis. Ganoderma angustisporum is characterised by its sessile basidiomata, white pore surface, almond-shaped, slightly truncate and narrow basidiospores. Holotype. CHINA. Fujian Prov., Pingtan County, on living tree of Casuarina equisetifolia, 18 August 2016, Cui 13817 (BJFC!).
Type Diagnosis. Ganoderma casuarinicola is characterised by its strongly laccate reddishbrown pileal surface, white pore surface, luminous yellow to yellowish-brown cutis.

Discussion
The two new Ganoderma species were found on living trees of Casuarina equisetifolia from the southeast coast of China. However, they are quite different from each other in morphology. Their main morphological differences are presented in Table 2. Phylogenetically, the two new species grouped together with some other laccate Ganoderma species in a well-supported clade.
In conclusion, both morphology and phylogeny inferred from the combined ITS, EF1-α and RPB2 sequences support that the specimens, collected on living trees of Casuarina equisetifolia from the southeast coast of China, are two new species within the Ganoderma genus.