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. 2026 Jul 27;137:299–316. doi: 10.3897/mycokeys.137.196582

Two new species from tree-hole humus in Guizhou Province, China: Leptobacillium doupengense (Hypocreales, Cordycipitaceae) and Metapochonia doupengshanensis (Hypocreales, Clavicipitaceae)

Wen-Kai Lin 1, Li-Li Lou 1, Xiao Zou 1,2, Ye-Ming Zhou 1,2,✉
PMCID: PMC13434372  PMID: 42553603

Abstract

Fungal strains were isolated from humus substrates collected from tree holes in Doupengshan Scenic Area, Duyun City, Guizhou Province, China, a region characterized by a typical karst ecosystem. Tree-hole humus represents a spatially restricted and nutrient-rich microhabitat formed by decomposed plant material and organic residues, yet hypocrealean fungi associated with this substrate remain poorly explored. Based on a combined five-gene dataset (ITS, nrSSU, nrLSU, tef-1α and rpb1), together with phylogenetic analyses and morphological comparisons, two new species of Hypocreales were identified: Leptobacillium doupengensesp. nov. and Metapochonia doupengshanensissp. nov. Both species formed distinct lineages in the phylogenetic tree and were clearly separated from other species within their respective genera. This study provides detailed morphological descriptions and illustrations of L. doupengense and M. doupengshanensis, along with comparisons with related species in their genera. The discovery of these two species highlights the potential of tree-hole humus in karst ecosystems as an overlooked source of hypocrealean fungal diversity. These findings provide morphological and molecular evidence for recognizing and distinguishing L. doupengense and M. doupengshanensis and contribute additional taxonomic data to clarify species diversity and phylogenetic relationships within Leptobacillium and Metapochonia.

Key words: Leptobacillium spp., Metapochonia spp., morphology, phylogenetic analysis

Introduction

Zare and Gams (2016) proposed the genus Leptobacillium Zare & W. Gams during their revision of the Albo-erecta section of the former genus Verticillium Nees. Phylogenetically, this genus belongs to Cordycipitaceae. The generic name Leptobacillium is derived from its slender conidia. Zare and Gams (2016) originally designated Leptobacillium leptobactrum (W. Gams) Zare and W. Gams as the type species of this genus. This species comprises two varieties, L. leptobactrum var. leptobactrum and L. leptobactrum var. calidius (W. Gams) Zare and W. Gams, which are distinguished by their optimal growth temperatures. Species within the genus Leptobacillium possess two morphologically distinct types of conidia. The first type, which is ellipsoidal to subglobose, is primarily found at the apex of the conidial chain, whereas the second type, cylindrical, bacilliform, or fusiform, is distributed along the remainder of the conidial chain (Zare and Gams 2016; Leplat et al. 2022). To date, 14 species have been accepted in the genus Leptobacillium. They have been isolated not only from insects and fungi, but also from plants, rocks, freshwater environments, mural paintings, and decaying wood (Liu and Cai 2012; Zare and Gams 2016; Crous et al. 2018; Gomes et al. 2018; Sun et al. 2019; Okane et al. 2020; Leplat et al. 2022).

In contrast to Leptobacillium, Metapochonia Kepler, S.A. Rehner & Humber was established to accommodate several species previously placed in Pochonia Bat. & O.M. Fonseca. Kepler et al. (2014) transferred five species previously assigned to Pochonia, P. bulbillosa (W. Gams & Malla) Kepler, S.A. Rehner and Humber, P. goniodes (Drechsler) Kepler, S.A. Rehner and Humber, P. microbactrospora (W. Gams & Zare) Kepler, S.A. Rehner and Humber, P. rubescens (Zare, W. Gams & López-Llorca) Kepler, S.A. Rehner and Humber, and P. suchlasporia (W. Gams & Dackman) Kepler, S.A. Rehner and Humber, to the newly established genus Metapochonia based on a multi-locus phylogenetic analysis. This genus is morphologically very similar to Pochonia, as both produce dictyochlamydospores and are parasitic on nematode eggs or cysts (Zare and Gams 2007; Kepler et al. 2014). Metapochonia currently comprises 11 species, which have been isolated from soil, plant roots, the sclerotia of Ophiocordyceps sinensis (Berk.) G.H.Sung, J.M.Sung, Hywel-Jones and Spatafora, river water, and nematode eggs (Huang et al. 2015; Zhang et al. 2017; Labuda et al. 2018; Kondo et al. 2020; Crous et al. 2024).

Guizhou Province, China, is characterized by a typical karst ecosystem and heterogeneous microhabitats that provide favorable conditions for fungal diversity. Numerous hypocrealean fungi have been reported from this region, indicating that Guizhou represents an important area for exploring the diversity of Hypocreales (Wen et al. 2017; Chen et al. 2019, 2022d, 2025). However, previous studies have mainly focused on fungi isolated from host surfaces, fruiting bodies, soil, or plant-associated substrates, whereas hypocrealean fungi from humus substrates accumulated within tree holes remain poorly investigated. Tree-hole humus represents a spatially restricted and nutrient-rich microhabitat formed by decomposed plant material and other organic residues, and may harbor previously undocumented fungal taxa. In this study, hypocrealean fungi were investigated from humus substrates collected from tree holes in Doupengshan Scenic Area, Duyun City, Guizhou Province. Based on morphological observations and multi-locus phylogenetic analyses, two new species belonging to Cordycipitaceae and Clavicipitaceae were identified. These findings contribute to the knowledge of hypocrealean fungal diversity in karst microhabitats and provide additional taxonomic data for Leptobacillium and Metapochonia.

Materials and methods

Material collection and isolation

Humus substrates were collected from tree holes in Doupengshan Scenic Area, Duyun City, Qiannan Buyi and Miao Autonomous Prefecture, Guizhou Province, China, in October 2024 and October 2025. The samples were placed in sterile plastic bags and transported to the laboratory at 4 °C. For fungal isolation, approximately 1 g of each humus sample was suspended in 9 mL of sterile distilled water and shaken thoroughly to obtain a humus suspension. The suspension was serially diluted, and aliquots of the appropriate dilutions were spread onto potato dextrose agar (PDA) plates supplemented with 0.1 g/L streptomycin and 0.05 g/L tetracycline using a sterile spreader. The plates were incubated at 25 °C in the dark for 14 days. Emerging fungal colonies were selected based on colony morphology and transferred onto fresh PDA plates. Pure cultures were obtained by repeated subculturing of hyphal tips onto fresh PDA plates. To prepare holotypes, the pure cultures were dried at 50 °C and deposited in the Institute of Fungus Resources, Guizhou University (GZUIFR) (Zhang et al. 2023b). Living cultures were preserved on PDA slants at 4 °C and in 30% glycerol at -80 °C.

Morphological characterization

The cultures were transferred to PDA and incubated at 25 °C for 14 days. Colony diameters and macromorphological characteristics were recorded, and photographs of the colony surface and reverse sides were taken using a Nikon D7500 camera (Nikon, Tokyo, Japan). To observe the micromorphological characteristics, hyphal fragments were mounted in sterile water. Morphological features, including conidiophores, phialides, and conidia, were examined using a light microscope (Axio Imager 2 Pol, ZEISS, Germany).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted using the Solarbio Fungal Genomic DNA Extraction Kit (Solarbio, D2300, China) and stored at -20 °C. Genetic markers were amplified by polymerase chain reaction (PCR). The primer pair ITS1/ITS4 was used to amplify the internal transcribed spacer (ITS) region (White et al. 1990), NS1/NS4 was used to amplify the small subunit ribosomal RNA (nrSSU) (White et al. 1990), LR0R/LR5 was used to amplify the large subunit ribosomal RNA (nrLSU) (Vilgalys and Hester 1990; Rehner and Samuels 1994), 983F/2218R was used to amplify the translation elongation factor-1 alpha (tef-1α) gene (Rehner and Buckley 2005), and CRPB1/RPB1Cr was used to amplify the largest subunit of RNA polymerase II (rpb1) (Castlebury et al. 2004). Amplification products were verified using 1% agarose gel electrophoresis. Purified PCR products were sent to Tsingke Biotechnology Co., Ltd. (Chongqing, China) for sequencing. The sequences obtained in this study have been deposited in GenBank (Table 1).

Table 1.

Information and GenBank accession numbers of the related species used for phylogenetic analyses in this study.

Species Strain ITS nrSSU nrLSU tef-1α rpb1 Reference
Keithomyces acicularis JCM 33284T LC435734 LC435738 LC435741 LC462188 NA (Iwasaki et al. 2019)
Keithomyces acicularis JCM 33285 LC463198 LC435739 LC435742 LC462189 NA (Iwasaki et al. 2019)
Keithomyces carneus CBS 399.59 MT078887 NA MH869445 NA NA (Sung et al. 2007a)
Keithomyces carneus CBS 239.32T AY624171 EF468988 EF468843 EF468789 EF468894 (Sung et al. 2007a)
Leptobacillium cavernicola LRMH C212 OM622523 OM628842 OM628781 OM654332 OM677781 (Leplat et al. 2022)
Leptobacillium cavernicola LRMH C216 OM622524 OM628843 OM628782 OM654333 OM677782 (Leplat et al. 2022)
Leptobacillium cavernicola LRMH C217 MF788211 OM628844 OM628783 OM654334 OM677783 (Leplat et al. 2022)
Leptobacillium chinense CGMCC 3.14969 JQ410323 NA JQ410321 NA NA (Okane et al. 2020)
Leptobacillium chinense CGMCC 3.14970T JQ410324 NA JQ410322 NA NA (Okane et al. 2020)
Leptobacillium coffeanum COAD 2057 MF066034 NA MF066032 NA NA (Okane et al. 2020)
Leptobacillium coffeanum COAD 2061 MF066035 NA MF066033 NA NA (Okane et al. 2020)
Leptobacillium doupengense GZUIFR DPS001T PX844695 PX844697 PX844704 PX893664 PX893665 This study
Leptobacillium doupengense GZUIFR DPS003 PX845559 PX845586 PX845587 PX893666 PX893667 This study
Leptobacillium filiforme URM 7918T NA NA MH979399 NA NA (Chen et al. 2021)
Leptobacillium latisporum TBRC 16288T OP856540 OP850838 OP856529 NA NA (Preedanon et al. 2023)
Leptobacillium leptobactrum ZJ14B02 PP385689 NA PP381743 NA NA (Zare and Gams 2016)
Leptobacillium leptobactrum AH17C05 PP384754 NA PP380808 NA NA (Zare and Gams 2016)
Leptobacillium leptobactrum var. calidius CBS 703.86 EF641866 EF641850 KU382226 NA NA (Zare and Gams 2016)
Leptobacillium leptobactrum var. leptobactrum CBS 771.69 EF641868 EF641852 KU382224 NA NA (Zare and Gams 2016)
Leptobacillium longiphialidum YFCC 23039272 PQ509282 PQ508806 PQ508808 PQ560997 PQ567240 (Lu et al. 2025)
Leptobacillium longiphialidum YFCC 24079491 PQ509281 PQ508805 PQ508807 PQ560996 PQ567239 (Lu et al. 2025)
Leptobacillium marksiae BRIP 70307aT PQ061114 NA PQ047739 NA NA (Tan and Shivas 2024)
Leptobacillium muralicola CGMCC 3.19014 MH379983 NA MH379997 NA NA (Sun et al. 2019)
Leptobacillium muralicola CGMCC 3.19015 MH379985 NA MH379999 NA NA (Sun et al. 2019)
Leptobacillium symbioticum NBRC 113865T LC485673 NA LC506046 NA NA (Okane et al. 2020)
Leptobacillium xianyushanense RCEF6793T OQ780699 NA OQ780702 NA NA (Chang et al. 2024)
Leptobacillium xianyushanense RCEF6795 OQ780930 NA OQ780931 NA NA (Chang et al. 2024)
Marquandomyces marquandii CBS 182.27T MH854923 EF468990 MH866418 EF468793 EF468899 (Sung et al. 2007a)
Marquandomyces marquandii CBS 128893 MH865143 NA MH876582 NA NA (Sung et al. 2007a)
Marquandomyces sp. CBS 127132 MT078882 MT078872 MH875873 MT078849 MT078865 (Sung et al. 2007a)
Marquandomyces sp. CBS 129413 NA MT078874 MH876751 MT078851 MT078867 (Sung et al. 2007a)
Metapochonia bulbillosa CBS 145.70T MH859529 AF339591 AF339542 EF468796 EF468902 (Kepler et al. 2014)
Metapochonia cordycipiticonsociata CGMCC 317366T KM263567 KM263570 KM263574 KM263582 KM263578 (Labuda et al. 2018)
Metapochonia cordycipiticonsociata CGMCC 317367 KM263568 KM263571 KM263575 KM263583 KM263577 (Labuda et al. 2018)
Metapochonia doupengshanensis GZUIFR DPS110T PX849742 PX849745 PX849746 PX893668 PX893669 This study
Metapochonia doupengshanensis GZUIFR DPS111 PX849747 PX849748 PX849749 PX893670 PX893671 This study
Metapochonia goniodes CBS 891.72T MH860626 AF339599 MH872319 DQ522354 DQ522401 (Kepler et al. 2014)
Metapochonia hahajimaensis JCM 34019T LC529951 LC529955 LC529959 LC529963 NA (Kondo et al. 2020)
Metapochonia lutea BiMM-F96/DF4T MF983717 NA NA MF983718 NA (Labuda et al. 2018)
Metapochonia microbactrospora CBS 101433T NA NA MH874343 KJ398794 NA (Kepler et al. 2014)
Metapochonia parasitica ARSEF 3436 FJ973068 EF468993 EF468848 EF468799 EF468904 (Barron 1980)
Metapochonia rubescens CBS 464.88T MH862138 NA AF339566 EF468797 EF468903 (Kepler et al. 2014)
Metapochonia simonovicovae BiMMF299T PP085431 NA PP085432 PP085550 NA (Crous et al. 2024)
Metapochonia suchlasporia var. catenata CBS 248.83T MH861579 NA MH873310 KJ398789 NA (Kepler et al. 2014)
Metapochonia suchlasporia var. suchlasporia CBS 251.83T MH861580 NA MH873311 KJ398790 NA (Kepler et al. 2014)
Metapochonia variabilis CGMCC 3.17925T NA KY883278 NA NA KY883214 (Zhang et al. 2017)
Metapochonia variabilis CGMCC 3.17926 NA KY883279 NA NA KY883215 (Zhang et al. 2017)
Metarhizium anisopliae BUM_1900 NA NA NA MH143854 MH143869 (Zimmermann 1993)
Metarhizium flavoviride CBS 125.65 MT078885 MT078869 MT078854 MT078846 MT078862 (Kepler et al. 2014)
Metarhizium flavoviride CBS 700.74 NA MT078870 MT078855 MT078847 MT078863 (Kepler et al. 2014)
Papiliomyces longiclavatus YC 20061403 MZ702080 MZ702112 MZ702101 MZ955880 MZ955880 (Zhang et al. 2023a)
Papiliomyces sinensis GMBC 3053T PQ636503 PQ636500 PQ636506 PQ660655 PQ660658 (Chen et al. 2025)
Papiliomyces sinensis GMBC 3054 PQ636504 PQ636501 PQ636507 PQ660656 PQ660659 (Chen et al. 2025)
Pochonia chlamydosporia CBS 101244 DQ522544 DQ518758 DQ518758 DQ522327 DQ522372 (Gams and Zare 2001)
Pochonia chlamydosporia var. catenulata JCM 18598 AB758248 AB709810 AB709810 AB758456 AB758659 (Gams and Zare 2001)
Pochonia chlamydosporia var. catenulata JCM 18600 AB758266 AB709812 AB709812 AB758474 AB758677 (Gams and Zare 2001)
Pochonia chlamydosporia var. chlamydosporia CM 18605 AB758261 AB709817 AB709817 AB758469 AB758672 (Gams and Zare 2001)
Pochonia chlamydosporia var. chlamydosporia JCM 18607 AB758270 AB709819 AB709819 AB758478 AB758681 (Gams and Zare 2001)
Pochonia chlamydosporia var. ellipsospora JCM 18609T AB758257 AB709821 AB709821 AB758465 AB758668 (Nonaka et al. 2013b)
Pochonia chlamydosporia var. ellipsospora JCM 18611 AB758265 AB709823 AB709823 AB758473 AB758676 (Nonaka et al. 2013b)
Pochonia chlamydosporia var. spinulospora JCM 18613T AB758258 AB709827 AB709827 AB758466 AB758669 (Nonaka et al. 2013b)
Pochonia chlamydosporia var. spinulospora JCM 18619 AB758272 AB709830 AB709830 AB758480 AB758683 (Nonaka et al. 2013b)
Pochonia sinensis ZY 22.009T OQ709251 OQ709263 OQ709263 OQ719629 NA (Zhang et al. 2023b)
Pochonia sinensis ZY 22.010 OQ709252 OQ709264 OQ709264 OQ719630 NA (Zhang et al. 2023b)
Samsoniella anhuiensis RCEF2830 OM268837 OM268843 OM268848 OM483864 OM751889 (Wang et al. 2024)
Samsoniella anhuiensis RCEF2590 NA OR978313 OR978316 OR966516 OR989964 (Wang et al. 2024)
Samsoniella antleroides YFCC 6016 OQ476471 MN576747 MN576803 OQ506162 MN576863 (Wang et al. 2020)
Samsoniella antleroides YFCC 6113 OQ476472 MN576748 MN576804 OQ506163 MN576864 (Wang et al. 2020)
Simplicillium aogashimaense JCM 18167T AB604002 NA LC496874 LC496904 NA (Nonaka et al. 2013a)
Simplicillium aogashimaense JCM 18168 AB604004 NA LC496875 NA NA (Nonaka et al. 2013a)
Simplicillium calcicola LC5586T KU746706 NA KU746752 KX855252 NA (Zhang et al. 2017)
Simplicillium calcicola LC5371 KU746705 NA KU746751 KX855251 NA (Zhang et al. 2017)
Simplicillium lamellicola JC-1 MT807906 MT807908 MT807907 NA NA (Zare and Gams 2001)
Simplicillium lamellicola CBS 116.25T AJ292393 NA NA DQ522356 DQ522404 (Zare and Gams 2001)
Simplicillium minatense JCM 18176T AB603992 LC496893 LC496878 LC496908 NA (Nonaka et al. 2013a)
Simplicillium minatense JCM 18178 AB603993 LC496894 LC496879 LC496909 NA (Nonaka et al. 2013a)
Simplicillium obclavatum CBS 311.74T AJ292394 NA AF339517 EF468798 NA (Gams 2017)
Simplicillium obclavatum SUF81 NA NA MK788174 NA NA (Gams 2017)
Simplicillium scarabaeoidea DY101391 MT453842 MT453843 NA MT471335 MT471343 (Chen et al. 2021)
Simplicillium scarabaeoidea DY101392 MT453845 NA NA MT471336 NA (Chen et al. 2021)
Simplicillium subtropicum JCM 18180T AB603990 NA LC496880 LC496910 NA (Nonaka et al. 2013a)
Simplicillium subtropicum JCM 18181 AB603995 NA LC496881 LC496911 NA (Nonaka et al. 2013a)
Simplicillium sympodiophorum JCM 18184T AB604003 NA LC496882 LC496912 NA (Nonaka et al. 2013a)
Sungia yongmunensis EFCC 2131T JN049856 EF468977 EF468833 EF468770 EF468876 (Sung et al. 2007b)
Sungia yongmunensis EFCC 2135 NA EF468979 EF468834 EF468769 EF468877 (Sung et al. 2007b)
Yosiokobayasia kusanagiensis TNS F18494 JN049873 JF415954 JF415972 JF416014 JN049890 (Kepler et al. 2012)
Yosiokobayasia kusanagiensis BUM 1307 OM955151 OM951246 OM951251 OM988199 NA (Sung et al. 2007a)
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Phylogenetic analyses

The DNA sequences generated in this study were assembled and edited using DNASTAR Lasergene (v. 6.0). Sequences of five loci (ITS, nrSSU, nrLSU, tef-1α and rpb1) retrieved from GenBank, together with the newly obtained sequences, were aligned and trimmed using MEGA (v. 6.06) (Tamura et al. 2013). PhyloSuite (v. 2) was used for concatenation and analysis of the combined dataset (Zhao et al. 2025). The optimal nucleotide substitution model for each gene partition was selected based on the corrected Akaike Information Criterion (AICc) using ModelFinder (v. 3.0.1) (Kalyaanamoorthy et al. 2017).

Phylogenetic analyses of the concatenated loci were performed using both Bayesian inference (BI) and maximum likelihood (ML) methods. BI was conducted using MrBayes v. 3.2.7a to infer phylogenetic trees from the combined datasets (Ronquist et al. 2012). The Markov chain Monte Carlo (MCMC) analysis was run for 10 million generations, sampling every 500 generations, resulting in 20,000 trees. The first 25% of trees were discarded as burn-in, and the remaining trees were used to calculate posterior probabilities for the majority-rule consensus tree. Maximum likelihood analysis was performed using IQ-TREE v. 3.0.1 (Nguyen et al. 2015). Branch support was assessed using the ultrafast bootstrap method with 5,000 replicates (Minh et al. 2013). The resulting phylogenetic trees were visualized using the Interactive Tree of Life (iTOL) v. 6 (Letunic and Bork 2016).

Results

Phylogenetic analyses

Phylogenetic trees were constructed based on five gene loci (ITS, nrSSU, nrLSU, tef-1α and rpb1). The trees generated by ML and BI analyses (Figs 1, 2) were largely congruent in topology. These results are consistent with the established phylogenetic framework of related hypocrealean fungi (Kondo et al. 2020; Chang et al. 2024; Crous et al. 2024; Lu et al. 2025).

Figure 1.

Figure 1.

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Phylogenetic analysis of the new strains and their related species based on a multi-locus sequence dataset (ITS, nrSSU, nrLSU, tef-1α and rpb1). Statistical support values (≥75% ML / 0.75 PP) are indicated at the nodes, representing maximum likelihood bootstrap support and Bayesian inference posterior probabilities, respectively. Isolates obtained in this study are shown in bold.

Figure 2.

Figure 2.

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Phylogenetic analysis of the new strains and their related species based on a multi-locus sequence dataset (ITS, nrSSU, nrLSU, tef-1α and rpb1). Statistical support values (≥75% ML / 0.75 PP) are shown at the nodes, representing maximum likelihood bootstrap support and Bayesian inference posterior probabilities, respectively. Isolates obtained in this study are shown in bold.

In the phylogenetic tree of Leptobacillium (Fig. 1), Samsoniella antleroides (YFCC 6016 and YFCC 6113) was selected as the outgroup. The combined dataset comprised 40 sequences and 1,449 characters. The optimal model for maximum likelihood analysis was TN+I+G4, and the log-likelihood value of the best-scoring tree was -5310.456. The parameters of the GTR model were estimated as follows: substitution rates AC = 1.00000, AG = 1.68881, AT = 1.00000, GC = 1.00000, CT = 4.84415, GT = 1.00000. The optimal model for Bayesian inference was GTR+I+G. The strains GZUIFR DPS001 and GZUIFR DPS003 clustered together and formed an independent lineage within Leptobacillium with strong support (100% ML / 1 PP), phylogenetically close to but clearly separated from L. xianyushanense.

In the phylogenetic analysis of Metapochonia (Fig. 2), Samsoniella anhuiensis (RCEF 2590 and RCEF 2830) was selected as the outgroup. The combined dataset comprised 47 sequences and 3,801 characters. Based on the multi-locus phylogenetic analysis, the optimal model for maximum likelihood was TIM2+R2, and the log-likelihood value of the best-scoring tree was -13350.881. The parameters of the GTR model were estimated as follows: substitution rates AC = 1.50683, AG = 2.65597, AT = 1.50683, GC = 1.00000, CT = 7.51043, GT = 1.00000. The optimal model for Bayesian inference was GTR+I+G4. The two strains GZUIFR DPS110 and GZUIFR DPS111 clustered together and formed a distinct, well-supported lineage within Metapochonia (100% ML / 1 PP). This lineage was phylogenetically close to M. rubescens (CBS 464.88) but clearly separated from it and other related species in the genus.

Taxonomy

Kingdom Fungi

Division Ascomycota

Order Hypocreales

Family Cordycipitaceae

Genus Leptobacillium

Leptobacillium doupengense

W.K. Lin, Y.M. Zhou & L.L. Lou sp. nov.

6506C2DE-A039-5E31-89FB-E81B14D37B57

862246

Fig. 3

Figure 3.

Figure 3.

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Morphology of Leptobacillium doupengense on PDA after 14 days of growth at 25 °C. A, B. Front and reverse colony views on PDA; C–J. Phialides; K, L. Conidia. Scale bars: 10 mm (A, B); 10 μm (C–L).

Etymology.

The specific epithet refers to Doupengshan Scenic Area, the locality where the species was collected.

Type.

China, • Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Duyun City, Doupengshan Scenic Area (26°22'17"N, 107°23'31"E), from humus substrates accumulated within tree holes, October 2024, collected by Wen-Kai Lin (holotype: GZUIFR DPS001, dried culture specimen; ex-type culture: GZUIFR DPS003, derived from the holotype material).

Description.

Sexual morph: Not observed. Asexual morph: On PDA after 14 days at 25 °C, colonies reached 35–40 mm in diameter. Colony surface white, raised, circular with entire margin, mycelium velvety to floccose, reverse smooth, pale yellow, orange-yellow to cream-yellow, with yellow pigment diffusing into the agar. Hyphae septate, hyaline, smooth-walled, 1–2 μm wide. Conidiophores arising from aerial hyphae, hyaline, septate, smooth-walled. Phialides mostly solitary, rarely verticillate, borne terminally on conidiophores or lateral branches, 15–23 × 1.8–2.2 μm (n = 30), tapering toward the apex. Conidia formed in slender chains, hyaline, fusiform to narrowly cylindrical, 3–4 × 0.5–1 μm (mean = 3.5 × 1.0 μm, n = 30). Chlamydospores absent.

Habitat.

On humus substrates collected from tree holes of unidentified tree species.

Distribution.

China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Duyun City.

Notes.

Phylogenetic analyses based on multi-locus sequence data revealed that Leptobacillium doupengense forms a distinct and well-supported clade. Phylogenetically, L. doupengense is placed in a subclade with L. coffeanum, L. symbioticum, L. filiforme, and L. xianyushanense. Pairwise sequence comparisons in the ITS region revealed that L. doupengense differs from L. coffeanum (COAD 2057) by 28 bp with 4 gaps (93.9% similarity), from L. filiforme (URM 7918) by 25 bp with 3 gaps (94.6%), from L. symbioticum (NBRC 113865) by 22 bp with 1 gap (95.2%), and from L. xianyushanense (RCEF 6793) by 15 bp with 1 gap (96.7%). Species delimitation in this study was based on an integrative taxonomic approach, including an independent and well-supported placement in the multi-locus phylogeny, ITS sequence divergence from closely related species, and diagnosable morphological differences.

Morphologically, L. doupengense is similar to most species of Leptobacillium in producing long, slender chains of conidia. However, it can be distinguished from its closely related species by several morphological characteristics. Compared with L. coffeanum, L. doupengense produces conidia arranged in chains, whereas the conidia of L. coffeanum are produced in slimy heads (Gomes et al. 2018). Although L. doupengense is morphologically similar to L. xianyushanense, it differs from the latter in colony growth and colony surface morphology. On PDA at 25 °C after 14 days, colonies of L. doupengense reached 35–40 mm in diameter and were slightly raised, smooth, and circular, without obvious divergent cracks. In contrast, colonies of L. xianyushanense grow more slowly, reaching 20–36 mm in diameter after 14 days at 25 °C, and have a white, irregular floccose surface characterized by divergent cracks. Additionally, L. doupengense tends to produce shorter conidia (3–4 × 0.5–1 μm) than L. symbioticum (4.0–6.9 × 0.7–1.6 μm), L. filiforme (7.2–12.5 × 1.0 μm), and L. xianyushanense (3.8–5.6 × 0.5–1.2 μm) (Okane et al. 2020; Chen et al. 2021; Chang et al. 2024). Therefore, the combination of colony morphology, conidial size, ITS sequence divergence, and independent phylogenetic placement supports the recognition of L. doupengense as a distinct species within Leptobacillium.

Family Clavicipitaceae

Genus Metapochonia

Metapochonia doupengshanensis

W.K. Lin, Y.M. Zhou & L.L. Lou sp. nov.

8804837B-5693-59AB-AFDE-79E3A39B10FB

862247

Fig. 4

Figure 4.

Figure 4.

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Morphology of Metapochonia doupengshanensis on PDA after 14 days of growth at 25 °C. A, B. Front and reverse colony views on PDA; C–H. Phialides; I, J. Conidia. Scale bars: 10 mm (A, B); 10 μm (C–J).

Etymology.

The specific epithet refers to Doupengshan Scenic Area, the locality where the species was collected.

Table 2.

Morphological comparison of the new species with closely related species in the genus Leptobacillium.

Species Phialides Conidia Substrate References
L. doupengense Predominantly solitary, rarely verticillate, tapering from the base to the apex. 15–23 × 1.8–2.2 μm Fusiform, narrowly cylindrical. 3–4 × 0.5–1 μm humus substrate within tree holes This study
L. coffeanum Predominantly solitary, rarely in whorls of 2–3. 11–44 (–70) × 1.0–2.4 μm Two types of conidia: larger, 5.3–8.8 × 1.0–1.6 μm; smaller, 2.2–3.8 × 0.8–1.5 μm. Coffee branches Gomes et al. (2018)
L. symbioticum Predominantly solitary, rarely branched. Slender, tapering toward the apex. 7.1–30.6 × 1.6–3.5 μm Slightly fusiform to narrowly cylindrical. 4.0–6.9 × 0.7–1.6 μm Prostigmata, sori of Phakopsora pachyrhizi Okane et al. (2020)
L. filiforme Solitary, gradually tapering toward the apex. 9–18 × 1 μm Fusiform to filiform. 7.2–12.5 × 1.0 μm Citrullus lanatus Crous et al. (2018)
L. xianyushanense Phialides are predominantly solitary, gradually tapering toward the apex. 24.7–28.5 × 1.5–1.9 μm Narrowly cylindrical to slightly fusiform. 3.8–5.6 × 0.5–1.2 μm Rhizosphere of Camellia oleifera Chang et al. (2024)
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Table 3.

Morphological comparison of the new species with closely related species in the genus Metapochonia.

Species Phialides Conidia Conidial mass References
M. doupengshanensis 15–24.5 × 1–2.5 μm Globose to ellipsoidal. 2–2.5 × 1.5–2 μm Conidia produced in slimy heads. This study
M. lutea 22–25 × 1.5 μm The first type is ellipsoidal to ovoid. 3.0–3.5 × 2.0 μm The second type is slightly curved or reniform. 3.5 × 2.5 μm Spherical pseudocapitate structures. Labuda et al. (2018)
M. microbactrospora – Ellipsoidal to bacilliform. 2–2.5 × 0.7–1.0 μm Slimy spore heads. Zare et al. (2001)
M. parasitica 8.5–15 × 7–10 μm Spherical to subglobose. 3.0–4.5 × 2.5–3.0 μm – Barron (1980)
M. rubescens 18–25 × 0.7–1.0 μm Spherical to subglobose. 2.5–3.5 × 2.0–3.0 μm Dry spore heads. Zare et al. (2001)
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Type.

China, • Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Duyun City, Doupengshan Scenic Area (26°21'31"N, 107°25'16"E), from humus substrates accumulated within tree holes, 23 October 2025, collected by Li-Li Lou (holotype: GZUIFR DPS110, dried culture specimen; ex-type culture: GZUIFR DPS111, derived from the holotype material).

Description.

Sexual morph: Not observed. Asexual morph: On PDA after 14 days at 25 °C, colonies were slow-growing, reaching 38–41 mm in diameter, surface white, floccose, subcircular, with an entire margin and raised center; reverse smooth, orange-yellow to yellow. Hyphae hyaline, smooth-walled, branched, septate, 1–2 μm wide. Conidiophores arising from aerial hyphae, mostly prostrate, rarely erect, hyaline, smooth-walled. Phialides solitary or occasionally in pairs, slender, with a cylindrical basal portion, tapering toward the apex, 15–24.5 × 1–2.5 μm (n = 30). Conidia produced at the tips of phialides, solitary or in globose to subglobose heads, unicellular, hyaline, globose to ellipsoidal, 2–2.5 × 1.5–2 μm (mean = 2.0 × 1.5 μm, n = 30). Chlamydospores and crystals not observed.

Habitat.

On humus substrates collected from tree holes of unidentified tree species.

Distribution.

China, Guizhou Province, Qiannan Buyi and Miao Autonomous Prefecture, Duyun City.

Notes.

Phylogenetic analyses based on multi-locus sequence data revealed that Metapochonia doupengshanensis forms a distinct and well-supported clade within the genus Metapochonia (100% ML / 1 PP). It is phylogenetically placed in a subclade together with M. lutea, M. microbactrospora, M. parasitica, and M. rubescens, indicating a close relationship with this group of species. Morphologically, the genus Metapochonia is characterized by producing conidia on slender phialides, which may be verticillate or solitary, and most species are also known to produce chlamydospores. Metapochonia doupengshanensis is morphologically most similar to M. rubescens, but can be distinguished by its globose to ellipsoidal conidia (2–2.5 × 1.5–2 μm), whereas M. rubescens produces globose to subglobose conidia (2.5–3.5 × 2.0–3.0 μm) (Zare et al. 2001). Additionally, pairwise sequence analysis of the tef-1α gene revealed that M. doupengshanensis differs from its close relatives by 11–25 bp with 1–3 gaps. Specifically, it showed 98.8% similarity with M. rubescens (11 bp, 3 gaps), 98.6% with M. lutea (13 bp, 1 gap), 97.8% with M. microbactrospora (20 bp, 3 gaps), and 97.4% with M. parasitica (25 bp, 1 gap). Species delimitation in this study was based on an integrative taxonomic approach, including an independent and well-supported placement in the multi-locus phylogeny, tef-1α sequence divergence from closely related species, and diagnosable morphological differences. Therefore, both morphological and molecular data support the recognition of M. doupengshanensis as a new species within Metapochonia.

Discussion

Sequence data for several loci of Leptobacillium, such as nuclear ribosomal small subunit DNA (nrSSU), the translation elongation factor 1-α gene (tef-1α), and the largest subunit of RNA polymerase II (rpb1), remain limited in public databases. This lack of data may restrict the resolution of phylogenetic relationships within Leptobacillium and between this genus and other closely related genera in Cordycipitaceae. Therefore, the accumulation of multi-locus sequence data from newly collected taxa is important for improving species delimitation and phylogenetic resolution within the genus. The recently reported taxon Leptobacillium hepiali X. Zou, J. Shen & G.X. Fei from Guizhou Province was also considered because of its geographical relevance to the present study. However, its nomenclatural status appears to be uncertain, and the available sequence data suggest that it is not positioned within the clade containing L. doupengense and its closely related species. Therefore, it was not treated as a key comparative taxon in the present study.

Several species closely related to L. doupengense, including L. coffeanum, L. symbioticum, L. filiforme, and L. xianyushanense, have been reported from plants or plant-derived substrates (Gomes et al. 2018; Okane et al. 2020; Chang et al. 2024). In the present study, L. doupengense was isolated from humus accumulated within a tree hole, a substrate mainly derived from decomposed plant material. These observations suggest a possible ecological association between this lineage and plant-derived organic substrates. Given that tree-hole humus is mainly composed of decomposed plant material, L. doupengense may be involved in the colonization or turnover of plant-derived organic matter in this microhabitat, although this ecological role requires further experimental confirmation. However, because only a limited number of isolates are currently available and no ecological experiments were conducted, this possible substrate association should be interpreted cautiously. Further sampling from different substrates and habitats is needed to clarify the ecological preferences of this lineage.

Unlike the substrate associations observed in Leptobacillium, species of Metapochonia are commonly found on animal-derived substrates, such as nematode eggs or cysts (Kepler et al. 2014). In this study, M. doupengshanensis was isolated from tree-hole humus rather than directly from animal-derived substrates. This finding expands the known substrate range of Metapochonia and suggests that tree-hole humus may provide suitable microhabitats for hypocrealean fungi with different ecological affinities. Considering that some members of Metapochonia are associated with nematode eggs or cysts, the occurrence of M. doupengshanensis in tree-hole humus may also reflect potential interactions with microfauna or organic-matter-associated microbial communities; however, this hypothesis remains to be tested, and the ecological role of this species in tree-hole humus remains unknown.

The karst region of Guizhou Province, China, is characterized by complex topography, heterogeneous microhabitats, and rich fungal diversity. Numerous species of Hypocreales have been discovered recently in the region (Chen et al. 2021, 2022a, 2022b, 2022c, 2022d, 2025; Bu et al. 2025; Xu et al. 2025). Tree holes in karst forests often accumulate humus and represent spatially discrete, relatively stable, and nutrient-rich microhabitats. However, hypocrealean fungi in such microhabitats remain poorly studied. The discovery of L. doupengense and M. doupengshanensis from tree-hole humus highlights the potential importance of this overlooked microhabitat as a reservoir of fungal diversity in karst ecosystems. Further surveys combining broader sampling, culture-dependent isolation, high-throughput sequencing, and ecological observations are needed to better understand the diversity and ecological functions of hypocrealean fungi in tree-hole humus.

Supplementary Material

XML Treatment for Leptobacillium doupengense
XML Treatment for Metapochonia doupengshanensis

Acknowledgements

We are grateful to the editor and reviewers for their careful evaluation, constructive comments, and valuable suggestions that helped improve this manuscript. We also thank Dr. Wei Deping for her kind assistance with morphological observations.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Artificial Intelligence (AI) use

The authors accept full responsibility for the content of the manuscript, including the disclosure of any use of AI.

Artificial intelligence (AI) tools were used only for language polishing during manuscript preparation. No AI tools were used for image processing, data analysis, phylogenetic analysis, taxonomic decision-making, or scientific interpretation. The authors reviewed and edited all AI-assisted text and take full responsibility for the content of the manuscript.

Funding

This study was supported by the Natural Science Research Project of Colleges and Universities of Guizhou Provincial Department of Education (Youth Science and Technology Talent Development Project) (Project number: Qian Jiao Ji [2024]34), Guizhou Provincial Basic Research Program (Natural Science) (Project number: Qian Ke He Ji Chu – ZK[2024] Yi Ban 002), Natural Science Project of Guizhou University (Special Post) (Project number: Gui Da Te Gang He Zi [2022] 51).

Author contributions

Conceptualization: W-KL, Y-MZ. Study design: W-KL, Y-MZ. Microscopic observations: W-KL, L-LL. Writing – original draft: W-KL, L-LL. Methodology: W-KL, L-LL. Data analysis: W-KL, L-LL. Funding acquisition: Y-MZ, XZ. Writing – review and editing: Y-MZ, XZ. All authors have read and approved the final version of the manuscript.

Author ORCIDs

W.-K. Lin https://orcid.org/0009-0007-3198-7986

L.-L. Lou https://orcid.org/0009-0000-1072-3120

X. Zou https://orcid.org/0000-0002-3666-5536

Y.-M. Zhou https://orcid.org/0000-0001-8563-8000

Data availability

All of the data that support the findings of this study are available in the main text.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Leptobacillium doupengense
mycokeys.137.196582-treatment1.xml (19.3KB, xml)
XML Treatment for Metapochonia doupengshanensis
mycokeys.137.196582-treatment2.xml (28.4KB, xml)

Data Availability Statement

All of the data that support the findings of this study are available in the main text.

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