The genus Termitomyces: Outline, phylogeny, and divergence times estimation with description of a new edible species from Arabian Peninsula

doi:10.21203/rs.3.rs-3599241/v1

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The genus Termitomyces: Outline, phylogeny, and divergence times estimation with description of a new edible species from Arabian Peninsula

https://doi.org/10.21203/rs.3.rs-3599241/v1

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The termite cultivated mushroom genus Termitomyces belongs to the family Lyophyllaceae (Agaricales, Basidiomycota). In this study, we presented a phylogeny of Termitomyces based on internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), large subunit of nuclear ribosomal DNA (28S) and translation elongation factor 1 alpha (TEF-1α). The ancestral age of Termitomyces was estimated around 23 million years. Molecular clock analyses also indicate the presence of two clades, each clade with approximate stem age of 18 million years. We think that these clades can be treated for the infrageneric classification of Termitomyces. Furthermore, we also described a new species in the genus, Termitomyces dhofarensis from Dhofar region, located in southern Oman. The new species is edible. Local people collect it for generations for food and even trade it in the local markets. The species is described here based on a combination of morphological features of basidiomata and multigene phylogenetic analyses. The divergence time is estimated to be around 1.2 million years for Termitomyces dhofarensis. This is the first report of the genus from Arabian Peninsula.

Dhofar

Oman

edible mushroom

molecular phylogeny

taxonomy

1 new species

The genus Termitomyces R. Heim belongs to the family Lyophyllaceae Jülich (Moncalvo et al. 2000). The mushroom genera in the family have diverse ecologies, encompassing saprotrophic, parasitic to symbiotic lifestyles (Hofstetter et al. 2014). The generic name ‘Termitomyces’ introduced by Roger Heim refers to the mutualistic symbiotic association of the fungus and termite species (Heim 1942). Species of termites taking part in symbiosis with Termitomyces belong to the subfamily Macrotermitinae. It is estimated that this symbiosis evolved approximately 30 million years ago in the rain forests of central Africa (Heim 1942; Aanen et al. 2002; Aanen & Eggleton 2005; Roberts et al. 2016). Since all the known species of Termitomyces form a monophyletic lineage in Lyophyllaceae, indicating a single successful domestication event likely occurred and no instances of these fungi reverting to a free-living state have been reported (Aanen et al. 2002; van de Peppel et al. 2021). This suggests a stable and long-standing symbiotic association between the fungus and the termites.

Over time, the fungus-growing termites have expanded their distribution range from central Africa to sub-Saharan Africa (Bignell et al. 2010). Among the eleven known genera of fungus-growing termites, four of them have been established in south and southeastern Asia (Nobre & Aanen 2012), leading to the extension of the Termitomyces fungi to Asia as well. It could be summarized that Termitomyces fungi have developed a remarkable and ancient mutualistic relationship with certain termite species, and this association has had a significant impact on the distribution and ecology of both organisms across Africa and Asia.

Termitomyces are cultivated by termites on a specific structure similar to honeycomb called the fungus comb or fungus garden which is made from the faeces of termites. It is hypothesized that symbiosis is initiated when wood debris containing the basidiospores of the fungus is consumed by the termites (van de Peppel et al. 2021). Upon excretion of faeces, the basidiospores germinate into asexual structures the nodules. The nodules are consumed by the termites. Nodules have asexual spores, conidia, upon consumption the conidia present in the nodules are mixed with the plant material in the gut of termites (Leuthold et al. 1989). The fungus comb constructed from the faeces having the conidia which propagates the fungus throughout the fungus garden. The nodules provide nutrition to the termite in the form of essential amino acids and also contain digestive enzymes which help in wood digestion (Martin & Martin 1978; Nobre & Aanen 2012; Chiu et al. 2019).

The genus Termitomyces typified with T. striatus (Beeli) R. Heim, with more than 100 records listed in the databases such as IndexFungorum (https://www.indexfungorum.org/names/) and MycoBank (https://www.mycobank.org/ e; access on July 4, 2023), respectively. However, it is unclear how many biological species are reported due to the presence of synonyms and illegitimate names in the records.

Species of Termitomyces are characterized by slimy cap surface, usually with a pointed disc called the perforatorium, free lamellae, pinkish spore print, a stipe with a long root-like structure called the pseudorhiza (Heim 1941; van de Peppel et al. 2021). However, morphological variation exists, particularly in the size of basidiomata. For example, Termitomyces titanicus Pegler & Piearce is considered the humongous mushroom in the world with a pileus diameter up to one meter, on the other hand, T. microcarpus (Berk. & Broome) R. Heim which has a cap diameter of less than two centimeters (Pegler 1977; Piearce 1987).

Taxonomy of Termitomyces primarily relies on morphological features of the basidiomata, including a cap size, shape, and color, a perforatorium shape, and a pseudorhiza color. However, in some species, the basidiomata rarely develop or even lost the ability to form a fruiting body (Wood & Thomas 1989, Darlington 1994, Bingham 2002). In such instances the actual diversity could only be explained by the combination of morphological features observed in laboratory culture and molecular data (de Fine Licht et al. 2006; Aanen et al. 2009, Poulsen et al. 2014), e.g., Termitomyces cryptogamus van de Peppel, a recently described species based on morphology of laboratory culture and sequence analyses of internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) of nuc ribosomal DNA (van de Peppel et al. 2021, 2022a). Recent studies have reported several species of Termitomyces from Africa and Asia based on a combination of morphological characters of basidiomata and molecular sequence data, specifically employing the large subunit of nuclear ribosomal DNA (28S) region for phylogenetic analyses (Froslev et al. 2003; Izhar et al. 2020; Jannual et al. 2020; Maity et al. 2020; Tang et al. 2020; Usman & Khalid, 2020; van de Peppel et al. 2021, 2022a; Ashraf et al. 2022; Kobayashi et al. 2022; Das et al. 2023; Tang et al. 2023).

All the known species of Termitomyces are safely edible (Li et al. 2021) and widely traded as food in the markets of tropical and subtropical areas (Mondal et al. 2004; Chandra et al. 2007; Ye et al. 2019; Paloi et al. 2023).

Divergence time estimation is a powerful tool in evolutionary biology that helps us understand the temporal aspects of species and group divergence, providing insights into the evolutionary history and relationships among different organisms including the mushroom-forming fungi (Zhao et al. 2016, 2022; Cui et al. 2018; He et al. 2019; Varga et al. 2019). In a recent study, using the small subunit of ribosomal DNA (rSSU) sequences, the genus Termitomyces has shown to diverged approximately 17 million years ago (Palma & Federizo 2022). However, in this study, we used two DNA regions (ITS, 28S) of larger number of species of Termitomyces, using the same internal calibration points to estimate the ancestral age of the genus.

During the macrofungal exploration of Oman in the years 2018 and 2022, we encountered some interesting specimens of Termitomyces. Upon morphological characterization of basidiomata, multigene phylogenetic analyses, and time divergence estimation, these collections represent an undescribed species, which is described in detail in this study.

Sampling and morphology

Fruiting bodies of Termitomyces were collected during the rainy season (Khareef), in the months of August and September, in Dhofar, the southern region of Sultanate of Oman. The climate in the area is described as arid with a hot desert climate (BWh) based on the Köppen climate classification system (Peel et al. 2007; Belda et al. 2014). This classification suggests that the region experiences high temperatures throughout the year and has very little precipitation. However, the Khareef season (from mid-June to mid-September) brings a notable increase in rainfall in the area. The vegetation in Dhofar region mainly consists of deciduous trees (Miller 1994).

The basidiomata were found on the termite nest, the whole fruiting body including the pseudorhiza was dug out using a digging tool, tagged, and photographed in their natural habitat. For color determination, we used the Munsell soil color charts (Munsell 1992). Specimens were dried using food dehydrator with temperature adjusted at 45°C, and then kept in zipper. The samples were stored at -80°C for two weeks to kill all the insect eggs/larvae. For microscopic study, small sections were cut using a sterile blade. Sections were mounted in 5% aqueous KOH (w/v). For contrast appearance, the sections were rehydrated in 1% Congo red (w/v). Anatomical characters such as basidiospores, basidia, cheilocystidia, pleurocystidia, pileipellis, etc. were noted under a compound microscope (ECLIPSE Ni-U, Nikon Co., Ltd., Japan). At least, 50 structures were measured in each instance. For measurements of microscopic structures Piximetre (http://ach.log.free.fr/Piximetre/) was used. The specimens are deposited in Oman Animal and Plant Genetic Resources Center (Mawarid), and a personal herbarium at Department of Plant Sciences, College of Agricultural and Marine Science, Sultan Qaboos University, Sultanate of Oman (AA).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from dried specimens using X-AMP DNA reagent kit (Dubuque, Iowa, USA). Internal transcribed spacer (ITS), large subunit of nuc rDNA (28S) and translation elongation factor 1 alpha (TEF-1α) gene were amplified. The primer combinations were: ITS1F and ITS4 (White et al. 1990), LR0R and LR5 (Cubeta et al. 1991), EF1-983 and EF1-1567R (Rehner & Buckley 2005) for the amplification of ITS, 28S and TEF-1α, respectively. The PCR reactions were performed using PuReTaqTM Ready-To-Go PCR beads (GE Healthcare UK Limited, Buckinghamshire, UK), with 1.0 µL of each primer (10 µmol/L), 2 µL DNA template, and 22 µL Nuclease free water. The condition and amplification protocols (polymerase chain reaction PCR) were as described in Chen et al. 2017). The PCR products were purified and sequenced using the same primers at Macrogen Inc. © (Seoul, Republic of Korea).

Sequence alignment, phylogenetic analyses, and divergence times estimation

Sequences generated in this study were inspected and both forward and reverse primer reads were converted into a consensus sequence using BioEdit v. 7.2.5 (Hall 1999). Only a few sequences of TEF-1α region are available in GenBank, therefore, for phylogenetic analyses, we constructed a dataset comprised of ITS-28S sequences. The combined ITS-28S dataset consisting of those sequences which were published in the recent studies of Termitomyces (Frøslev et al. 2003; Mossebo et al. 2017; Ye et al. 2019; Izhar et al. 2020; Usman & Khalid, 2020; van de Peppel et al. 2021, 2022a; Ashraf et al. 2022; Kobayashi et al. 2022; Das et al. 2023; Ediriweera et al. 2023; Tang et al. 2020, 2023). Alignment of the dataset was done using MAFFT algorithm at http://www.ebi.ac.uk/goldm an − srv/webpr ank/ (Madeira et al. 2019) using default settings. The dataset was comprised of 60 specimens, representing 35 species of Termitomyces and Arthromyces claviformis (DR-2983) was chosen as the outgroup taxon as it belongs to the Termitomycetoid clade (van de Peppel et al. 2022b). Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic analyses were tested. The ML phylogeny was analyzed using RAxML-VI-HPC, implemented on Cipres Science Gateway (Miller et al. 2010). The ModelFinder software (Kalyaanamoorthy et al. 2017) was used to choose the best-fit model (TIM2 + F + G4) for the dataset. Bayesian Inference (BI) analysis was done using MrBayes v. 3.1.2 (Ronquist & Huelsenbeck 2003). Two independent chains were run, each consisting of six Markov chains. The chains were run for one million generations and sampled every 100th generations. Burn-in was determined by checking the likelihood trace plots in Tracer v. 1.6 (Rambaut et al. 2014) and subsequently discarded.

Time divergence was estimated in BEAST v. 1.8.2 (Drummond et al. 2012) using the ITS-28S dataset. The XML input file was generated in BEAUti v. 1.8.2 (Bayesian Evolutionary Analysis Utility; (Drummond et al. 2012). Prior’s parameters were adjusted according to the fossil-calibrated analysis of Palma & Federizo (2022). A lognormal relaxed molecular clock was used, specifying uniform distribution for the ulcd.mean parameter with initial values 0.033, upper set 1.0, lower set 0.0. On the calibrated nodes, a normal distribution was specified for treeModel.rootHeight parameter with a mean age of 30 My (million years) and a standard deviation of 1 My. Four independent Monte Carlo Markov Chain of 20 million generations each were run, logging states every 5000 generations by BEAST v. 1.8.2 (Drummond et al. 2012). The log files were inspected in Tracer v. 1.6 (Rambaut et al. 2014) to ensure ESS (effective sample sizes) values for all parameters were well over 200. The tree files were combined using the LogCombiner 1.8.2 (Drummond et al. 2012). An ultrametric maximum-clade-credibility (MCC) tree was summarized using TreeAnnotator 1.8.2 (Drummond et al. 2012), discarding 10% of states as burn-in and annotating clades with ≥ 0.9 posterior probability.

FigTree 1.4.2 (Rambaut 2012) was used for phylogenetic tree visualization, followed by tree annotation using Adobe Illustrator CC2019. The alignment is submitted to TreeBase (TB2:S30563). Sequences employed in the phylogeny of this study are given in Table 1.

Table 1

Taxa used in phylogenetic analyses are presented as appeared as in the phylogenetic tree, with herbarium number, GenBank accessions, country of origin, and references, bold fonts are the sequences of the new species.
Fungal species	Specimen Voucher	ITS	28S	Country	Ref.
Termitomyces fragilis	HKAS 88912	KY214475	-	China	Ye et al. (2019)
T. fragilis	HKAS 88909	KY214476		China	Ye et al. (2019)
T. sheikhupurensis	SKP124	MT192217	MT192228	Pakistan	Izhar et al. (2020)
T. pakistanicus	strain-S3	OP688123		Pakistan	Razaq et al. (2023)
T. pakistanicus	strain-AN	OP688124		Pakistan	Razaq et al. (2023)
T. umkowaan	HUH-SH5	KJ703245		Pakistan	Hussain et al. (2015)
T. acriumbonatus	LAH36362	MT179687	MT179690	Pakistan	Usman & Khalid (2020)
T. acriumbonatus	LAH36363	MT179688	MT179689	Pakistan	Usman & Khalid (2020)
T. robustus	KM142418		KY809265	Tanzania	Mossebo et al. (2017)
T. robustus	DM436		KY809271	Cameroon	Mossebo et al. (2017)
T. yunnanensis	HKAS_124501	OP179295	OP168083	China	Tang et al. (2023)
T. yunnanensis	HKAS_124502	OP179296	OP168084	China	Tang et al. (2023)
T. subumkowaan	DM260F		KY809239	Cameroon	Mossebo et al. (2017)
T. subumkowaan	DM260B		KY809275	Cameroon	Mossebo et al. (2017)
T. islamabadensis	LAH36788	MW520178	OM100949	Pakistan	Ashraf et al. (2022)
T. islamabadensis	LAH36789	MW520179	OM100950	Pakistan	Ashraf et al. (2022)
T. heimii	JM-leg.S.Muid.s.n		AF042586		Moncalvo et al. (2002)
T. intermedius	HKAS_117638	ON557369	ON556484	China	Tang et al. (2023)
T. intermedius	HKAS_117639	ON557370	ON556485	China	Tang et al. (2023)
T. tigrinus	HKAS_107560	MT683156	MT679729	China	Tang et al. (2023)
T. tigrinus	HKAS_107561	MT683157	MT679730	China	Tang et al. (2023)
T. radicatus	MRNo173	LC068787		Thailand	Ye et al. (2019)
T. clypeatus	TERM062	MN160316		Thailand	Maity et al. (2020)
T. striatus	BR5020169404421	OP179299	OP168080	Congo	Tang et al. (2023)
T. sagittaeformis	KM109566		KY809260	South Africa	Mossebo et al. (2017)
T. striatus	BR5020212704478V	OP179298	OP168082	Mali	Tang et al. (2023)
T. assamicus	GUBH20038	OQ346313		India	Das et al. (2023)
T. assamicus	GUBH20039	OQ976999		India	Das et al. (2023)
T. striatus f. bibasidiatus	DM280B		KY809241	Cameroon	Mossebo et al. (2017)
T. striatus f. subclypeatus	DM370B		KY809268	Cameroon	Mossebo et al. (2017)
T. striatus f. subclypeatus	DM151C		KY809242	Cameroon	Mossebo et al. (2017)
T. striatus	BR5020168468769	OP179297	OP168081	Rwanda	Tang et al. (2023)
T. entolomoides	MU67	EF091679		Thailand	Sawhasan et al. (2011)
T. dhofarensis	GOB-18-001	OR297690	-	Oman	This study
T. dhofarensis	GOB-18-008	OR297691	-	Oman	This study
T. dhofarensis	GOB-18-010	OR297692	-	Oman	This study
T. dhofarensis	QRN-18-005	OR297693	-	Oman	This study
T. dhofarensis	NHZ-22-001	OR297694	-	Oman	This study
T. dhofarensis	RAK-22-0025	OR297695	OR338597	Oman	This study
T. dhofarensis	JRZ2-22-0020	OR297696	OR338598	Oman	This study
T. brunneopileatus	DM392		KY809273	Cameroon	Mossebo et al. (2017)
T. brunneopileatus	DM394		KY809244	Cameroon	Mossebo et al. (2017)
T. globulus	DM770		KY809252	Cameroon	Frøslev et al. (2003)
T. upsilocystidiatus	MFLU 19-1289	MT683160	MN636637	China	Tang et al. (2020)
T. medius f. ochraceus	DM602B		KY809246	Cameroon	Mossebo et al. (2017)
T. floccosus	MFLU 19-1312	MT683161	MN633305	Thailand	Tang et al. (2020)
T. cylindricus	TERM045	MN160299		Thailand	Jannual et al. (2020)
T. bulborhizus	KM128338		KY809261	China	Mossebo et al. (2017)
T. gilvus	BORH-FUMS-A03	MK472701		Malaysia	Seelan et al. (2020)
T. mammiformis	DM25E		KY809229	Cameroon	Mossebo et al. (2017)
T. mammiformis	DM25G		KY809230	Cameroon	Mossebo et al. (2017)
T. singidensis	tgf74		AY232713	Tanzania	Frøslev et al. (2003)
T. mboudaeinus	DM223E		KY809237	Cameroon	Mossebo et al. (2017)
T. aurantiacus	DM152E		KY809234	Cameroon	Willis (2007)
T. aurantiacus	tgf 82		AY127804	Tanzania	Mossebo et al. (2017)
T. striatus	KM142436		KY809267	Malawi	Mossebo et al. (2017)
T. le-testui	DM150G		KY809231	Cameroon	Mossebo et al. (2017)
T. le-testui	KM128346		KY809263	China	Mossebo et al. (2017)
T. schimperi	DM24E		KY809228	Cameroon	Mossebo et al. (2017)
Arthromyces claviformis	DR-2983	OM905928	OM905980	Dominican Republic	van de Peppel et al. (2022b)

Phylogenetic analyses and divergence times estimation

The combined ITS-28S dataset consisted of 1197 aligned characters, of which 657 characters were constant, 399 were parsimony-informative, and 141 uninformative sites. The ML and BI phylogenies revealed similar topologies. The ML phylogeny is represented (Fig. 1), with values from both ML and BI analyses above the node. The clades at higher rank received a moderate to significant statistical support, however, clades representing individual species were significantly supported. The proposed new species Termitomyces dhofarensis represented by seven specimens (7 ITS and 2 28S sequences) formed a unique lineage in a clade consisted of Central African and Asian species. The phylogenetic relationship among the species of Termitomyces are not well-resolved using ML and BI phylogeny. However, the time divergence analyses reflected a better phylogeny of the genus (Fig. 2). In the MCC tree the stem of age of Termitomyces is estimated around 23 My. Species of the genus recovered in two clades, T-I and T-II (T refers to Termitomyces). Both these clades evolved approximately 18 My ago. Sequences representing the new species Termitomyces dhofarensis fall in clade T-II. The divergence time of Termitomyces dhofarensis is estimated around 1.2 My ago, possibly in the middle Pleistocene Epoch.

Taxonomy

Termitomyces dhofarensis S. Hussain, Al-Yahya’ei, A. Al-Owaisi & Al-Sadi, sp. nov. – Figs. 3–5.

MycoBank no.: MB 849497

Holotype. – SULTANATE of OMAN. Dhofar: Salalah, Wadi Naheez, on termite nests, 07 August 2022, S. Hussain, Al-Yahya’ei, A. Al-Owaisi & Al-Sadi, NHZ-22-001 (holotype Mawarid-NHZ-22-001), GenBank accessions: ITS = OR297694.

Description. – Basidiomata medium-sized. – Pileus 50–100 mm in diam, at first convex later on plano-convex, at maturity becoming plano-concave with a depressed to umbilicate center, in some cases remain convex with a papillate center at maturity; surface smooth and shiny, in some specimens radially fibrillose, light olive (7.5Y 7/4–10Y 5/4) to moderate olive (10Y 5/8–10Y 5/12) or pale brownish (10YR 7/4), at center olive brown (2.5Y 5/2– 2.5Y 3/4); margin inflexed when young, becoming straight or reflexed when mature; perforatorium an umbo with obtuse or acute tip, grayish olive (10Y 5/2–10Y 6/2); context 2–3 mm thick half-way to the margin, tough, white. – Lamellae free, creamy to pinkish, ventricose, crowded with 1–4 series of lamellulae, 3–5 mm wide, lamellar edge fimbriate to eroded. – Stipe 40–120 × 10–15 mm, central, cylindrical, slightly sub-bulbous at the base, sparsely scabrous, pale brownish (10YR 7/4), context solid. – Annulus absent. – Pseudorhiza 30–70 mm long, terete, tapering downwards, surface moderate yellowish-brown (10YR 6/4), longitudinally striate, context solid. – Order mushroomy, taste pleasant.

– Basidiospores 6.5–8.0 × 4.5–5.0 µm, on average 7.0 ± 0.3 × 4.5 ± 0.2 µm, Q = 1.43–1.7, av. Q = 1.6, ellipsoid to broadly ellipsoid, thick-walled, apiculus visible, germ pore absent, smooth, hyaline in KOH. – Basidia 24–30 × 7–10 µm, on average 27.5 × 8.0 µm, clavate to cylindrical, with small oil droplets, hyaline in KOH, mostly with 4-spored, rarely 2-spored. – Lamellar trama regular, parallel, 100–200 µm in diam, comprised of thin-walled, clavate to ellipsoid inflated cells 15–26 µm in diam, filamentous hyphae abundant, 4–9 µm in diam. – Subhymenium 10–15 µm in diam, comprised of 1–2 layers of fusiform to subglobose, or ellipsoid cells, measuring 8–12 × 4–7 µm. – Cheilocystidia abundant, 35–50 × 13–20 µm, on average 42.3 × 16.8 µm, fusiform to broadly fusiform, with a leptocystidiate neck, neck sometimes septate measuring 15–23 × 4–6 µm, smooth, thin-walled, hyaline in KOH. – Pleurocystidia 33–45 × 15–25 µm, on average 39 × 21 µm, ellipsoid to broadly clavate, smooth, thin-walled, hyaline in KOH. – Pileipellis intricate trichoderm to irregular trichoderm, hyaline to pale brownish in KOH, terminal elements 22–40 × 5–10 µm, with obtuse apex. – Clamp connections absent.

Etymology. –The specific epithet ‘dhofarensis’ refers to Dhofar, the type locality the new species.

Habitat or host plant. – Occurring solitary on termite nests, basidiomata emerge in summer, from July to September.

Distribution. – Since this is the new species, therefore, currently only known Dhofar region, located in southern part of Oman.

Other materials examined. – SULTANATE of OMAN. Dhofar: Salalah, Wadi Jarzeez, on termite nests, 11 August 2022, S. Hussain, Al-Yahya’ei & A. Al-Owaisi, JRZ2-22-0020 (Mawarid-JRZ2-22-0020), GenBank accession: ITS = OR297695, 28S = OR338598, TEF-1α = OR360511; Salalah, Rakhyoot, on termite nests, 10 August 2022, S. Hussain, Al-Yahya’ei & A. Al-Owaisi, RAK-22-0025 (Mawarid-RAK-22-0025), GenBank accession: ITS = OR297696, 28S = OR297697, TEF-1α = OR351393; Salalah, Googeb, on termite nests, 02 September 2018, Al-Sadi & Al-Kharousi, GOB-18-001 (AA GOB-18-001), ITS accession = OR297690; same area, same date, Al-Sadi & Al-Kharousi, GOB-18-008 (AA GOB-18-008), ITS accession = OR297691; same area, same date Al-Sadi & Al-Kharousi, GOB-18-010 (AA GOB-18-010), ITS accession = OR297692; Salalah, Qiron, on termite nests, 04 September 2018, Al-Sadi & Al-Kharousi, QRN-18-005 (AA QRN-18-005), ITS accession = OR297693.

Comments. – The new species Termitomyces dhofarensis is characterized by medium-sized basidiomata, a pale olive pileus, a slightly pointed perforatorium, a stipe scaly, a pseudorhiza comparatively shorter than the stipe, and microscopically with leptocystidiate cheilocystidia. Using ML analyses based on combined ITS-28S sequence data, the new species Termitomyces dhofarensis is similar to T. brunneopileatus Mossebo & Essouman, T. globulus R. Heim & Gooss.-Font., and T. upsilocystidiatus S.M. Tang, Raspé & K.D. Hyde. Termitomyces brunneopileatus, originally reported from Cameroon (Central Africa), shares the pileus morphology to the new species. However, T. dhofarensis can be differentiated from T. brunneopileatus by several characters. The pileus margin is radially plicate to sulcate striate, basidiospores are smaller (4.5–6.0 × 2.6–3.5 µm) and subellipsoid, and pseudorhizae are longer (up to 600 mm) in T. brunneopileatus (Mossebo et al. 2017). However, in T. dhofarensis, a pileus margin is reflexed, basidiospores larger (6.5–8.0 × 4.5–5.0 µm) and a pseudorhiza is substantially smaller (70 mm), respectively. Termitomyces globulus originally reported from Cameroon (Heim 1951), differs from the new species by its large-sized basidiomata, (i) a broader pileus (100–200 mm in diam), (ii) a longer stipe (100–200 × 15–20 mm) and smaller basidiospores (5.0–7.0 × 3.5–4.5 µm). Similarly, Termitomyces upsilocystidiatus can be differentiated from the new species by its characteristic Y-shaped cheilocystidia and smaller basidiospores (5.0–6.7 × 3.6–4.1 µm; Tang et at. 2020). The new species is differing from Termitomyces cylindricus S.C. He as the later has a medium to large-sized basidiomata with a larger pileus (up to 120 mm in diam), a greenish pseudorhiza which is comparatively longer (125–170 mm), cheilocystidia clavate, measuring 25–40 × 7–13 µm (Pegler & Vanhaecke 1994). Another interesting species recently reported from Thailand Termitomyces floccosus S.M. Tang, Raspé & S.H. Li, has much smaller basidiomata, with pileus 50–60 mm in diam, cap surface with floccose squamules, with a broadly umbonate perforatorium (Tang et al. 2020). Termitomyces medius f. ochraceus Mossebo & Essouman is a small-sized taxon with cap 20–40 mm in diam, perforatorium is almost absent or present in a form of pseudo-umbo, thin-walled basidiospores measuring 5.8–7.5 × 3.8–5 µm (Mossebo et al. 2017).

All the known species of Termitomyces are symbiotically domesticated by a single group of termites of the subfamily Macrotermitinae. To date, no species of Termitomyces has been reported as a free-living fungus, which suggests that these mushrooms may have single symbiotic origin (De Duve 2005; Burnetti & Ratcliff 2020). This single domestication origin of Termitomyces may attribute to these five features: these mushrooms have a (i) reduced potential to degrade plant-cell wall components, (ii) the presence of pseudorhiza, (iii) the formation of asexual spores (conidia), (iv) the insect-faecal association, and (v) the loss of clamp connections (van de Peppel et al. 2021). Some of these features are shared by other members of Termitomycetoid clade, suggesting that the termitomycetoid mushrooms have a predisposition to domestication. Moreover, some of these features are reported in other mushrooms, such as formation of conidia in Asterophora Ditmar and the presence of pseudorhiza in Tricholomella Zerova ex Kalamees (Buller 1924; Kalamees 1992). It is suggested that these features allowed the ancestor of Termitomyces to successfully colonize the comb created by the most recent common ancestor of fungus-growing termites (van de Peppel et al. 2021).

In the last couple of years, several new species of Termitomyces have been reported from Asia, based on combination of morphological characters of basidiomata and molecular phylogeny (Izhar et al. 2020; Jannual et al. 2020; Tang et al. 2020, 2023; Usman & Khalid, 2020; Ashraf et al. 2022; Kobayashi et al. 2022; Das et al. 2023). It seems that the tropical and subtropical regions of Asia are rich in species diversity of the genus. Interestingly, all the known species of Termitomyces are considered edible, can be safely consumed as food, and has been classified as E1 (which means safely edible) according to system devised for the edibility of mushrooms (Li et al. 2021). These mushrooms are vital source of nutrition, widely traded as food in the markets of tropical and subtropical areas (Mondal et al. 2004; Chandra et al. 2007; Ye et al. 2019; Paloi et al. 2023).

Palma & Federizo (2022) estimated the ancestral species of Termitmyces originated at a mean stem of 17.47 My ago, suggested that the genus diverged in the Late Miocene. Our molecular clock analysis showed that Termitomyces emerged at a mean stem of 23.12 My ago (95% HPD of 12.75–30.82 My), which overlapped previous study (Palma & Federizo 2022). The two clades recovered in time divergence analyses of Termitomyces, clade T-I diversified with an estimated mean stem age of 18.23 My (95% HPD of 8.98–27.28 My) and clade T-II with mean age of 18.04 My (95% HPD of 8.08–27.35 My), respectively. It is hypothesized that species in both clades are emerged simultaneously, in congruent with the previous study (Palma & Federizo 2022). We think that these two clades could be use for the infrageneric classification of Termitomyces.

In this study, we also described a new species Termitomyces dhofarensis from Dhofar region, located in southern part of Sultanate of Oman. Locally, it is called “Qampa”. In the region this mushroom is recognized as a very precious mushroom due to its nutritional value and consumed as food for centuries. It is considered the main meal in the rainy season for the mountain communities. The fruiting season starts from late July to early September, with the onset of the monsoon rains in the region. Local people collect it, consume and even trade it in the markets. According to the local people, this mushroom was more abundant in the recent past, but now its population is declining. This decline may be due to anthropogenic activities. We think some legislation is necessary to protect this remarkable mushroom for future generations.

It is interesting to note that despite the arid climate of the Arabian Peninsula, the monsoon rainy season in Dhofar region provides a window of opportunity for various organisms, including unique mushrooms, to flourish and complete their life cycles.

Competing interests: The authors declare no competing interests.

Acknowledgements

We would like to thank Bader Al Quyudhi and Shamsa Al Balushi, Maryam Al Hinai for their invaluable help in the sampling trip to the Dhofar region which was essential to the success of our research. We would also like to thank Amer Qattan for his guidance and support throughout the project. His expertise in the region was invaluable, and his advice was always helpful. Thanks to the members of the Hiking Team Nabil Al Dhanki, Saif Al Salami, Mohammed Al Jahwari, and Ibrahim Al Sabahi for their help in the collection of mushroom Specimens in 2018. This research was funded by Agriculture and Fisheries Development Fund, Sultanate of Oman (grant number 104/1/1).

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The genus Termitomyces: Outline, phylogeny, and divergence times estimation with description of a new edible species from Arabian Peninsula

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