Four new species of Tremella (Tremellales, Basidiomycota) based on morphology and DNA sequence data

Abstract In the present study, a total of 33 Tremella specimens in China were collected and examined using molecular phylogenetic analysis based on a combined sequence dataset of the nuc rDNA internal transcribed spacer (ITS) region and nuc 28S rDNA D1/D2 domain in conjunction with the morphological characters. Four new species, namely Tremellabasidiomaticola, T.cheejenii, T.erythrina, and T.salmonea, are newly described based on their distinct phylogenetic relationships and the comparison of morphological characters with known Tremella species. Our results indicate a high species diversity of Tremella waiting to be discovered.


Introduction
Tremella Pers. has been traditionally considered to be the largest and most polyphyletic genus in Tremellaceae (Fell et al. 2000;Scorzetti et al. 2002;Sampaio et al. 2004;Boekhout et al. 2011;Millanes et al. 2011;Weiss et al. 2014;Liu et al. 2015a). The members of Tremella sensu lato are dimorphic fungi that contain both a haploid unicellular yeast stage and a dikaryotic filamentous stage. This genus is characterized by its mycoparasitic lifestyle and comprises species growing on the hymenium of Corticiales,

Sampling and morphological examination
Specimens were collected from Guangdong, Guangxi, Heilongjiang, Jilin, Qinghai, Tibet, and Yunnan provinces in China. The specimens were air dried immediately after their collection. Macromorphological descriptions were based on field observations. Micromorphological examination followed the studies by Chen (1998) and Millanes et al. (2014). Microscopic structures, features, and measurements were observed using handmade sections stained with 1% Phloxin after pretreatment with 5% KOH and photographed with Zeiss AXIO Imager A2 coupled to an AxioCam MRc5 digital camera. Basidiospores and conidia measurements are present as follows: length range × width range, L = mean spore length (arithmetic average of all spores), W = mean spore width (arithmetic average of all spores), Q = variation in the L/W ratios and n = number of spores measured. All specimens were preserved in the XZL culture collection (personal culture collection of Xin-zhan Liu housed in the Institute of Microbiology, Chinese Academy of Sciences). Type specimens were deposited in Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS). The cultures were deposited in China General Microbiological Culture Collection Center (CGMCC) and the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands.

DNA extraction, PCR amplification and sequencing
DNA was extracted directly from the specimens examined. A very small amount of dry tissue was soaked in sterile water for 30 min and dried with sterile filter papers. The tissue was taken into 2 ml eppendorf tube with quartz sand (1-2 mm), lyophilized using liquid nitrogen and immediately crushed with tissue grinder for 2 min using SCIENTZ-48 at 70 Hz (SCIENTZ, China). The sample was homogenized in 1 ml 5% CTAB preheated at 65 °C. The mixture was warmed up at 65 °C for 1 h and centrifuged by 15000 rpm for 15 min. The supernatant was purified with phenol:chloroform:isoamyl alcohol (25:24:1) for twice of which the second purification step without phenol. The supernatant was incubated for 30 min at 37 °C with 25 μl RNAase (20 mg/ml) and then purified again. The precipitation with 3 M sodium acetate and ethyl alcohol absolute was conducted. Finally, the DNA was washed twice with 70% (w/v) ethanol and then dissolved in 50 μl of pure water. The nuc rDNA ITS region and D1/D2 domain of nuc 28S rDNA were amplified using the protocols described previously (Liu et al. 2015a). PCR products were observed on 1% Agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of PCR products were carried out at TSINGKE Biological Technology, Beijing, China.

Phylogenetic analyses
Phylogenetic analyses were performed as described previously with modification (Millanes et al. 2011;Liu et al. 2015a, b). Vishniacozyma carnescens CBS 973 T was chosen as outgroup because the genera Vishniacozyma is the sister group of Tremellaceae (Liu et al. 2015a, b). Four partitions, i.e., ITS1, 5.8S, ITS2 and D1/D2 domain, were chosen as the appropriate scheme (Millanes et al. 2011;Zamora et al. 2017). Multiple sequences were aligned using MAFFT algorithm and the G-INS-I option (Standley 2013). Major insertions and ambiguous regions were identified and eliminated with Gblocks version 0.91b (Castresana 2000) using a relaxed selection (minimum number of sequences for a conserved position = 36, minimum number of sequences for a flank position = 60, maximum number of contiguous non-conserved positions = 10, minimum length of a block = 5 and allowed gap positions = 'with half'), following Talavera and Castresana (2007). PartitionFinder V2.1.1 (Lanfear et al. 2017) was used to determine the best-fit evolutionary model for each partition, with the following settings: the 'all' search algorithm, the corrected Akaike Information Criterion (AICc) for model selection and either the 'raxml' or 'mrbayes' set of models.
Dataset congruence was assessed manually by analyzing the datasets separately by maximum likelihood bootstrapping. Conflict among clades was considered significant if a significantly supported clade (bootstrap support ≥ 70%; Hillis and Bull 1993) for one marker was contradicted with significantly supported by another. Incongruence was found between topologies derived from ITS1, 5.8S, ITS2, and D1/D2 domain.
Maximum likelihood (ML) analyses of single gene were performed in RAxML-HPC V.8 (Stamatakis 2014) on the CIPRES Science Gateway (Miller et al. 2010). The GTR+G, GTR+G, GTR+I+G and GTR+I+G models were applied to each partition. The bestscoring tree was obtained using rapid bootstrap analysis by running 1000 replicates. Four single-gene trees estimated above were then used as input to infer the species tree with the coalescent-based approach implemented in the ASTRAL program v5.6.3 (Mirarab and Warnow 2015). The bootstrapping option of ASTRAL was used for 1000 replicates.
Bayesian analyses were conducted by Markov Chain Monte Carlo (MCMC) sampling for combined nucleotide sequences using MRBAYES 3.2.2 (Ronquist et al. 2012) on the CIPRES Science Gateway (Miller et al. 2010). Likelihood models were selected for each of the four gene partitions among the 24 models implemented in MrBayes. A HKY+I+G model was selected for the ITS1, a K80+G model was selected for the 5.8S, a SYM+I+G was selected for the ITS2 and a GTR+I+G model was selected for D1/D2 domain. Two independent runs were executed, each with four chains, three of which were incrementally heated. The analysis was conducted for 5 million generations with trees sampled every 5000 generations. The first 25% trees, which represent the burn-in phase of the analysis, were discarded after checking for stability on the log-likelihood curves and the split-frequencies of the runs in Tracer v.1.7 (Rambaut et al. 2018). The remaining trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree.
Branches that received bootstrap values (BP) for Maximum likelihood and Bayesian posterior probabilities (BPP) greater than or equal to 50% (BP) and 0.95 (BPP) were considered as significantly supported. The GenBank accession numbers for the sequences of the ITS region and D1/D2 domain used in this study are listed in Table 1.

Phylogenetic analyses
The combined dataset consisted of ITS1 region (44 bp), 5.8S region (156 bp), ITS2 region (168 bp), and D1/D2 domain (532 bp) (a total of 900 bp) for 57 specimens and 13 strains in genus Tremella with Vishniacozyma carnescens CBS 973 T as the outgroup. Two methods for phylogenetic tree construction resulted in a similar topology. Therefore, only the best scoring RAxML tree is shown with BP and BPP values simultaneously in Figure  Type. CHINA, Fujian Province, Ningde city, Gutian county, on the basidioma of Tremella fuciformis, July 2017, X.Z. Liu (holotype strain: CGMCC 2.5724 T , ex-holotype strain: CBS 15261 T ). Etymology. Basidiomaticola refers to the species isolated from the basidioma of T. fuciformis.
Habitat. On wood of deciduous tree, in forest dominated by Rosaceae, Moraceae, Lauraceae, and Theaceae.
Notes. Only one specimen representing T. salmonea formed a distinct clade closely related to T. taiwanensis with 96.8%-98.3% sequence identities in D1/D2 domain and 95.4%-96.6% in ITS region, respectively. The affinity of T. salmonea to T. taiwanensis lacked high support by the coalescent-based method (Fig. 1). Tremella salmonea differs from T. taiwanensis in its larger basidia ( In addition, hyphae-like conidiogenous cells and dikaryotic conidia were observed in T. salmonea compared to monokaryotic conidia produced from apex of sterigmata (Chen 1998). Swollen cells were located in the hymenium in T. salmonea whereas they were absent in T. taiwanensis (Chen 1998).

Discussion
Tremella s. s. is characterized by their tremella-like basidiomata. Many morphological characteristics have been used in taxonomic studies of Tremella, including the shape, colour, and size of basidiomata, basidia, and basidiospores, as well as other features such as length of the stalks and sterigmata, spore formation of the basidia, conidia, swollen cells, and hyphidia (Chen 1998). However, morphology-based taxonomy of Tremella species is very complicated. Almost 30 macromorphological and micromorphological characters need to be checked for identification at the species level (Chen 1998). Morphological taxonomy cannot provide enough evidence of phylogenetic relationship. Morphologically, Tremella globispora resembles species in the Indercorata group by its pyriform to capitates basidia and its spores that are broader than long (Chen 1998). Nevertheless, it is more related to species in the Fuciformis group, based on molecular data (Chen 1998;Fell et al. 2000;Scorzetti et al. 2002;Liu et al. 2015b). The application of molecular phylogenetics has significantly benefited the systematics and taxonomy of Tremella. In the present study, four new species of genus Tremella are described from China based on both morphological and molecular data.
The fruiting bodies of fungi harbour diverse microbial community including bacteria, yeasts and filamentous fungi (Buzzini et al. 2005;Barbieri et al. 2007;Pacioni et al. 2007). Microbial habitants could have roles in the development of the fruiting body, such as mycelium growth, nutrition supply, antifungal activity, and flavour formation (Sbrana et al. 2002;Barbieri et al. 2007;Antony-Babu et al. 2013;Seung-Yoon et al. 2018). There have been a new bacterial species found in the fruiting body of T. fuciformis which can cause infection (Wen et al. 2016). Tremella basidiomaticola was isolated from the fruit body of T. fuciformis and their relationship and contributions to the growth of fruiting body remain unknown.
Tremella salmonea is highly supported as belonging to the Mesenterica group. Microscopically, T. salmonea and T. mesenterica are similar in that both species share loose a hymenial structure with abundant hyphidia. However, these two species have different basidiomata colour: in T. salmonea basidiomata are salmon-orange, whereas in T. mesenterica they are yellowish. Other species in the T. mesenterica group with similar basidiomata colour include T. roseolutescens (basidia 20-27 μm × 18-27 μm) and T. tropica (basidia 19-21 μm × 15-17 μm), but these are clearly different in the shape of their basidiomata and size of their basidia (Bandoni et al. 1996;Chen 1998;Roberts 2008).
A total of 33 specimens of Tremella s. s. were collected from seven provinces (Guangdong, Guangxi, Heilongjiang, Jilin, Qinghai, Tibet, and Yunnan), which span a large portion of China and have different climates, humidity, and vegetation types. This implies the genus is really diverse beyond current knowledge. Tremella s. s. showed a significant deviation from the optimal range calculated for the genus rank using the phylogenetic rank boundary optimization (RPBO) analysis that indicates great genetic variation between different species in Tremella s. s. (Liu et al. 2015b). Two subclades, namely Mesenterica and Fuciformis, are included in this genus and can be featured by distinct ecological and morphological characters (Chen 1998, Liu et al. 2015b). They could probably be reclassified as two separate genera in the future. Further studies with additional fresh collections will clarify the systematic of this genus and enrich the knowledge of distribution, abundance, and ecology of Tremella species.
Key to the whitish species in Tremella s. s.