﻿Morphological and molecular analyses reveal two new species of Termitomyces (Agaricales, Lyophyllaceae) and morphological variability of T.intermedius

﻿Abstract Two new species, Termitomycestigrinus and T.yunnanensis are described based on specimens collected from southwestern China. Termitomycesyunnanensis is morphologically characterized by a conspicuously venose pileus surface that is grey, olive grey, light grey to greenish grey at center, light grey towards margin, and a cylindrical white stipe. Termitomycestigrinus is morphologically characterized by a densely tomentose to tomentose-squamulose pileus showing alternating greyish white and dark grey zones, and a stipe that is bulbous at the base. The two new species are supported by phylogenetic analyses of combined nuclear rDNA internal transcribed spacer ITS1-5.8S-ITS2 rDNA (ITS), the mitochondrial rDNA small subunit (mrSSU) and the nuclear rDNA large subunit (nrLSU). The morphological variability of T.intermedius, including five specimens newly collected from Yunnan Province, China, is also discussed. The collections showed variability in colour of the stipe surface and in the shape of cheilocystidia when compared to the original description. Full descriptions of the two new species and of T.intermedius, as well as a taxonomic key to the 14 Termitomyces species reported from China are provided.


Introduction
Termitomyces R. Heim (1942a) was established based on the type species T. striatus (Beeli) R. Heim (Heim 1942a). Termitomyces species are characterized by their obligate symbiotic association with termites (Aanen et al. 2002). Most of species in this genus present a pseudorhiza connected to the termite nests, a usually conspicuous perforatorium (differentiated structure at the centre of the pileus, often in the form of a papilla or umbo), pinkish spore deposit, and smooth broadly ellipsoid to ellipsoid basidiospores (Mossebo et al. 2017;Izhar et al. 2020;Seelan et al. 2020;Usman and Khalid 2020). To date, 59 species of Termitomyces have been described worldwide (based on Index Fungorum, accessed on 17 January 2023), of which 14 are reported from China (Wei et al. 2004;Huang et al. 2017;Ye et al. 2019;Tang et al. 2020).
Termitomyces species are ecmically important and widely traded as food in the markets of tropical and subtropical areas (Parent and Thoen 1977;Mondal et al. 2004;Chandra et al. 2007;Ye et al. 2019). In India, Termitomyces species such as T. microcarpus (Berk. & Broome) R. Heim and T. heimii Natarajan have also been used for the treatment of diseases such as cold, fever, and fungal infections (Venkatachalapathi and Paulsamy 2016).
Recently, molecular phylogenetic approaches have increasingly been applied to investigate phylogenetic relationships among genera and species of Agaricales . Through these studies, Termitomyces was strongly supported as a genus in Lyophyllaceae, with close relationship to the genera Calocybe Kühner, Tephrocybe Donk, and Lyophyllum P. Karst. (Bellanger et al 2015;He et al. 2019). Sinotermitomyces M. Zang, originally described in southwestern China (Zang 1981), was also proven to be a synonym of Termitomyces based on the study of type material (Wei et al. 2006).
For the past 70 years, a number of new Termitomyces species have been described based only on morphological characteristics. The lack of good illustrations and/or of detailed descriptions made the taxonomy of Termitomyces complicated, until the advent of molecular phylogeny. Mossebo et al. (2017) provided molecular markers (nrLSU and mrSSU), bringing more evidence for the classification of Termitomyces species. Since then, a series of new Termitomyces species have been described from Asia based on combined molecular and morphological data (Ye et al. 2019;Izhar et al. 2020;Seelan et al. 2020;Tang et al. 2020;Usman and Khalid 2020).
During investigations of Termitomyces across southwestern China and Thailand, several Termitomyces collections were made. Amongst them, two Termitomyces species from Yunnan, China, are newly described herein. In addition to the morphological descriptions and illustrations, molecular phylogenetic analyses based on the ITS1-5.8S-ITS2, mrSSU and nrLSU supported the two new species.

Studied specimens
Eleven specimens were collected from Southwestern China. Collection locations were subtropical broad-leaved forests in Yunnan Province, where the annual average temperature is 12-22 °C, and the elevation is 1,000-3,500 m (Xiwen and Walker 1986). Three additional specimens were obtained on loan from the Herbarium of Meise Botanic Garden, Belgium (BR).

Morphological studies
Descriptions of macro-morphological characteristics and habitats were obtained from the photographs and notes. Colour codes were based on Kornerup and Wanscher (1978). Once the macromorphological characteristics were noted, specimens were dried at 40 °C in a food dryer until no more moisture was left, and stored in sealed plastic bags. For microscopy study, dried mushroom materials were sectioned and mounted in 5% KOH solution and 1% Congo red. Microscopic characters such as basidia, basidiospores, and cystidia were observed and photographed using a light microscope (Nikon eclipse 80i) equipped. For microscopic characters' descriptions, 60-100 basidiospores, 20 basidia, and 10 cystidia were randomly measured, the abbreviations [x/y/z] denote x basidiospores measured from y basidiomata of z collections, (a-) b-c (-d) denote basidiospore dimensions, where the range b-c represents 95% of the measured values while "a", and "d" are extreme values, L m and W m , the average length and width are also given with their standard deviations; Q refers to the length/width ratio of individual basidiospore while Q m refers to the average Q value ± standard deviation. Specimens of the two new Termitomyces species were deposited at the herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS) and Mae Fah Luang University herbarium (MFLU).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from dry specimens using Ezup Column Fungi Genomic DNA extraction Kit following the manufacturer's protocol. PCR amplification, PCR product purification, and sequencing. The primers used for nrLSU amplification were LR0R and LR5 (Vilgalys and Hester 1990). The mrSSU region was amplified with Termitomyces specific primer pairs viz. SSUFW105 and SSUREV475 (Aanen et al. 2002). The ITS gene region was amplified using the primers ITS1 or ITS5, and ITS4 (White et al. 1990).

Sequence alignment and phylogenetic analyses
A total of 29 newly generated sequences and 66 sequences from GenBank were used as ingroup and twelve sequences of Lyophyllum shimeji (Kawam.) Hongo, L. decastes (Fr.) Singer, Asterophora lycoperdoides (Bull.) Ditmar, and A. parasitica (Bull.) Singer retrieved from GenBank were used as outgroup (see Table 1). The outgroup taxa were selected based on the phylogeny in Hofstetter et al. (2014). The sequences were aligned with MAFFT version 7 (Katoh and Standley 2013) and checked in Bioedit version 7.0.5 (Hall 2007 Phylogenies and node support were first inferred by Maximum Likelihood (ML) from the three single-gene alignments separately, using RAxML-HPC2 version 8.2.12 (Stamatakis 2014) with 1,000 rapid bootstraps, as implemented on the Cipres portal (Miller et al. 2010). Since no supported conflict (bootstrap support value (BS) ≥ 70%) was detected among the topologies, the three single-gene alignments were concatenated using SequenceMatrix (Vaidya et al. 2011). Partitioned Maximum Likelihood (ML) analysis was performed on the concatenated data set, as described above. For Bayesian Inference (BI), the best substitution model for each character set was determined with the program MrModeltest 2.3 (Nylander 2004) on Cipres. The selected models were GTR+I+G for nrLSU, GTR+G for mrSSU, GTR+G for ITS1+ITS2, and K80 for 5.8S. Bayesian analysis was performed using MrBayes version 3.2.7a (Ronquist et al. 2011) as implemented on the Cipres portal (Miller et al. 2010). Two runs of six chains each were conducted by setting generations to 50,000,000 and using the stoprul command with the stopval set to 0.01; trees were sampled every 200 generations. A clade was considered to be strongly supported if showing a BS ≥ 70% and a posterior probability (PP) ≥ 0.90.

Phylogenetic analyses
The alignments of the nrLSU, mrSSU, 5.8S and ITS1+ITS2 sequences were 538, 354, 157, and 464 characters long after trimming, respectively. The combined data set had an aligned length of 1,516 characters, of which 946 characters were constant, 570 were variable but parsimony-uninformative, and 400 were parsimony-informative.
ML and BI analyses generated nearly identical tree topologies with little variation in statistical support. Thus, only the ML tree is displayed (Fig. 1). Phylogenetic data together with thorough morphological analysis (see below) showed that the two newly described taxa in this study are significantly different from other known Termitomyces species. Table 1. Names, specimen vouchers, origin, and corresponding GenBank accession numbers of the sequences used in this study. New species are shaded in gray and newly generated sequences are in bold; "*" following a species name indicates that the specimen is the type of that species and "N/A" refers to the unavailability of data.  Figure 1. Strict consensus tree illustrating the phylogeny based on the combined nrLSU, mrSSU, 5.8S and ITS1+ITS2 data set. Maximum likelihood bootstrap proportions equal to or higher than 70%, and Bayesian posterior probabilities equal to or higher than 0.90 are indicated at nodes. The two Asterophora species and two Lyophyllum species were used as the outgroup. The two newly described species are in red. Holotype specimens are in bold.
Habitat and distribution. Basidiomata scattered on soil with decaying litter under which termites have built their nest. Occurring in summer. So far only known from southwestern China.
Notes. Termitomyces tigrinus is distinguished from other Termitomyces by densely tomentose to tomentose-squamulose pileus with regularly alternating greyish white and dark grey zones, and a small, dark grey perforatorium as an acute papilla, two conspicuously different types of basidia, broadly ellipsoid to ellipsoid basidiospores, clavate, thin-walled cheilocystidia that are rare (HKAS 107560), or absent (in HKAS 107561).
Habitat and distribution. Solitary above underground termite nests; basidiomata occurring in summer. Known from southwestern China. Notes. Termitomyces yunnanensis is distinguished from other Termitomyces species by its clearly striated pileus surface, medium grey, olive grey, light grey to greenish grey at center, light grey towards margin on the pileus surface; perforatorium dark grey and umbonate, thin-walled or thick-walled basidia, ellipsoid, obovoid to broadly clavate cheilocystidia and pleurocystidia.

Key to species of Termitomyces reported from China
To date, 14 Termitomyces species have been reported from China. However, the identification of some species, namely T. aurantiacus, T. eurrhizus, T. entolomoides, T. globulus, T. mammiformis and T. tylerianus, was based on morphology only. Further studies using DNA sequence analyses are required to confirm or inform the presence of those species in China.

Discussion
In this study, we combined sequences of three non-translated loci (nrLSU, mrSSU and ITS) to carry out phylogenetic analyses of Termitomyces species in order to investigate the phylogenetic relationships between the two new species we described and other Termitomyces species.
Most Termitomyces species have uniform morphology, although some show extensive variability. In this study, five T. intermedius specimens were collected from Yunnan Province, China and showed differences in stipe surface colour and cheilocystidia shape when compared to the holotype of T. intermedius from Japan (Terashima et al. 2016). However, the latter, and our collections had identical DNA sequences (see above notes), which indicates their conspecificity. Termitomyces le-testui (Pat.) R. Heim, T. microcarpus (Berk. & Broome) R. Heim, T. striatus, and T. schimperi were also reported to be morphologically variable, with multiple formae described (See Index Fungorum). However, some specimens identified as T. striatus (DM280, DM151, BR5020212704478V, BR5020168468769 and BR5020169404421), despite showing similar morphology, clustered in different species-level clades in our phylogeny. Because of this morphological variability in some Termitomyces species, species identification or delineation should not be based only on morphology. Molecular analyses are also necessary to resolve the relationship between Termitomyces species.
In China, Termitomyces species are considered as delicacies, widely collected and consumed by local people, usually stir-fried with chili, bacon and garlic. They are called "Jizongjun" in Chinese, which means the taste of chicken. Termitomyces species are considered nutritious (a good source of proteins, lipids, crude fibres and minerals) for a daily healthy diet (Kansci et al. 2003). Termitomyces are an important source of income for people from rural areas of China. Termitomyces tigrinus, T. intermedius and T. yunnanensis are commonly found in mushroom markets from July to September and often sold around 120-200 RMB/kg.
To  (Wei et al. 2004;Huang et al. 2017;Ye et al. 2019;Tang et al. 2020). These species are mainly distributed in southern part of China.
Termitomyces tigrinus and T. yunnanensis are widely distributed in the subtropical broad-leaved forests of Dali, Yuxi, Baoshan, and Chuxiong in Yunnan, where the annual average temperature is 12-22 °C, and the elevation is between 1,000-3,500 m (Xiwen and Walker 1986). Termitomyces species form symbiotic relationships with termites in the subfamily Macrotermitinae, and their distribution thus depends on the presence of termites. In China, Yunnan, Guangxi and Hainan provinces have a tropical to subtropical climate suitable for termites, hence the abundance of Termitomyces species in those provinces.
"Yunnan Province Technology Innovation Talents Objects" (Project ID 2017HB084) and edible fungi industry system of China , and YCJ[2020]323. The authors appreciate the support given by Thesis Writing Grant of Mae Fah Luang University, Thailand, to S.-M. Tang. J. Degreef, A. Mukandera and the herbarium of Meise Botanic Garden, Belgium (BR) are also thanked for the loan of specimens.