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A Survey of Termitomyces (Lyophyllaceae, Agaricales), Including a New Species, from a Subtropical Forest in Xishuangbanna, China

Abstract A survey of mushrooms was conducted in Xishuangbanna, Yunnan Province, China, in the rainy season (May to October) of 2012, 2013, and 2014, during which 16 specimens of Termitomyces were collected. Preliminary macro- and micro-characteristics, together with ITS sequence data, showed that four of the specimens belonged to a new species (Termitomyces fragilis), while the other 12 belonged to T. aurantiacus, T. eurrhizus, T. globules, T. microcarpus, and T. bulborhizus. In this paper, T. fragilis is introduced as a species new to science based on morphological characterization and phylogenetic analyses. Macro- and micro- morphological descriptions, color photographs and line drawings of the new species, and a phylogenetic tree to show the placement of the new species are provided. T. fragilis is then compared with other closely related taxa in the genus Termitomyces.

Various species of Termitomyces are widely distributed in Africa and Southeast Asia [16][17][18][19], and several taxa have also been recorded in Central America [20]. Molecular phylogenetic analysis reveals that Termitomyces forms a monophyletic clade in Agaricales [21][22][23], although genetically they exhibit a degree of differentiation between Asia and Africa [6]. The southern part of the Yangzi River basin in China is rich in wild fungal resources such as Termitomyces [13,[24][25][26]. In the southwestern province of Yunnan, Termitomyces mushrooms are locally famed for their taste and association with termites. Locally they are known as "Jizong", meaning chicken-mushroom (due to the texture of the mushroom flesh), or "Yizong", meaning ant-planted mushroom [25,[27][28][29].
Xishuangbanna is a tropical county located in south Yunnan Province, China, with abundant tropical vegetation resources providing suitable habitats for a wide range of insects, and more specifically, termites [30]. Xishuangbanna is also home to a high diversity of Termitomyces species, which are collected for consumption and for sale in markets, providing additional income to rural households [31][32][33].
As a result of previous studies, more than 400 samples of Termitomyces have been deposited in different fungal herbaria in China [13], but sequence data, especially nuclear ribosomal internal transcribed spacer (ITS) gene, is largely unavailable in GenBank, and there are few publications relating to this genus. Many of the past research studies investigating Termitomyces have aimed to study their relationship with termites, ignoring the taxonomy and phylogeny of this genus [22,[34][35][36][37][38][39] Based on the need for further research on the Termitomyces genus, we set out to study the composition of Termitomyces species in a subtropical rainforest in Xishuangbanna, Yunnan Province, China. In this study, we provide molecular based descriptions of the new species; morphological descriptions of all the collected specimens; and a record of the related habitats.

Specimen collection and macro morphological descriptions
All 16 specimens were collected during the rainy season (May to October), from 2012 to 2014, in Mengsong, Xishuangbanna, Yunnan Province, China. The vegetation cover at the collection sites was dominated by Camellia sinensis var. assamica, Castanopsis mekongensis, Schima wallichii, and Quercus acutissima. Specimens were photographed in situ, then gathered and wrapped in aluminum foil, or kept separately in a collecting box in order to avoid mixing or crushing of specimens, and finally returned to the laboratory for further analysis and characterization. Odor and color changes upon bruising were recorded at the time of collection. Description of macro-characteristics, chemical testing, and further photographing of fresh samples were carried out soon after returning to the field station each day. This was done in accordance with the methodology described by Largent [43,44]. The Kornerup and Wanscher method was used to describe color terms, where a specific code is assigned to an observed color (chromotaxy) [45]. Specimens were dried below 40 C in a food drier, sealed in plastic bags, and deposited in the Kunming Institute of Botany herbarium (HKAS). Facesoffungi numbers and Index Fungorum numbers were obtained as detailed in Jayasiri et al. and Index Fungorum [46,47].

Micro morphological description
Micro-morphological features were documented by examining dried specimens following the methods of Largent [48]. For micro-morphological examination, sections were cut with a razor blade from dried specimens and mounted on slides in 5﹪ KOH and Congo red, and then observed, measured and illustrated using a compound microscope (Zeiss Axioskop 40). In the description of the basidiospores, "n" indicates the number of basidiospores (20 basidiospores per collection) which were measured; [a/b/c] in the taxonomic description refer to the following: a ¼ number of basidiospores measured, b ¼ number of fruiting bodies and c ¼ number of collections; Lm ¼ mean basidiospore length over a population of basidiospore; Wm ¼ mean basidiospore width over a population of basidiospore; Q¼"Length/width ratio" (l/W) of a basidiospore in side view; Qm ¼ average Q of all basidiospores measured.

DNA extraction, PCR, and sequencing
Genomic DNA was extracted from three dried specimens of the new species using a Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Ltd., Hangzhou, P. R. China). The ITS gene region of the new species was amplified. Amplification was performed in 25 ml volumes containing 1.0 ml template DNA, 9. The primers used for sequencing the whole ITS region were the same as those used in White et al. [49]. Amplified products were confirmed with 1% agarose gel electrophoresis stained with ethidium bromide. The amplified PCR fragments were sent to a commercial sequencing provider (Beijing Bai Mai Hui Kang Biological Engineering Technology Co., Beijing, P.R. China). The newly generated sequence data of the new species were deposited in GenBank.

Sequence alignment and phylogenetic analyses
The taxon information and GenBank accession numbers used in the molecular analyses are listed in Table 1. All the available reliable nrITS sequences were retrieved from the GenBank. Sequences for each species were aligned using Clustal X [50]. Alignments were manually adjusted to allow for maximum sequence similarity. Gaps were treated as missing data. Unweighted maximum parsimony (MP) analysis was performed using PAUP 4.0b10 [51]. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max trees were unlimited, branches of zero length were collapsed, and all multiple parsimonious trees were saved. Clade stability of the trees resulting from the parsimony analyses were assessed by bootstrap analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa [52].
Bayesian phylogenetic analyses were performed with MrBayes 3.2.2 [53][54][55]. Four Markov chains were run for 1,000,000 generations and sampled every 100th generation, resulting in 1000 trees. Those trees sampled prior to searches reaching a split deviation frequency value reaching 0.01 were discarded as the burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities (PP) of the individual clades. Trees were viewed in Treeview and exported to Photoshop [56].

Taxonomy of termitomyces fragilis
Among the 16 specimens that were collected from the study plots (see Table 2), four specimens are proposed to be a new species to science; of these, all four specimens were shown to be the same species, as described below.
Termitomyces fragilis L. Ye, Karun, J.C. Xu, K. D. Hyde and Mortimer sp. nov. As shown in Figure 1, Figure 2 and Figure 3. Diagnosis: The new species T. fragilis is characterized by small basidiocarps, with a hollow and fragile stipe. It grows in shallow soils that are rich in organic matter, and is associated with termites.   Fruiting on soil substrates, with rich tree leaf-litter covering, local name is "big Jizong" or "round-root Jizong". Found in mixed tea plantation dominated by Camellia sinensis var. assamica and Castanopsis calathiformis trees. T. microcarpus HKAS88901 HKAS88907 Fruiting on soil substrates, with mosses covering, local name is "Small Jizong" or "Jizong flower". Found in secondary forest dominated by Schimawallichii, Dendrocalamus membranaceus and Pueraria trees. Notes: The new species T. fragilis morphologically closely resembles the small basidiocarps of T. microcarpus, T. tylerianus, and T. clypeatus (see discussion).

Discussion
DNA from the 16 Termitomyces specimens was extracted and sequenced several times using primers (ITS (ITS1, ITS5, ITS4); SSU (SSUFW105, SSUREV4757); nLSU (LROL, LR5)) and PCR protocols based on recent references. However, only the ITS sequences from the collection of new species proved successful. The fresh samples collected in the first and second years gave good sequences, while the other older samples did not provide good sequence results, so we used only morphological descriptions to identify those specimens. The morphological characteristics clearly showed the remaining 12 specimens as belonging to five distinct, previously described species.
Termitomyces fragilis is described as having small to medium basidiocarps. We made morphological comparisons between our new species and three closely related species that also have small basidiocarps: T. microcarpus [25], T. tylerianus [57], and T. clypeatus [58]. Basidocarps of T. fragilis are morphologically very similar to T. microcarpus; basidiocarps of both species are small and generally, the pseudorhiza are absent. The colors of the pileus in the two species are very similar, but the color in the center part of the pileus of T. fragilis turns to reddish-gray upon bruising. Lamellae of T. microcarpus are white at first, becoming pinkish when mature, but in T. fragilis the lamella is white when young, becoming pinkish-white when mature. The stipe of T. microcarpus is solid and strong, but the stipe of T. fragilis is fistulose to broadly fistulose, and fragile. Basidiospores of T. microcarpus (5.5-8.0 Â 3.5-5.5 mm) are ovoid to ellipsoid, and smaller than in T. fragilis. The basidia of T. fragilis are bigger than those of T. microcarpus (16.0-26 Â 6.0-9.0 mm). The cystidia of the two species are similar, but T. fragilis has bigger cystidia than T. microcarpus (cheilocystidia 15.0-48 Â 9.0-20.0 mm; pleurocystidia 23-44 Â 9.0-26 mm). The diameter of pileipellis top cells of T. fragilis is the same as T. microcarpus (2.0-5.0 mm), but the second layer of cells are always wider [13]. Termitomyces fragilis is phylogenetically closely related to T. microcarpus (As shown in Figure 5), but the comparison of the ITS sequences shows there are 25 base pair differences between the two species.
Termitomyces clypeatus, another small basidiocarp species in Yunnan Province, is morphologically similar to T. fragilis. The pileus of Termitomyces clypeatus has a diameter of 2.5 cm, whereas in T. fragilis the pileus can be 2-5 cm. The stipe of T. clypeatus is usually longer and wider than that of T. fragilis (4.0-13.0 Â 0.5-1.8 cm), and the stipe of T. clypeatus is solid. Termitomyces clypeatus [59] has a long (up to 22 cm), cylindrical bottom stipe, which is much more developed than that of T. fragilis. Basidiospores of T. clypeatus (6.0-9.0 Â 4.0-6.0 mm) are smaller than those of T. fragilis. Basidia of T. clypeatus (15-28 Â 5.5-9.0 mm) are smaller than those of T. fragilis. The cystidia of T. clypeatus are similar in shape to those of T. fragilis, but smaller than those of T. Phylogenetic analyses also show that T. fragilis is closely related to T. microcarpus (as shown in Figure 5). According to the phylogenetic tree, the species most closely related to T. fragilis in the clade is an unidentified Termitomyces species from Japan. However, there are no descriptions, photo plates, or drawings available for the unidentified Termitomyces from Japan. This reflects the knowledge gaps which exist in Termitomyces research due to the limited availability of specimens, literature, and GenBank information. Additional termitomyces specimens collected Termitomyces aurantiacus [26], T. eurrhizus, T. globulus [59], T. microcarpus [25], T. bulborhizus [60] (as shown in Table 2), and T. fragilis were all found in Mengsong. Apart from T. fragilis and T. microcarpus, all of the aforementioned species (Figure 4) have large, solid basidiocarps, which are highly valued as edible mushrooms.
There are more than 20 Termitomyces species recorded in China, of which most of them have been found in Yunnan [61][62][63]. According to Zhang the Termitomyces species of Yunnan account for more than 80% of the records in China, and is close to half of the world's records, while the Termitomyces fruiting habitat in Yunnan is less than 10% of the world's total habitat, giving context to the degree of abundance and diversity of Termitomyces in Yunnan [63]. This high level of abundance and diversity is likely due to the high diversity of termite species in Yunnan Province, and more specifically, Xishuangbanna County, which has the greatest diversity of termites in China [64]. Bootstrap support values for maximum parsimony equal or higher than 50% are defined as right side above the nodes and Bayesian posterior probabilities equal or higher than 0.90 are given left side, above the nodes. Data were analyzed with random addition sequence and unweighted parsimony, and gaps were treated as missing data. The tree is rooted with Lyophyllum infumatum (JF908334) and L. connatum (HE819396).