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Three Novel Entomopathogenic Fungi From China and Thailand.

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Affiliations

  • 1. CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.
      Authors
    • Wei DP 1, 2, 3
    • Wanasinghe DN 1
    • Xu JC 1
    • Mortimer PE 1
    • Karunarathna SC 1, 6
    • Tibpromma S 1, 6
    (6 authors)
  • 2. Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand.
      Authors
    • Wei DP 1, 2, 3
    • To-Anun C 2
    (2 authors)
  • 3. Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand.
      Authors
    • Wei DP 1, 2, 3
    • Hyde KD 3
    (2 authors)
  • 4. Department of Botany and Microbiology, College of Sciences, King Saud University, Riyadh, Saudi Arabia.
      Authors
    • Elgorban AM 4
    (1 author)
  • 5. Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka.
      Authors
    • Madawala S 5
    (1 author)
    • Show all (6)

ORCIDs linked to this article

Frontiers in Microbiology, 08 Jan 2021, 11:608991
https://doi.org/10.3389/fmicb.2020.608991 PMID: 33584571 PMCID: PMC7873960

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Abstract 

Entomopathogenic fungi are ubiquitous in tropical rainforests and feature a high level of diversity. This group of fungi not only has important ecological value but also medicinal value. Nevertheless, they are often ignored, and many unknown species have yet to be discovered and described. The present study aims to contribute to the taxonomical and phylogenetic understanding of the genus Paraisaria by describing three new species collected from Guizhou and Yunnan Provinces in China and Krabi Province in Thailand. The three novel species named Paraisaria alba, P. arcta, and P. rosea share similar morphologies as those in the genus Paraisaria, containing solitary, simple, fleshy stroma, completely immersed perithecia and cylindrical asci with thickened caps and filiform ascospores that often disarticulate at maturity. Phylogenetic analyses of combined LSU, SSU, TEF1-α, RPB1, RPB2, and ITS sequence data confirm their placement in the genus Paraisaria. In this study, the three entomopathogenic taxa are comprehensively described with color photographs and phylogenetic analyses. A synopsis table and a key to all treated species of Paraisaria are also included.

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Front Microbiol. 2020; 11: 608991.
Published online 2021 Jan 8. https://doi.org/10.3389/fmicb.2020.608991
PMCID: PMC7873960
PMID: 33584571

Three Novel Entomopathogenic Fungi From China and Thailand

De-Ping Wei,1,2,3 Dhanushka N. Wanasinghe,1,4,5 Jian-Chu Xu,1,4,5 Chaiwat To-anun,2 Peter E. Mortimer,1,5,6 Kevin D. Hyde,3,7,8,9 Abdallah M. Elgorban,10 Sumedha Madawala,11 Nakarin Suwannarach,12,13 Samantha C. Karunarathna,1,5,6,12,13,* Saowaluck Tibpromma,1,5,6,12,13,* and Saisamorn Lumyong12,13,14,*
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Abstract

Entomopathogenic fungi are ubiquitous in tropical rainforests and feature a high level of diversity. This group of fungi not only has important ecological value but also medicinal value. Nevertheless, they are often ignored, and many unknown species have yet to be discovered and described. The present study aims to contribute to the taxonomical and phylogenetic understanding of the genus Paraisaria by describing three new species collected from Guizhou and Yunnan Provinces in China and Krabi Province in Thailand. The three novel species named Paraisaria alba, P. arcta, and P. rosea share similar morphologies as those in the genus Paraisaria, containing solitary, simple, fleshy stroma, completely immersed perithecia and cylindrical asci with thickened caps and filiform ascospores that often disarticulate at maturity. Phylogenetic analyses of combined LSU, SSU, TEF1-α, RPB1, RPB2, and ITS sequence data confirm their placement in the genus Paraisaria. In this study, the three entomopathogenic taxa are comprehensively described with color photographs and phylogenetic analyses. A synopsis table and a key to all treated species of Paraisaria are also included.

Keywords: Insect fungi, Ophiocordycipitaceae, Paraisaria alba, Paraisaria arcta, Paraisaria rosea, taxonomy, Yunnan Province

Introduction

Entomopathogenic fungi are a group of unicellular or multicellular, heterotrophic, eukaryotic microorganisms that can enter into a parasitic relationship with parasitized insects, killing or otherwise disabling their hosts (Samson et al., 1988). They reproduce via sexual or asexual spores, or both (Mora et al., 2017). It is of global importance to survey and describe insect pathogens (Hyde et al., 2019). Entomopathogenic fungi can act as natural enemies of agricultural pests and play an important role in maintaining ecological balance (Fernández-Grandon et al., 2020; Sobczak et al., 2020). For example, fungal pathogens such as, Coelomomyces, Culicinomyces, and Lagenidium have the capacity to kill larva and adult mosquitoes, reducing their host population (Scholte et al., 2004). Some entomopathogenic fungi, e.g., Beauveria bassiana, Beauveria brongniartii, Metarhizium anisopliae, and Verticillium lecanii, have been developed as biocontrol agents usable against agricultural pests like aphids, locusts, grasshoppers and cockchafer in Africa and Europe (Roberts and Hajek, 1992; Shah and Pell, 2003). Beauveria bassiana and B. brongniartii were found to be especially safe bioinsecticides (Zimmermann, 2007). Additionally, some insect pathogens with pharmacological activities are frequently studied, such as Cordyceps militaris extract, which exhibits antitumor properties (Li et al., 2020). Cordyceps spp. have been utilized as therapeutic agents for metabolic-related disorders (Cao et al., 2020). Cordyceps cicadae has renoprotective effects on hypertensive renal injuries (Huang et al., 2020). Entomopathogenic fungi have important biotechnological applications (Hyde et al., 2019) and Paraisaria is no exception. Several studies have explored the importance of Paraisaria species, such as their antioxidative activity (Ma et al., 2012), nucleoside components (Suo et al., 2013), intracellular polysaccharide composition (Wang et al., 2019) and AGS gastric cancer cells anti-proliferation effects (Ye et al., 2015). Additionally, P. heteropoda reportedly produces anti-bacterial and anti-fungal compounds (Krasnoff et al., 2005). Experiments into optimal cultural conditions and nutritional sources were conducted by Sung et al. (2011). Applications of other species in this genus have been poorly studied.

Entomopathogenic fungi are phylogenetically diverse and taxonomically distributed in Ascomycota, Basidiomycota, Chytridiomycota, Entomophthoromycota, Microsporidia, Oomycota and Zygomycota (Vega et al., 2012; Araújo and Hughes, 2016; Mora et al., 2017). Different groups of entomopathogens usually develop respectively unique strategy to colonize their hosts (Mora et al., 2017). It is worth to mention that entomopathogenic taxa in Entomophthorales (Entomophthoromycota) enter into biotrophic relationships with their insect hosts, while those in Hypocreales (Ascomycota) can be hemibiotrophic at earlier stages and transform into saprophytism (Shah and Pell, 2003). The diversity, taxonomy and phylogeny of entomopathogenic fungi have been extensively studied recently (Aung et al., 2008; Mora et al., 2017; Hyde et al., 2018). Most insect pathogens are known from three families: Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae. They are found in the Hypocreales, Hypocreomycetidae, Sordariomycetes, Ascomycota (Sung et al., 2007a; Maharachchikumbura et al., 2016; Wijayawardene et al., 2018). The generic composition of Ophiocordycipitaceae underwent several changes over time (Sung et al., 2007a; Quandt et al., 2014; Maharachchikumbura et al., 2016; Shrestha et al., 2017; Wijayawardene et al., 2018), and currently ten genera are accepted (Hyde et al., 2020). New combinations of these genera were proposed for Polycephalomyces by Kepler et al. (2013), Tolypocladium by Quandt et al. (2014), Perennicordyceps by Matočec et al. (2014) and Drechmeria, Harposporium, Ophiocordyceps, and Purpureocillium by Spatafora et al. (2015). The genus Paraisaria was recently recovered in Ophiocordycipitaceae (Mongkolsamrit et al., 2019).

The genus Paraisaria was established by Samson and Brady (1983), with P. dubia as the type species, whose sexual morph was known as Ophiocordyceps gracilis (syn. Cordyceps gracilis). The sexual morph of this genus is characterized by solitary stromata with a stipe terminating in a globose or ellipsoid fertile head, completely immersed, ostiolate, gregarious perithecia, cylindrical asci and hyaline, filiform, multi-septate ascospores, which break into aseptate fragments when mature. Its asexual morphs are characterized by verticillate branched conidiophores, phialidic, flask-shaped, usually sympodially proliferating conidiogenous cells, which terminate in 1–4 necks, and aseptate, hyaline, smooth-walled conidia, which usually aggregate in slimy heads (Samson and Brady, 1983). Li et al. (2004) synonymized Isaria gracilioides under P. gracilioides and linked its sexual morph to Ophiocordyceps gracilioides. Evans et al. (2010) found the asexual morph of P. myrmicarum from a red ant host (Myrmica rubra) in a natural environment in the United Kingdom. Quandt et al. (2014) have dropped the genus Paraisaria and used its sexual genus Ophiocordyceps according to the ‘one fungus one name’ principle. Mongkolsamrit et al. (2019) resurrected Paraisaria on the basis of three new species, e.g., P. orthopterorum, P. phuwiangensis, and P. yodhathaii as well as eight new combinations, e.g. P. amazonica (Sanjuan et al., 2015), P. blattarioides (Sanjuan et al., 2015), P. coenomyiae (Ban et al., 2015), P. gracilioides (Kobayasi, 1941; Pérez-Villamares et al., 2017), P. gracilis (Samson and Brady, 1983; Pérez-Villamares et al., 2017), P. heteropoda (Sung et al., 2011; Mongkolsamrit et al., 2019), P. paramyrmicarum (= P. myrmicarum) (Evans et al., 2010) and P. tettigonia (Wen et al., 2016). So far, together with the three new species in this study, 14 species are accepted in Paraisaria.

This study is part of a larger survey of fungi in the Greater Mekong Subregion where we came across numerous new taxa (Hyde et al., 2018). In this study, three specimens of entomopathogenic fungi were collected from disturbed forests in China and Thailand, and the typical macro- and micro- morphological characteristics indicate that they are of the Paraisaria species. The multigene phylogenetic analysis of LSU, SSU, TEF1-α, RPB1, RPB2, and ITS confirmed their placement within Paraisaria as three distinct new species.

Materials and Methods

Sample Collection, Isolation, and Morphological Studies

In this study, a total of four fungal specimens were collected. One specimen (HKAS 102484) was collected from Krabi Province in Thailand on an adult cricket. Two specimens (HKAS 102553 and HKAS 102552) on dead larvae of Lepidoptera sp. were collected from Guizhou Province of China. One specimen (HKAS 102546) was collected from Yunnan Province in China on Coleoptera sp. larva. Among them, the hosts of specimens HKAS 102484, HKAS 102553 and HKAS 102552 were found completely immersed into soil with the stroma protruding from the ground in a forest. Specimen HKAS 102546 was found in a similar condition, but differed in that it was found under a karst stone formation. Macro-morphological characteristics of fresh collections were recorded with a camera (iPhone XS Max) in the field and then the specimens were transported to the laboratory in plastic boxes for subsequent studies. The culture of the specimen HKAS 102546 was created by transferring a small mass of mycelium inside the body of the host into potato dextrose agar (PDA, 1% w/v peptone) using a burned needle and incubated at room temperature (25°C). The pure culture was stored in twice-sterilized water, a 15% glycerinum solution and PDA medium, and deposited in the KUMCC culture collection of the Kunming Institute of Botany (KIB), Chinese Academy of Sciences (CAS). The fruiting bodies were dried with allochroic silica gel and deposited in KUN herbarium of KIB. Facesoffungi numbers were registered as outlined in Jayasiri et al. (2015).

The fresh fruiting bodies were examined and hand-sectioned under an Optec SZ660 stereo dissecting microscope. The key fungal structures viz. ascomata, perithecia, peridium, asci and ascospores were mounted in sterilized water or cotton blue solution slides and observed and photographed using a compound microscope (Nikon ECLIPSE Ni) with a digital camera (Canon EOS 600D) fitted on to the top of the microscope. These important fungal structures were measured with the Tarosoft (R) Image Frame Work program and the images used were processed with Adobe Photoshop CS3 Extended v. 10.0 (Adobe®, San Jose, CA, United States).

DNA Extraction, PCR Amplification, and Sequencing

The total DNA was extracted from stromal tissue of specimens HKAS 102552, HKAS 102553, HKAS 102484 and from fresh mycelium of KUMCC 20-0001 (ex-type culture of isolate HKAS 102546) using DNA extraction kit (Omega Fungus Genomic DNA Extraction Kit, China), following the protocol of the manufacturer. The obtained DNA was stored at −20°C in a refrigerator. The PCR amplification was performed in 25 μL volumes consisting 12.5 μL PCR mixture (2 × Taq PCR Master Mix, red dye) which contains Taq DNA polymerase, dNTPs, MgCl2, a reaction buffer, a PCR reaction enhancer, an optimizer and stabilizer, 8.5 μL of twice-sterilized water, 1 μL of each primer and 2 μL of 30 μg/μl DNA template. The internal transcribed spacer (ITS1-5.8S-ITS2, ITS), large subunit ribosomal RNA (LSU rRNA), small subunit ribosomal RNA (SSU rRNA), translation elongation factor 1-alpha gene (TEF1-α) and RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) were amplified with the primers and procedures mentioned in Table 1. The PCR products were sent to Tsingke company, Yunnan Province, China, for sequencing the above genes. The generated sequences were submitted to GenBank, and the accession numbers have been shown in Table 2.

TABLE 1

Gene and primers used in the phylogenetic analyses.

Gene (reference)PrimerSequencesPCR condition
LSU (Vilgalys and Hester, 1990)LRORACCCGCTGAACTTAAGC(1) Initialization at for 3 min at 94°C. (2) 40 cycles of denaturation at 94°C for 45 s, annealing at 56°C for 50 s, and extension at 72°C for 1 min. (3) final elongation at 72°C for 10 min and (4) storage at 4°C.
LR5TCCTGAGGGAAACTTCG
SSU (White et al., 1990)NS1GTAGTCATATGCTTGTCTC
NS4CTTCCGTCAATTCCTTTAAG
ITS (White et al., 1990)ITS4TCCTCCGCTTATTGATATGC
ITS5GGAAGTAAAAGTCGTAACAAGG
RPB1 (Castlebury et al., 2004)CRPB1AfCAYCCWGGYTTYATCAAGAA(1) Initialization at 94°C for 2 min, (2) 10 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 1 min, and extension at 72°C for 1 min, (3) followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 1 min, and extension at 72°C for 1 min and (4) final elongation at 72°C for 3 min. (5) storage at 4°C.
CRPB1CrCCNGCDATNTCRTTRTCCATRTA
TEF1-α (Rehner and Buckley, 2005)983FGCYCCYGGHCAYCGTGAYTTYAT
2218RATGACACCRACRGCRACRGTYTG
RPB2 (Liu et al., 1999; Sung et al., 2007b)RPB2-5F RPB2-7cRGAYGAYMGWGATCAYTTYGG CCCATRGCTTGTYYRCCCAT(1) Initialization at 95°C for 3 min. (2) 40 cycles of denaturation at 95°C for 1 min, annealing at 52°C for 2 min, and extension at 72°C for 90 s. (3) final elongation at 72°C for 10 min and (4) storage at 4°C.

TABLE 2

GenBank accession numbers of the taxa used in the phylogenetic analyses.

SpeciesSpecimen numberSSULSUTEF1-αRPB1RPB2ITSReferences
Ophiocordyceps highlandensisHKAS 83206KM581282––KM581274KM581278–Yang et al., 2015
Ophiocordyceps highlandensisHKAS 83207KM581284––KM581276KM581280–Yang et al., 2015
Ophiocordyceps konnoanaEFCC 7295EF468958––EF468862EF468915–Araújo et al., 2018
Ophiocordyceps konnoanaEFCC 7315EF468959–EF468753EF468861EF468916–Araújo et al., 2018
Ophiocordyceps melolonthaeOSC 110993DQ522548DQ518762DQ522331DQ522376––Sung et al., 2007a
Ophiocordyceps melolonthaeOphgrc679–KC610768KC610744KF658666––Araújo et al., 2018
Ophiocordyceps nigrellaEFCC 9247EF468963EF468818EF468758EF468866EF468920–Araújo et al., 2018
Ophiocordyceps raveneliiOSC 110995DQ522550DQ518764DQ522334DQ522379DQ522430–Araújo et al., 2018
Ophiocordyceps raveneliiOSC 151914KJ878932–KJ878978KJ879012KJ878950–Araújo et al., 2018
Ophiocordyceps superficialisMICH 36253EF468983––EF468883––Sung et al., 2007a
Ophiocordyceps variabilisARSEF 5365DQ522555DQ518769DQ522340DQ522386DQ522437–Araújo et al., 2018
Ophiocordyceps variabilisOSC 111003EF468985EF468839EF468779EF468885EF468933–Araújo et al., 2018
Paraisaria albaHKAS 102484MN943843MN943839MN929085MN929078MN929082MN947219This study
Paraisaria amazonicaHUA 186143KJ917562KJ917571KM411989KP212902KM411982–Ban et al., 2015
Paraisaria amazonicaHUA 186113KJ917566KJ917572–KP212903KM411980–Ban et al., 2015
Paraisaria arctaHKAS 102553MN943845MN943841MN929087MN929080–MN947221This study
Paraisaria arctaHKAS 102552MN943844MN943840MN929086MN929079MN929083MN947220This study
Paraisaria blattarioidesHUA186093KJ917559KJ917570KM411992KP212910––Ban et al., 2015
Paraisaria blattarioidesHUA 186108KJ917558KJ917569–KP212912KM411984–Ban et al., 2015
Paraisaria coenomyiaeNBRC 106964AB968385AB968413AB968571–AB968533AB968397Ban et al., 2015
Paraisaria coenomyiaeNBRC 108993AB968384AB968412AB968570–AB968532AB968396Ban et al., 2015
Paraisaria gracilioidesHUA 186095KJ917556–KM411994KP212914––Li et al., 2004
Paraisaria gracilioidesHUA 186092KJ917555KJ130992–KP212915––Mongkolsamrit et al., 2019
Paraisaria gracilisEFCC 3101EF468955EF468810EF468750EF468858EF468913–Araújo et al., 2018
Paraisaria gracilisEFCC 8572EF468956EF468811EF468751EF468859EF468912–Araújo et al., 2018
Paraisaria heteropodaOSC 106404AY489690AY489722AY489617AY489651––Araújo et al., 2018
Paraisaria heteropodaEFCC 10125EF468957EF468812EF468752EF468860EF468914JN049852Araújo et al., 2018
Paraisaria orthopterorumBBC 88305–MK332583MK214080MK214084–MH754742Mongkolsamrit et al., 2019
Paraisaria orthopterorumTBRC 9710–MK332582MK214081MK214085–MH754743Mongkolsamrit et al., 2019
Paraisaria phuwiangensisBBH 43491–MK192058–MH211351–MH188542Mongkolsamrit et al., 2019
Paraisaria phuwiangensisTBRC 9709–MK192057MK214082MK214086–MK192015Mongkolsamrit et al., 2019
Paraisaria phuwiangensisBBH 43492–MH201169MH211355MH211352–MH188541Mongkolsamrit et al., 2019
Paraisaria roseaHKAS 102546MN943846MN943842MN929088MN929081MN929084MN947222This study
Paraisaria tettigoniaGZUH CS14062709KT345955–KT375440KT375441–KT345954Wen et al., 2016
Paraisaria yodhathaiiBBH 43163–MK332584MH211353MH211349–MH188539Mongkolsamrit et al., 2019
Paraisaria yodhathaiiTBRC 8502–MH201168MH211354MH211350–MH188540Mongkolsamrit et al., 2019
Polycephalomyces formosusARSEF 1424KF049615KF049634KF049689KF049651KF049671KF049661Xiao et al., 2018
Polycephalomyces nipponicusBCC 2325KF049622KF049640KF049696KF049655KF049677KF049665Xiao et al., 2018
Polycephalomyces ramosopulvinatusEFCC 5566KF049627KF049682KF049645–KF049658Xiao et al., 2018
Polycephalomyces ramosusMFLU 18-0162MK863043MK863050–––MK863250Xiao et al., 2018
Purpureocillium lilacinumCBS 284.36––EF468792EF468898–AY624189Mongkolsamrit et al., 2019
Purpureocillium lilacinumCBS 431.87–EF468844EF468791EF468897–AY624188Mongkolsamrit et al., 2019
Purpureocillium takamizusanensisNHJ 3497EU369096EU369033EU369014EU369053EU369074–Sung et al., 2007a
Tolypocladium capitatumNBRC 106327JN941737JN941404–JN992471–JN943317Mongkolsamrit et al., 2019
Tolypocladium inflatumCBS 567.84–MH873477–––MH861779Mongkolsamrit et al., 2019
Tolypocladium inflatumCBS 127142–MH875875–––MH864435Mongkolsamrit et al., 2019
Tolypocladium japonicumOSC 110991DQ522547DQ518761DQ522330DQ522375DQ522428JN049824Mongkolsamrit et al., 2019
Tolypocladium ophioglossoidesNBRC 106331JN941733JN941408–JN992467–JN943320Mongkolsamrit et al., 2019
Drechmeria gunniiOSC 76404AF339572AF339522AY489616AY489650DQ522426JN049822Mongkolsamrit et al., 2019
Drechmeria balanoidesCBS 250.82AF339588AF339539DQ522342DQ522388DQ522442MH861495Mongkolsamrit et al., 2019
Harposporium anguillulaeARSEF 5407–AY636080––––Mongkolsamrit et al., 2019
Harposporium anguillulaeARSEF 5593–AY636081––––Mongkolsamrit et al., 2019
Harposporium helicoidesARSEF 5354AF339577AF339527––––Mongkolsamrit et al., 2019
Perennicordyceps prolificaNBRC 100744JN941709JN941432–JN992443––Mongkolsamrit et al., 2019
Perennicordyceps prolificaNBRC 101750JN941708JN941433–JN992442–JN943340Ban et al., 2009
Perennicordyceps prolificaNBRC 103838JN941707JN941434–JN992441–JN943339Ban et al., 2009
Perennicordyceps cuboideaNBRC 100941–AB378646–––AB378666Ban et al., 2009
Perennicordyceps cuboideaNBRC 101742–AB378648–––AB378667Ban et al., 2009
Cordyceps militarisOSC 93623AY184977AY184966DQ522332DQ522377–JN049825Kepler et al., 2013
Cordyceps kyusyuensisEFCC 5886EF468960EF468813EF468754EF468863EF468917–Kepler et al., 2013
The new species generated in this study are in black bold.

Sequence Alignment and Phylogenetic Analyses

The generated sequences were assembled with Sequencing Project Management (SeqMan) (Clewley, 1995). The sequences for the combined alignment were selected based on the blast results of LSU, SSU, ITS, TEF, RPB1, and RPB2 as well as the recent references listed in Table 2. The individual gene alignment was aligned in MAFFT v. 7 web server1 (Kuraku et al., 2013; Katoh et al., 2019). The alignments of each locus were improved by manually removing uninformative gaps and ambiguous regions using BioEdit v. 7.0.9.1 (Hall, 1999) and were concatenated in Sequence Matrix v. 1.7.8 (Vaidya et al., 2011). The final combined alignment was converted to a NEXUS file (.nex) with ClustalX2 v. 1.83 (Thompson et al., 1997) and was used for Bayesian inference (BI) analysis and Maximum parsimony analysis (MP). The optimum nucleotide substitution model of each gene was selected by MrModeltest v.2.3 (Nylaner, 2004) using the Akaike information criterion (AIC) method and was applied to Bayesian inference (BI) analysis that was performed using MrBayes on XSEDE (2.2.7a) (Ronquist and Huelsenbeck, 2003) on CIPRES Science Gateway2. The Bayesian posterior probability (BYPP) was estimated by the Markov Chain Monte Carlo (MCMC) technique. Six simultaneous Markov Chains were run for 2,000,000 generations with sampling every 1,000 generation. The first 25% of sampled trees were discarded during the burn-in period. Maximum likelihood analysis was carried out using RAxML-HPC2 on XSEDE (8.2.10) in CIPRES Science Gateway V. 3.3 (Miller et al., 2010), with default algorithm and bootstrap iterations were set to 1,000 and substitution model was set to GTRGAMMA + I. Maximum parsimony analysis was implemented in PAUP v. 4.0b10 (Swofford, 2002) through heuristic search with 1,000 random replicates of stepwise addition and tree-bisection-reconnection (TBR) of branch-swapping algorithm. Gaps were treated as missing data and max trees was set to 1,000. Branches collapsed when minimum branch length was zero. The consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for the maximum parsimony tree. For the delimitation of new species based on nucleotide comparison, we follow the suggestion of Jeewon and Hyde (2016).

The tree topologies were visualized in FigTree v1.4.0 (Rambaut, 2006) and edited in Microsoft power point (2016) and Adobe Photoshop CS3 Extended v. 10.0 (Adobe®, San Jose, CA, United States). The final alignment and trees were submitted to TreeBASE with submission number 256643.

Results

Phylogenetic Analyses

Phylogenetic analyses were constructed with combined LSU, SSU, TEF1-α, RPB1, RPB2, and ITS sequences data of 58 representative taxa in Ophiocordycipitaceae. Trees were rooted to Cordyceps militaris (OSC 93623) and C. kyusyuensis (EFCC5886) in Cordycipitaceae. The alignment contains 5239 characters, including gaps (LSU: 918, SSU: 1027, TEF1-α: 906, RPB1: 664, RPB2: 1024, ITS: 700). Parsimony analysis of this dataset produced the 20 most parsimonious trees of 4833 steps in length, of which 3436 characters were constant, 380 variable characters parsimony-uninformative and 1423 characters parsimony-informative. The first parsimonious tree was represented as the best tree, with CI = 0.549, RI = 0.777, RC = 0.426 and HI = 0.451. The RAxML analysis of the combined dataset yielded a best scoring tree with a final ML optimization likelihood value of −30766.070218. The matrix had 2305 distinct alignment patterns, with 41.28% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.236752, C = 0.277080, G = 0.283017, T = 0.203151; substitution rates AC = 1.485223, AG = 3.851975, AT = 0.915108, CG = 1.456245, CT = 6.890167, GT = 1.000000; gamma distribution shape parameter α = 0.465094.

In the phylogenetic analyses (Figure 1), eight genera are included in Ophiocordycipitaceae labeled on the tree. With the exception of Ophiocordyceps, the other remaining genera are monophyletic and individually they received strong statistical support. The three novel entomopathogenic fungi grouped with the taxa in Paraisaria with significant statistical support (1.00 PP/100% ML/98% MP). Paraisaria alba (HKAS 102484) constitutes a sister phylogenetic affiliation to P. yodhathaii with 0.96 PP/98% MP statistical support. Paraisaria rosea (HKAS 102546) is closely related to P. amazonica and P. blattarioides, but this is statistically not supported in all three formats. Two strains of P. arcta grouped as an intermediate clade with close phylogenetic connection to P. coenomyiae, P. gracilioides, and P. heteropoda.

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Phylogram generated from maximum likelihood analysis based on combined LSU, SSU, TEF1-α, RPB1, RPB2, and ITS sequence data. Bootstrap values for BI equal to or higher than 95%, ML and MP equal to or greater than 60% are placed on the notes. The newly generated sequences are indicated in blue bold. The host order of Paraisaria species and the generic names are labeled in the right side.

Taxonomy

Paraisaria alba D. P. Wei and K. D. Hyde, sp. nov. Figure 2

An external file that holds a picture, illustration, etc. Object name is fmicb-11-608991-g002.jpg

Paraisaria alba (HKAS 102484, holotype). (a) Habitat. (b) Host imbedded into the soil with the stroma emerging from the ground. (c) Stroma arising from host. (d) Fertile head. (e) Longitudinal section through fertile head. (f,g) Perithecia. (h) Peridium. (i–k) Asci. (l) Ascus cap. (m) Part of the asci. (n–p) Secondary ascospores. Scale bars: (f) 500 μm, (g) 200 μm, (h) 20 μm, (i–k) 50 μm, (m) 30 μm, (l,n–p) 5 μm. (k–m mounted in cotton blue reagent.).

Etymology: alba refers to the white fertile head.

MycoBank number: MB 833999

Facesoffungi number: FoF 07239

Parasitic on an adult cricket (Orthoptera). Sexual morph: Stroma up to 26 mm in tall, single, unbranched, growing from the flank of the host. Fertile head 3.5 mm in diam., globose, white when fresh, yellow brown when dry. Stipe 22.5 × 1.2 mm, slightly flexuous, fleshy, white, glossy, not hollow. Perithecia 200–500 × 100–220 (x¯ = 325 × 145, n = 20) μm, immersed, ovoid. Asci 160–250 × 2.5–5 (x¯ = 200 × 3.5, n = 10) μm, unitunicate, hyaline, narrow cylindrical, attenuated toward the base, with thickened cap. Peridium 10–40 (x¯ = 20, n = 30) μm in thick, comprising hyaline, thick-walled cell of textura angularis. Apical cap 4.6–7.4 × 3.2–4.9 (x¯ = 6 × 3.8, n = 30) μm, with a narrow tunnel throughout the center. Ascospores filiform, equal to the asci in length, when mature, breaking into numerous secondary ascospores. Secondary ascospores 3–5 × 0.5–1.5 (x¯ = 4 × 1, n = 30) μm, cylindrical, hyaline, smooth, one-celled, straight, with truncated ends.

Material examined

Thailand, Krabi, Plai Phraya (N: 8°24′410′′, E: 98°45′34′′). On an adult cricket, 20 December 2018, Deping Wei, 211-1(HKAS 102484– holotype). We tried to culture P. alba by transferring a small piece of inner stroma tissue into a PDA medium using a sterilized needle, but growth was not observed.

Notes

The multigene phylogenetic analysis showed that P. alba groups with P. yodhathaii with fairly good statistical support (0.96 PP/98% MP, Figure 1). This relationship is, however, not supported by the ML analysis. Paraisaria alba differs from P. yodhathaii in having solitary stroma, a white fertile head, and smaller perithecia, asci and secondary ascospores, whereas P. yodhathaii has paired stromata, grayish yellow fertile head, larger perithecia and larger asci and secondary ascospores (Table 3). The comparison of the nucleotide sequences between P. alba and P. yodhathaii show 10 (including 6 gaps) out of 410 bp (2.4%), 6 out of 746 bp (0.8%), 5 out of 881 bp (0.56%) and 8 out of 534 bp differences (1.5%) in ITS, LSU, TEF1-α and RPB1 sequences, respectively. SSU and RPB2 sequences data of P. yodhathaii are not available in GenBank. Henceforth, we describe our collection as a new species in Paraisaria according to the guidelines of Jeewon and Hyde (2016).

TABLE 3

Synopsis of Paraisaria species discussed in this study.

SpeciesHostDistributionStroma (mm)Fertile part (mm)Perithecia (μm)Asci (μm)Part-ascospores (μm)Asexual morphs
P. albaAdult cricket (Orthoptera)Thailand: Krabi ProvinceSolitary, 26 longGlobose, white, 3.5 in diam.Ovoid, 200–500 × 100–220160–250 × 2.5–53–5 × 0.5–1.5Absent
P. amazonicaa,d,hAdult or imago of Acrididae (Orthoptera)Colombia and EcuadorGregarious, 20–45 longSubglobose to spherical, reddish brown, 2.5–5.5Ovoid-ellipsoidal, 760–1100 × 220–400325–450 × 59–17 × 0.5–2Absent
P. arctaLarva of LepidopteraChina: Guizhou ProvinceSolitary, 16 longSubglobose with constriction at center, white, 2 × 3Ampulliform to ellipsoidal, 230–530 × 70–180100–180 × 2–42.6– 4.2 × 0.5–1.3Absent
P. blattarioidesc,hAdult of Blattaria (Dictyoptera)Belize, Colombia and EcuadorGregarious, 14–20 longOvoid, subglobose, chestnut brown, 2–3 × 1.5–2.5Ellipsoidal, 650–800 × 220–300180–250(–300) × 4–56–16 × 1.5Absent
P. coenomyiaebLarva of Coenomyia (Diptera)JapanSolitary, 30–35 longOvoid, subglobose, chestnut brown, 8 × 10Lanceolate, 700–750 × 200–220500–750 × 7.8–8.08–15 × 1.8–2.5Absent
P. gracilioidesb,e,hLarva of Elateridae (Coleoptera)Bolivia, China, Colombia, Japan and MexicoUsually solitary, 20–90 longSpherical, pale rufous, 4–5.5Ellipsoidal to naviform, 680–900 × 200–280450–700 × 5–6.57–12 × 1–2Present
P. gracilisd,g,hLarva of Hepialidae (Lepidoptera)Africa, America, Asia, Europe, and OceaniaUsually solitary, 40– 90 longGlobose to ellipsoidal, red ochreous to pale orange, 4–9 × 4–7Elongate to oviform, (320–)560–840 × 200–360(200–)400–528 × 5–85–9 × 1.5–2Present
P. heteropodaeNymph of Cicadidae (Hemiptera)Australia, JapanSolitary, 120 longOvoid, cinnamon buff, 7–9 × 6–7Ampulliform, 610–660 × 210250–300 × 5.2–76–7.7 × 0.9–1Present
P. myrmicarumMyrmica rubra (Hymenoptera)United Kingdom–––––Present
P. orthopterorumfNymph of OrthopteraThailand: Trat ProvinceSolitary, 10–45 longGlobose, gray orange, 2–4 × 3Obclavate, 520–650 × 150–250400 × 55–10 × 1–1.5Present
P. phuwiangensisfLarva of Elateridae (Coleoptera)Thailand: Khon Kaen ProvinceSolitary, 30–50 longGlobose to subglobose, light brown, 4–8 × 4–7Obpyriform, 800–1200 × 300–380500 × 3–55–10 × 1–2Present
P. roseaLarva of ColeopteraChina: Yunnan ProvinceSolitary, 14.5 longSubglobose, pale pink, 4.5 × 4Ampulliform, 500–900 × 150–350230–390 × 3.5–64–11 × 1.5–2.5Present
P. tettigoniafAdult of Tettigonia (Orthoptera)China: Guizhou ProvincePaired, 32.5–37.5 longGlobose, white, 2–2.5Elongated to ampulliform, 520–680 × 205–275530–615 × 6.5–9.36.7–9.4 × 1.5–2.3Absent
P. yodhathaiifLarva of Elateridae (Coleoptera)Thailand: Khon Kaen ProvinceGregarious, 20–35 longGlobose to subglobose, grayish yellow, 2–4 × 2–5Obclavate, 650–800 × 160–250480 × 5–65–10 × 1–2Present
The new species generated in this study are in bold.aBan et al., 2009; bBan et al., 2015; cHennings, 1904; dKobayasi, 1941; eMains, 1940; fMongkolsamrit et al., 2019; gSamson and Brady, 1983; hSanjuan et al., 2015; iWen et al., 2016.

Paraisaria arcta D. P. Wei and K. D. Hyde, sp. nov. Figure 3

An external file that holds a picture, illustration, etc. Object name is fmicb-11-608991-g003.jpg

Paraisaria arcta (HKAS 102553, holotype). (a) Substrate. (b–d) Stromata emerging from host mouth. (e) Fertile head. (f–h) Perithecia. (i) Ostiole. (j) Peridium. (k,l) Asci. (m) Parts of ascus. (n) Ascus cap. (o,p) Secondary ascospores. Scale bars: (d,e) 2000 μm, (f) 500 μm, (g,h) 200 μm, (i,k) 50 μm, (j,m) 15 μm, (l) 30 μm, (n–p) 3 μm.

Etymology: arcta refers to the constricted fertile head.

MycoBank number: MB 834000

Facesoffungi number: FoF 07240

Parasitic on larva of Lepidopteran larva. Sexual morph: Stroma 16 mm long, single, arising from the mouth of host larva. Fertile head 2 mm long, 3 mm wide, white, nearly globose, constricted at the center, with sticky and crystal-like substance on the surface. Stipe 14 mm long, 2 mm wide, straight, fleshy, white, glossy. Perithecia 230–530 × 70–180 (x¯ = 387 × 113, n = 20) μm, completely immersed, ampulliform to ellipsoid. Peridium 14–20 (x¯ = 17, n = 30) μm wide, composed of hyaline, thick-walled, smooth-walled cells of textura angularis. Asci 100–180 × 2–4 μm (x¯ = 137 × 2.9, n = 15), unitunicate, hyaline, narrow cylindrical, tapering toward the base, 8-spored, with thickened cap. Apical cap 3.5–4.5 × 2–3.6 μm thick (x¯ = 4 × 2.8, n = 20), with a narrow tunnel throughout the center. Ascospores hyaline, narrow filiform, equal to the asci in length, when mature, breaking into numerous secondary ascospores. Secondary ascospores 2.6–4.2 × 0.5–1.3 μm (x¯ = 3.3 × 0.9, n = 60), cylindrical, with truncated ends, hyaline, smooth, one-celled, straight.

Material examined

China, Guizhou Province, Qianxinan Buyei and Miao Autonomous Prefecture, Ceheng County, Gaofeng Villige (N: 24°57′33′′, E: 105°50′1′′), on dead larva of Lepidoptera sp., 6 August 2018, Deping Wei, GFC604 (HKAS 102553–holotype); GFC603 (HKAS 102552 – paratype). The culturing of P. arcta was tried by transferring a mass of mycelium found inside body of the larva host to a PDA medium using a sterilized needle. However, mycelium growth was not observed.

Notes

Paraisaria arcta resembles P. alba found in Krabi Province, Thailand and P. tettigonia discovered in Guizhou Province, China in having white fertile heads but differs from P. alba in its associated host and number of stromata are distinct from P. tettigonia (Wen et al., 2016). Paraisaria arcta can also be distinguished from the other species in Paraisaria by the color and shape of its fertile head. A conspicuous ravine throughout the center of the fertile head is present in P. arcta, which is lacking in the other species in this genus. The detailed comparisons are shown in Table 3. Multigene phylogenetic analysis showed P. arcta constitutes a distant clade from other species in Paraisaria, with strong statistical support (100% ML, 100% MP, 1.00 PP, Figure 1). Herein, we introduce this collection as a new species of Paraisaria.

Paraisaria rosea D. P. Wei and K. D. Hyde, sp. nov. Figures 4, ​,55

An external file that holds a picture, illustration, etc. Object name is fmicb-11-608991-g004.jpg

Sexual morph of Paraisaria rosea (HKAS 102546, Holotype). (a) Stroma emerging from host. (b) Fertile head. (c) Transverse section of the fertile head. (d–f) Perithecia. (g) Peridium. (h,k,m) Asci. (i,j) Asci cap. (l) Part of ascus. (n–p) Secondary ascospores. Scale bars: (d) 1000 μm, (e) 300 μm, (f) 200 μm, (g,m) 30 μm, (h,k) 50 μm, (l) 20 μm, (i,j, n–p) 5 μm. (k mounted in Melzer’s reagent).

An external file that holds a picture, illustration, etc. Object name is fmicb-11-608991-g005.jpg

Asexual morph of Paraisaria rosea (KUMCC 20-0001, ex-type). (a,d) Upper and lower views of cultures on PDA after 50 days. (b,e) Upper and lower views of cultures on PDA after 16 months incubation in dark environments. (c,f) Enlargement of aerial synnemata produced on culture. (g) Synnema bearing conidiophores. (h–l) Phialides. (m) Conidia. (n,o) Irregularly aggregated conidia. Scale bars: (g) 100 μm, (h–l) 30 μm, (m–o) 5 μm. (h–k,m mounted in cotton blue reagent.).

Etymology: rosea refers to its pink fertile head.

MycoBank number: MB834001

Facesoffungi number: FoF 07241

Parasitic on a larva of Coleoptera. Host buried in the soil, with the stroma erumpent from the ground. Sexual morph: Stroma up to 14.5 mm long, laterally emerging from the middle part of the larva body, simple, erect. Fertile head 4.5 × 4 mm, subglobose, pale pink at top and paler toward the base when fresh, pale yellow-brown when dry. Stipe 10 × 1.5 mm, white, straight, unbranched, glossy, cylindrical, inside not hollow. Perithecia 500–900 × 150–350 (x¯ = 762 × 256, n = 30) μm, completely immersed, ampulliform, ostiolate. Peridium 9–15 (x¯ = 12, n = 30) μm wide, composed of hyaline, thick-walled cells of textura angularis to textura globulosa to textura prismatica. Asci 230–390 × 3.5–6 (x¯ = 280 × 5, n = 15) μm, hyaline, cylindrical, unitunicate, eight-spored, possessing a prominent apical cap. Apical cap 5–7 × 2–6 (x¯ = 6 × 4, n = 20) μm, with a conspicuous tunnel throughout the center. Ascospores filiform, hyaline, breaking into secondary ascospores when mature. Secondary ascospores 4–11 × 1.5–2.5 (x¯ = 7.5 × 2, n = 30) μm, hyaline, cylindrical with truncate ends, smooth-walled, aseptate. Asexual morph: Hyphomycetous. Synnemata producing from the center of culture after 16 months incubation in dark environment, composed of loose, septate hyphae, white, filamentous, aerial, straight, branched, fasciculate, bearing shining droplets and conidiophores. Mycelium 2.4–3.7 (x¯ = 3, n = 10) μm in wide, septate, hyaline, smooth-walled. Conidiophores 33–48 (x¯ = 41, n = 10) μm in height, irregularly differentiate from the synnemata, sparse, gregarious, branched. Phialides 5.8–11.5 × 3–5.5 (x¯ = 8.6 × 4, n = 30) μm, ampulliform, 1-necked, hyaline, aseptate, enteroblastic, phialidic, monophialidic. Conidia 8–12 × 2–2.6 (x¯ = 9.8 × 2.3, n = 50) μm, hyaline, cylindrical, smooth-walled, aseptate, with round ends.

Culture characteristics

Culture was made from mycelium inside body of the host larva, slowly growing on PDA, reaching 1.3 cm in diam after incubated at room temperature (25°C) for 50 days, convex, dense, with undulate edges, smooth surface become filamentous after forming aerial synnemata. The shooting conidia land on the surrounding culture and develop new colonies.

Material examined

China, Yunnan Province, Kunming, Western hill Park (N: 24°57′28′′, E: 102°38′17′′), on larva of Coleoptera sp. buried in soil, 27 July 2018, Deping Wei, XS2712 (HKAS 102546 – Holotype); (KUMCC 20-0001 – ex-type living culture).

Notes

Paraisaria rosea is closely related to P. amazonica and P. blattarioides, without any statistical support (Figure 1). However, P. rosea can be distinguished from these related species based on the number of stromata, the color of the fertile head and the size of asci and secondary ascospores (Table 3). The ITS sequence of P. amazonica and P. blattarioides are not available in GenBank database; the nucleotide differences in the TEF1-α, RPB1 and RPB2 region between P. rosea and the two above species are greater than 1.5% (Table 4). Thereby, we introduced P. rosea as a new species in this genus based on the distinctive morphology and molecular support.

TABLE 4

The comparison of nucleotide sequences between Paraisaria rosea and two close species.

SpeciesTEF1-α (bp)RPB1 (bp)RPB2 (bp)
Paraisaria amazonica4.4% (38/862)5.7% (37/642)4.3% (31/711)
Paraisaria blattarioides1.6% (14/862)2.5% (16/629)–

Discussion

The sexual morph of Paraisaria species phenotypically share an erect or slightly flexuous, cylindrical, colorless, fleshy stipe that terminates in a subglobose to globose fertile head and completely immersed perithecia. Asci are cylindrical with a thickened apical cap. Ascospores are hyaline, multi-septate and usually break into numerous cylindrical, truncated fragments at maturity. However, they can be distinguished according to their associated host, the number of stroma and the color of the fertile head. Species in this genus usually infect several stages of insects, such as larvae of Coleoptera, Diptera, and Lepidoptera; nymphs of Hemiptera and Orthoptera; or adults of Dictyoptera, Hymenoptera (ant) and Orthoptera (Evans et al., 2010; Sanjuan et al., 2015; Mongkolsamrit et al., 2019). According to the number of stromata, species of Paraisaria can be divided into three groups: solitary stroma, paired stromata and multiple stromata (see the key below). The shape of their fertile head features little variation, though differing in color, ranging from white, pale pink, pale rufous, red ochreous to pale orange, chestnut, cinnamon buff, grayish, reddish brown to dark brown (see Table 3).

The asexual morphs of this genus are known in eight species, viz. P. myrmicarum (Evans et al., 2010), P. gracilis (Samson and Brady, 1983), P. gracilioides (Li et al., 2004), P. rosea (this study), P. heteropoda, P. orthopterorum, P. phuwiangensis, and P. yodhathaii (Mongkolsamrit et al., 2019). Their conidiophores are irregularly branched and generally develop from white, rope-like synnemata. Their phialides are flask-shaped, with a swollen base and narrow neck. Most species produce only one neck from the terminal phialides. Some species, e.g., P. gracilis, P. gracilioides, P. myrmicarum and P. orthopterorum produce 1–4 necks per phialides. Their conidia are cylindrical or ellipsoid or fusiform. Some species, e.g., P. orthopterorum and P. yodhathaii have both cylindrical and fusiform forms of conidia (Mongkolsamrit et al., 2019).

Sung et al. (2007a) have concluded that multi-gene phylogeny gave more deeper understanding of phylogenetic relationships of Cordyceps and Clavitipitaceae than that of single gene. Recently, the combined LSU-TEF1-α-RPB1 datasets (Mongkolsamrit et al., 2019), combined SSU-LSU-TEF-RPB2 datasets (Ban et al., 2015), and combined SSU-LSU-TEF1-α-RPB1-RPB2 datasets (Quandt et al., 2014; Sanjuan et al., 2015) were allowed for intraspecific and intergeneric identification within Ophiocordycipitaceae. However, individual gene phylogenies are rarely utilized for identification of species in Paraisaria.

Key to the Accepted Species in Paraisaria

  • (1)

    Host belong to Hymenoptera……………………P. myrmicarum

  • (1)

    Host not belong to Hymenoptera…………………………………….2

  • (2)

    Fertile part colorless…………………………………………………………3

  • (2)

    Fertile part pigmented……………………………………………………..4

  • (3)

    Fertile part constrict at the center………………………….P. arcta

  • (3)

    Fertile part is not constricted at the center………………………5

  • (4)

    Stromata gregarious…………………………………………………………6

  • (4)

    Stromata solitary……………………………………………………………..7

  • (5)

    Stromata branched………………………………………….P. tettigonia

  • (5)

    Stromata unbranched……………………………………………..P. alba

  • (6)

    Stromata equal or shorter than 20 mm………..P. blattarioides

  • (6)

    Stromata longer than 20 mm……………………………………………8

  • (7)

    Attack nymph stage of host……………………………………………..9

  • (7)

    Attack larva stage of host……………………………………………….10

  • (8)

    Fertile part reddish brown…………………………….P. amazonica

  • (8)

    Fertile part grayish yellow……………………………..P. yodhathaii

  • (9)

    Stromata long, 120 mm…………………………………P. heteropoda

  • (9)

    Stromata short, 10–45 mm………………………P. orthopterorum

  • (10)

    Host belong to Coleoptera……………………………………………..11

  • (10)

    Host belong to other order of insect………………………………12

  • (11)

    Stromata equal or shorter than 14.5 mm………………P. rosea

  • (11)

    Stromata longer than 14.5 mm………………………………………13

  • (12)

    Pathogenic on larva of Diptera (Coenomyia). ……………………………………………………………………P. coenomyiae

  • (12)

    Pathogenic on larva of Lepidoptera (Hepialidae) …………………………………………………………………………..P. gracilis

  • (13)

    Phialides solitary or in whorls of 2–3, with one neck………………………………………………………..P. phuwiangensis

  • (13)

    Phialides sympodially proliferating, with 1–4 necks…………………………………………………………….P. gracilioides

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, MN943843, MN943839, MN929085, MN929078, MN929082, and MN947219; https://www.ncbi.nlm.nih.gov/genbank/, MN943845, MN943841, MN929087, MN929080, and MN947221; https://www.ncbi.nlm.nih.gov/genbank/, MN943844, MN943840, MN929086, MN929079, MN929083, and MN947220.

Author Contributions

D-PW, DW, and SK: conceptualization. D-PW: data curation. D-PW and DW: formal analysis, methodology, and writing – original draft. SL, ST, and SK: funding acquisition. D-PW and DW: investigation. ST and SK: project administration. KH, J-CX, and PM: supervision. CT-a, AE, SM, ST, SK, KH, J-CX, PM, NS, and SL: writing – review and editing. All authors: contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We acknowledge Kunming Institute of Botany, Chinese Academy of Sciences for providing the laboratories and instruments for molecular work. We appreciate the Centre of Excellence in Fungal Research (Mae Fah Luang University) for providing funding for collecting trips and Dr. Shaun Pennycook is thanked for help in naming the new fungal species. Austin Smith at World Agroforestry (ICRAF), Kunming Institute of Botany, China, is thanked for English editing.

Funding. We appreciate Thailand Research Fund (TRF) grant no. DBG6080013 entitled “The future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species” for its financial support. We are grateful for the National Science Foundation of China (NSFC) project code 31750110478 for funding the sequencing cost. DW would like to thank CAS President’s International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008) and the 64th batch of China Postdoctoral Science Foundation (grant no. Y913083271). PM and DW thank the National Science Foundation of China for financial support under the following grants: 41761144055 and 41771063. SK thanks CAS President’s International Fellowship Initiative (PIFI) young staff under the grant number: 2020FYC0002 and the National Science Foundation of China (NSFC) for funding this work under the project code 31851110759. ST would like to thank the International Postdoctoral Exchange Fellowship Program (number Y9180822S1), CAS President’s International Fellowship Initiative (PIFI) (number 2020PC0009), China Postdoctoral Science Foundation and the Yunnan Human Resources, and Social Security Department Foundation for funding her postdoctoral research. The authors extend their appreciation to the researchers supporting project number (RSP-2021/56) King Saud University, Riyadh, Saudi Arabia. This work was partly supported by Chiang Mai University.

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