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Article

Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China

by
An-Hong Zhu
1,2,3,
Zi-Kun Song
1,4,
Jun-Fang Wang
1,4,
Hao-Wen Guan
1,5 and
Hai-Xia Ma
1,6,7,*
1
Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Hainan Institute for Tropical Agricultural Resources, Haikou 571101, China
2
School of Ecology and Nature Conservation, Beijing Forestry University, Beijing 100083, China
3
Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
4
College of Plant Protection, Jilin Agricultural University, Changchun 130118, China
5
School of Life Science, Liaoning University, Shenyang 110036, China
6
Haikou Key Laboratory for Protection and Utilization of Edible and Medicinal Fungi, Haikou 571101, China
7
Hainan Key Laboratory of Tropical Microbe Resources, Haikou 571101, China
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(1), 72; https://doi.org/10.3390/microorganisms12010072
Submission received: 8 November 2023 / Revised: 12 December 2023 / Accepted: 28 December 2023 / Published: 29 December 2023
(This article belongs to the Section Systems Microbiology)
Browse Figures
Versions Notes

Abstract

:
Hypoxylaceous fungi are abundant in China, but their discovery and report are uneven in various provinces, with more fungi in Yunnan and Hainan and fewer fungi in Tibet. During the investigation of macro-fungi in Motuo county, Tibet Autonomous Region, we collected a number of xylarialean specimens. Six hypoxylaceous specimens growing on dead angiosperm were collected from the forests of Motuo county, and they were described and illustrated as two new species in Hypoxylon based on a combination of morphological characters and molecular evidence. Hypoxylon diperithecium was characterized by its bistratal perithecia, purple-brown stromatal granules, citrine to rust KOH-extractable pigments, and light brown to brown ascospores ellipsoid-inequilateral with conspicuous coil-like ornamentation. Hypoxylon tibeticum was distinct from other species by having pulvinate and applanate stromata, surface vinaceous, with orange granules, orange KOH-extractable pigments, and brown ascospores with inconspicuous ornamentation. The multi-gene phylogenetic analyses (ITS-LSU-RPB2-TUB) supported the two new taxa as separate lineages in the genus Hypoxylon. A key to all known Hypoxylon taxa from China is provided.

1. Introduction

Motuo county, between 27°33′–29°55′ N and 93°45′–96°05′ E, is located in the southeastern Tibet Autonomous Region of southwestern China, and it covers an area of 34,000 square kilometers [1,2,3]. The area enjoys the tropical monsoon rainforest and subtropical humid monsoon climate and is one of the most abundant regions of light, heat and water [4,5]. Its complex topography and diverse habitat abound with different kinds of biological resources, and the area has long been reputed as the “world’s biological gene bank”. There are extremely abundant animal and plant resources, and more than 3000 plant species, 850 genera and 230 families have been reported in the county (http://www.motuo.gov.cn/, accessed on 18 September 2023) [6,7,8,9,10]. Due to severe climatic conditions and inconvenient transportation, few investigations and studies of macro-fungi diversity have been carried out in Motuo county. In the past, about 200 species of macro-fungi have been reported in Motuo county [11,12,13,14,15,16,17,18], among which four species are pyrenomycetous fungi [12]. In recent years, some new species and new records of pyrenomycetous fungi have been discovered in the area, e.g., Eutypella motuoensis Hai X. Ma & Z.E. Yang, Hypoxylon damuense Hai X. Ma, Z.K. Song & Y. Li, H. medogense Hai X. Ma, Z.K. Song & Y. Li, H. zangii Hai X. Ma, Z.K. Song & Y. Li, Annulohypoxylon leptascum (Speg.) Y.M. Ju, J.D. Rogers & H.M. Hsieh, Daldinia bambusicola Y.M. Ju, J.D. Rogers & F. San Martín, H. sublenormandii Suwann., Rodtong, Thienh. & Whalley, and so on [19,20,21].
In order to further understand the diversity of macro-fungi in Motuo county, we carried out a field survey focusing on xylarialean fungi in September 2021. We collected a number of xylarialean specimens, including Annulohypoxylon, Daldinia, Diatrype, Eutypella, Neoeutypella, Hypoxylon, Jackrogersella, and Xylaria. Hypoxylon Bull. (Hypoxylaceae, Ascomycota) was established by Bulliard in 1791 and typified with H. fragiforme (Pers.) J. Kickx f. [22,23]. The type genus is the largest genera in the family Hypoxylaceae, with more than 200 species accepted [24,25,26] and 1188 epithets in the Index Fungorum (http://www.indexfungorum.org/Names/Names.asp, accessed on 22 September 2023). Most taxa of the genus are mainly associated with angiosperm wood as saprotrophs and endophytes, and degrade cellulose and lignin, which play a key role in the substance circulation of a forest ecosystem [24,27,28,29,30,31]. Currently, the placement of Hypoxylon and related genera in Hypoxylaceae is confusing because many are polyphyletic [32,33,34,35,36]. In order to further understand the species diversity and phylogeny of Hypoxylaceae, we carried out complete morphological and multi-gene phylogenetic studies on these specimens from Motuo county. In this study, two new species are introduced based on morphological and phylogenetic evidence.

2. Materials and Methods

2.1. Morphological Studies

The studied specimens were deposited at the Fungarium of the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences (FCATAS). Morphological observations and measurements in this study followed Ma et al. (2018) [24] and Song et al. (2022) [37]. The morphology of stromata and perithecia were observed and measured by a VHX-6000 microscope (Osaka, Japan). Microscopic characteristics, measurements and photographs of the teleomorph were made from slide preparations of fresh stromata mounted in water, 10% KOH and Melzer’s reagent. Sections were observed at a magnification up to ×1000 by using an Olympus IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan). The ornamentation of ascospores were observed with a scanning electron microscope (SEM) (Phenom Corporation, Rotterdam, The Netherlands). The colors were described based on the color-codes by Rayner (1970) [38]. The following abbreviations were used: KOH = 10% potassium hydroxide, n = number of measuring objects, M = average of sizes of all measuring objects.

2.2. DNA Extraction and Sequencing

Total genomic DNA was extracted from fresh stromata using a rapid plant genome extraction kit (Aidlab Biotechnologies, Beijing, China) following the manufacturer’s instructions. Four loci, including nrITS, nrLSU, RPB2, and beta-tubulin (TUB), were amplified and sequenced using primers pairs ITS4/ITS5 [39], LR0R/LR5 [40], fRPB2-7CR/fRPB2-5F [41], and T1/T22 [42], respectively. The PCR procedures for ITS, LSU, RPB2 and beta-tubulin followed Ma et al. (2022) [35] in the phylogenetic analyses. Purification and sequencing were performed by the Beijing Genomics Institute (Shenzhen, China), and newly generated sequences were deposited in GenBank.

2.3. Phylogenetic Analysis

Phylogenetic analyses for Hypoxylon and related genera including Annulohypoxylon, Jackrogersella, Parahypoxylon, Pyrenopolyporus, Rhopalostroma and Thamnomyces were performed with maximum likelihood (ML) and Bayesian inference (BI) analyses based on the combined ITS-nrLSU-RPB2-TUB dataset (Table 1). Biscogniauxia nummularia (Bull.) Kuntze and Xylaria hypoxylon (L.) Grev. were used as outgroups [19].
The sequences were aligned using the online MAFFT tool (http://mafft.cbrc.jp/alignment/server/, accessed on 23 August 2023), and edited using BioEdit 7.0.5.3 [43] and ClustalX 1.83 [44]. Maximum likelihood (ML) analysis was conducted by raxmlGUI 2.0 using rapid bootstrapping with 1000 replicates, and GTRGAMMA+G as a substitution model [35]. Bayesian inference (BI) analysis was implemented in MrBayes 3.2.6 [45] using jModelTest 2 to conduct model discrimination. Six simultaneous Markov chains were run for 4,000,000 generations, from which every 100th generation was sampled as a tree. Phylogenetic trees were viewed in FigTree 1.4.2.
Table 1. GenBank accession numbers of sequences used in phylogenetic analyses are presented.
Table 1. GenBank accession numbers of sequences used in phylogenetic analyses are presented.
Species NameSpecimen No.LocalityGenBank Accession No.References
ITSLSURPB2β-tubulin
Annulohypoxylon annulatumCBS 140775USAKU604559KY610418KY624263KX376353[33,46,47]
A. truncatumCBS 140778USAKX376329KY610419KY624277KX376352[33,47]
Biscogniauxia nummulariaMUCL 51395FranceKY610382KY610427KY624236KX271241[33]
Hypomontagnella barbarensisSTMA 14081ArgentinaMK131720MK131718MK135891MK135893[34]
Hy. monticulosaMUCL 54604GuianaKY610404KY610487KY624305KX271273[33]
Hy. submonticulosaCBS 115280FranceKC968923KY610457KY624226KC977267[24,33]
Hypoxylon addisMUCL 52797EthiopiaKC968931--KC977287[24]
H. anthochroumYMJ 9MexicoJN660819--AY951703[24]
H. aveirenseCMG 29PortugalMN053021--MN066636[48]
H. baihualingenseFCATAS 477ChinaMG490190--MH790276[37]
H. baruenseUCH 9545PanamaMN056428--MK908142[49]
H. begaeYMJ 215USAJN660820--AY951704[32]
H. bellicolorUCH 9543PanamaMN056425--MK908139[49]
H. brevisporumYMJ 36Puerto RicoJN660821--AY951705[32]
H. carneumMUCL 54177FranceKY610400KY610480KY624297KX271270[33]
H. cercidicolaCBS 119009FranceKC968908KY610444KY624254KX271270[24,33]
H. chrysalidosporumFCATAS 2710ChinaOL467294OL615106OL584222OL584229[35]
H. crocopeplumCBS 119004FranceKC968907KY610445KY624255KC977268[33]
H. cyclobalanopsidisFCATAS 2714ChinaOL467298OL615108OL584225OL584232[35]
H. damuenseFCATAS 4207ChinaON075427ON075433ON093251ON093245[19]
H. dieckmanniiYMJ 89041203ChinaJN979413--AY951713[32]
H. diperitheciumFCATAS 4226ChinaON178671ON350864ON365561ON365565This study
H. diperitheciumFCATAS 4323ChinaON178672ON350865ON365562ON365566This study
H. duraniiYMJ 85ChinaJN979414--AY951714[32]
H. erythrostromaYMJ 90080602ChinaJN979416--AY951716[32]
H. eurasiaticumMUCL 57720IranMW367851-MW373852MW373861[50]
H. fendleriDSM 107927USAMK287533MK287545MK287558MK287571[51]
H. ferrugineumCBS 141259AustriaKX090079--KX090080[52]
H. fragiformeMUCL 51264GermanyKM186294KM186295KM186296KM186293[51]
H. fraxinophilumMUCL 54176FranceKC968938--KC977301[24]
H. fulvosulphureumMFLUCC 13-0589ThailandKP401576--KP401584[53]
H. fuscumCBS 113049FranceKY610401KY610482KY624299KX271271[33]
H. gibriacenseMUCL 52698GermanyKC968930---[24]
H. greideraeBRIP 72533USANR 182619OP598062--[54]
H. griseobrunneumCBS 331.73IndiaKY610402MH872399KY624300KC977303[24,33,55]
H. guilanenseMUCL 57726IranMT214997MT214992MT212235MT212239[56]
H. haematostromaMUCL 53301MartiniqueKC968911KY610484KY624301KC977291[34]
H. hainanenseFCATAS 2712ChinaOL467296OL616132OL584224OL584231[35]
H. hinnuleumMUCL 3621USAMK287537MK287549MK287562MK287575[51]
H. howeanumMUCL 47599GermanyAM749928KY610448KY624258KC977277[24,33,57]
H. hypomiltumMUCL 51845GuadeloupeKY610403KY610449KY624302KX271249[33]
H. invadensMUCL 51475FranceMT809133MT809132MT813037MT813038[58]
H. investiensCBS 118183MalaysiaKC968925KY610450KY624259KC977270[24,33]
H. isabellinumSTMA 10247MartiniqueKC968935--KC977295[24]
H. jaklitschiiJF13037Sri LankaKM610290--KM610304[24]
H. jecorinumYMJ 39MexicoJN979429--AY951731[32]
H. jianfengenseFACATAS845ChinaMW984546MZ029707MZ047260MZ047264[36]
H. larissaeFACATAS844ChinaMW984548MZ029706MZ047258MZ047262[36]
H. laschiiMUCL 52796GermanyJX658525---[59]
H. lateripigmentumMUCL 53304MartiniqueKC968933KY610486KY624304KC977290[24,33]
H. lenormandiiCBS 135869CameroonKY610390KY610453KY624262KM610295[33,60]
H. liviaeCBS 115282NorwayNR155154--KC977265[24]
H. lividicolorYMJ 70ChinaJN979432--AY951734[32]
H. lividipigmentumYMJ 233MexicoJN979433--AY951735[32]
H. macrosporumYMJ 47CanadaJN979434--AY951736[32]
H. medogenseFCATAS 4061ChinaON075425ON075431ON093249ON093243[19]
H. munkiiYMJ 90080403ChinaJN979436--AY951738[32]
H. musceumMUCL 53765GuadeloupeKC968926KY610488KY624306KC977280[24,33]
H. notatumYMJ 250USAJQ009305--AY951739[32]
H. olivaceopigmentumDSM 10792USAMK287530MK287542MK287555MK287568[51]
H. perforatumCBS 115281FranceKY610391KY610455KY624224KX271250[33]
H. petriniaeCBS 114746FranceNR155185KY610491KY624279KX271274[33]
H. pilgerianumSTMA 13455MartiniqueKY610412-KY624308KY624315[33]
H. porphyreumCBS 119022FranceKC968921KY610456KY624225KC977264[24,33]
H. pseudofendleriMFLUCC 11-0639ThailandKU940156KU863144--[61]
H. pseudofuscum18264GermanyMW367857MW367848MW373858MW373867[50]
H. pulicicidumCBS 122622MartiniqueJX183075KY610492KY624280JX183072[33,62]
H. rickiiMUCL 53309MartiniqueKC968932KY610416KY624281KC977288[33]
H. rubiginosumMUCL 52887GermanyKC477232KY610469KY624266KY624311[33,63]
H. rutilumYMJ 181France---AY951752[32]
H. samuelsiiMUCL 51843GuadeloupeKC968916KY610466KY624269KC977286[24,33]
H. sheariiYMJ 29MexicoEF026142--AY951753[32]
H. spegazzinianumSTMA 14082ArgentinaKU604573--KU604582[64]
H. sporistriatatunicumUCH 9542PanamaMN056426--MK908140[49]
H. subgilvumYMJ 88113007ChinaJQ009315--AY951755[32]
H. sublenormandiiJF 13026Sri LankaKM610291--KM610303[60]
H. teeravasatiPUFD4IndiaKY863509MF385274MG986895MG986894[65]
H. texenseDSM 107933USAMK287536MK287548MK287561MK287574[51]
H. tibeticumFCATAS4022ChinaOR654146OR654303ON254302ON230084This study
H. tibeticumFCATAS4371ChinaOR654263OR654304QQ303928QQ303964This study
H. tibeticumFCATAS4212ChinaOR654264OR654305ON254308ON254275This study
H. tibeticumFCATAS4373ChinaOR654265OR654306QQ303933QQ303965This study
H. ticinenseCBS 115271FranceJQ009317KY610471KY624272AY951757[32,33]
H. trugodesMUCL 54794Sri LankaKF234422NG066380KY624282KF300548[24,33]
H. ulmophilumYMJ 350RussiaJQ009320--AY951760[32]
H. vinosopulvinatumYMJ 90080707ChinaJQ009321--AY951761[32]
H. vogesiacumCBS 115273FranceKC968920KY610417KY624283KX271275[33]
H. wujiangenseGMBC0213ChinaMT568854MT568853MT585802MT572481[66]
H. wuzhishanenseFCATAS 2708ChinaOL467292OL615104OL584220OL584227[35]
H. zangiiFCATAS 6092ChinaOQ316425OQ348528OQ303910OQ303948[19]
Jackrogersella cohaerensCBS 119126GermanyKY610396KY610497KY624270KY624314[33]
J. multiformisCBS 119016GermanyKC477234KY610473KY624290KX271262[24,33]
Parahypoxylon papillatumATCC 58729USANR155153KY610454KY624223KC977258[24,33]
Pyrenopolyporus hunteriMUCL 52673Ivory CoastKY610421KY610472KY624309KU159530[33,47]
Py.laminosusMUCL 53305MartiniqueKC968934KY610485KY624303KC977292[24,33]
Py. nicaraguensisCBS 117739Burkina FasoAM749922KY610489KY624307KC977272[24,33,57]
Rhopalostroma angolenseCBS 126414Ivory CoastKY610420KY610459KY624228KX271277[33]
Thamnomyces dendroideaCBS 123578FrenchGuianaFN428831KY610467KY624232KY624313[33,67]
Xylaria hypoxylonCBS 122620SwedenKY610407KY610495KY624231KX271279[33]
Species in bold were derived from this study. “-” are not available.

3. Results

3.1. Phylogenetic Analysis

The phylogeny of Hypoxylon and related genera based on a combined ITS-nrLSU-RPB2-TUB dataset included 98 ITS, 64 nrLSU, 65 RPB2 and 95 TUB sequences from 97 specimens representing 93 taxa. There were 2852 character positions for ITS alignment, 3462 character positions for LSU alignment, 1288 character positions for RPB2 alignment, and 2225 character positions for TUB alignment. The dataset of four DNA loci had an aligned length of 3538 characters, of which 1520 characters were parsimony informative.
The topologies from BI and ML analyses are highly similar; the BI tree is shown in this study. Branches that received bootstrap support for maximum likelihood (ML) higher than or equal to 70% (ML-BS) and Bayesian posterior probabilities (BPP) higher than or equal to 0.95 (BPP) were showed in topologies. In phylogenetic analysis, the two new species were clearly separated from other sampled species of Hypoxylon. The two strains of H. diperithecium were closely related to H. anthochroum Berk. & Broome and H. griseobrunneum (B.S. Mehrotra) J. Fourn., Kuhnert & M. Stadler with high support (BS = 98, PP = 1.00, Figure 1), and four strains of H. tibeticum clustered with H. pseudofendleri D.Q. Dai, K.D. Hyde with high support (BS = 94, PP = 1.0, Figure 1).

3.2. Taxonomy

Hypoxylon diperithecium Hai X. Ma, Z.K. Song & A.H. Zhu, sp. nov., Figure 2.
MycoBank: MB850560
Diagnosis. Differs from H. griseobrunneum in its two layers of perithecia, smaller perithecia and asci with shorter stipes. Differs from H. subgilvum in its perithecial layer and color of KOH-extractable pigments and ascospores.
Etymology. The epithet diperithecium (Lat.) refers to the species has bistratal perithecia.
Holotype. China: Tibet Autonomous Region, Motuo County, Damu Township, Kabu Village, 29°38′42″ N, 95°37′44″ E, alt. 1280 m, saprobic on the bark of dead wood, 2 October 2021, Haixia Ma & Zikun Song, FCATAS 4226 (XZ226).
Teleomorph. Stromata pulvinate, 1.4–5 × 0.4–1.3 cm × 0.8–1.2 mm thick; with inconspicuous to conspicuous perithecial mounds; surface livid purple (81) to bay (6), exposing black subsurface layer when colored coating worn off; with purple-brown granules immediately beneath the surface and between perithecia; yielding citrine (13) to rust (39) KOH-extractable pigments; tissue below the perithecial layer dark brown, 0.1–0.7 mm thick. Perithecia ovoid to tubular, bilayer, black, 0.1–0.3 × 0.25–0.45 mm. Ostioles opening higher than the stromatal surface. Asci cylindrical with eight obliquely uniseriate ascospores, 78–139 µm total length, the spore-bearing portion 56–73 × 5.2–7.6 µm, and stipes 23–77 µm long, with amyloid apical apparatus bluing in Melzer’s reagent, discoid, 0.7–0.8 × 1.9–2.1 µm. Ascospores light brown to brown, unicellular, ellipsoid-inequilateral, with narrowly rounded ends, 9.2–11.6 × 4–5.7 µm (n = 60, M = 10.2 × 4.8 µm), with straight spore-length germ slit on the convex side; perispore dehiscent in 10% KOH, with conspicuous coil-like ornamentation in SEM; epispore smooth.
Additional specimens examined. China: Tibet Autonomous Region, Motuo County, Damu Township, Kabu Village, 29°37′45″ N, 95°37′50″ E, alt. 1300 m, saprobic on the bark of dead wood, 2 October 2021, Haixia Ma & Zikun Song, Col. XZ323 (FCATAS 4323).
Note. Some stromata of Hypoxylon diperithecium have two layers of perithecia visible, and the upper and the lower may be same species according to morphology of ascospore and perithecia; this feature is similar to H. subgilvum Berk. & Broome. Hypoxylon subgilvum has three stromatal layers with the basal layer an effete Biscogniauxia, and other two layers are considered the same species [23,68]. Morphologically, H. subgilvum can be distinguished from H. diperithecium by its orange red stromatal granules, KOH-extractable pigments orange, and brown to dark brown ascospores [23]. Moreover, molecular evidence supported H. diperithecium as a distinct species from H. subgilvum (Figure 1).
Although H. anthochroum and H. griseobrunneum were grouped with H. diperithecium (Figure 1), they differ from the new species proposed here because the former has only one layer of perithecia instead of two layers and has dull reddish brown or blackish granules immediately beneath surface and between perithecia, yielding isabelline (65), olivaceous (48), gray olivaceous (107), greenish olivaceous (90), or amber (47) KOH-extractable pigments [23]. While some stromata of H. griseobrunneum tend to develop multiple perithecial layers, it can be distinguished from H. diperithecium by having larger perithecia, with KOH-extractable pigments Fawn (87), and longer stipes of asci (76–86 µm) [24]. Therefore, H. diperithecium is proposed as a new species.
Hypoxylon tibeticum Hai X. Ma, Z.K. Song & A.H. Zhu, sp. nov., Figure 3.
MycoBank: MB850558
Diagnosis. Differs from H pseudofendler in its smaller perithecia and slightly larger ascospores. Differs from H. wuzhishanense in its brown vinaceous stromatal surface with orange granules between perithecia and perispore dehiscent in KOH. Differs from H. pilgerianum in its larger ascospores.
Etymology. The epithet tibeticum (Lat.) refers to the locality (Tibet Autonomous Region) of the type specimens.
Holotype. China: Tibet Autonomous Region, Motuo County, Damu Township, Kabu Village, the large bend of Linduo, 29°27′51″ N, 95°26′39″ E, alt. 781 m, saprobic on the stems of dead bamboo, 24 September 2021, Haixia Ma & Zikun Song, FCATAS 4022 (XZ22).
Teleomorph. Stromata effused-pulvinate, applanate, 1.4–11.1 × 0.2–1.5 cm × 0.2–0.35 mm thick, irregularly elongate, often coalescent; surface brown vinaceous (84) or dark vinaceous (85), pruinose, with inconspicuous to slightly conspicuous perithecial mounds; with orange granules immediately beneath the surface and between perithecia; yielding orange (7) KOH-extractable pigments; the tissue beneath the perithecia dark brown, 0.05–0.15 mm thick. Perithecia spherical, black, 0.1–0.23 mm diam. Ostioles umbilicate, opening lower than the stromatal surface, mostly fringed with white material forming a disc. Asci cylindrical, with eight obliquely uniseriate ascospores, 75–101 µm total length, the spore-bearing portion 64–91 × 7.8–11.5 µm, and stipes 9–17 µm long, with amyloid apical apparatus bluing in Melzer’s reagent, discoid, 0.89–1.54 × 2.1–2.95 µm. Ascospores brown, unicellular, ellipsoid-inequilateral, with narrowly to broad rounded ends, 9.8–13 × 5.1–6.9 µm (n = 60, M = 11.34 × 6.21 µm), with straight spore-length germ slit on the convex side; perispore dehiscent in 10% KOH, with faint inconspicuous coil-like ornamentation in SEM; epispore smooth.
Additional specimens examined. China: Tibet Autonomous Region, Motuo County, Damu Township, Kabu Village, the large bend of Linduo, 29°27′51″ N, 95°26′39″ E, alt. 780 m, saprobic on the stems of dead bamboo, 24 September 2021, Haixia Ma & Zikun Song, FCATAS 4371 (XZ324); Kabu Village, 29°37′45″ N, 95°37′50″ E, alt. 1280 m, saprobic on dead bamboo, 2 October 2021, Haixia Ma & Zikun Song, FCATAS4212 (XZ212), FCATAC4373 (XZ326).
Note. Based on the phylogenetic analyses, four species of Hypoxylon growing on dead bamboo culms grouped together (Figure 1), including H. pilgerianum Henn., H. pseudofendleri D.Q. Dai & K.D. Hyde, H. wuzhishanense Hai X. Ma & Z.K. Song, and the new species H. tibeticum.
In the phylogenetic tree (Figure 1), H. tibeticum is the sister species of H. pseudofendleri from Thailand with strong support values (BS = 94, PP = 1). Morphologically, both H. tibeticum and H. pseudofendleri have effused-pulvinate and purplish-brown stromata, with orange granules beneath the surface and between perithecia. However, H pseudofendleri differs in its larger perithecia (0.5–0.85 × 0.35–0.5 mm), ostioles slightly higher than the stromatal surface, and slightly smaller ascospores (9–11.5 × 4.5–6.5 µm, M = 10.2 × 5.7 µm) [61]. Hypoxylon wuzhishanense from Hainan tropical rainforest of China has similar stromatal morphology and ascospores size, but it has rust (39), livid purple (81) to dark brick (60) stromatal surface, with yellowish-brown granules beneath the surface and between perithecia, and most of perispore indehiscent in 10% KOH [35]. Hypoxylon pilgerianum was first described from Brazil on culms of Chusquea [69]; subsequently, many specimens on culms of dead bamboo were found from China, Madagascar, Malaysia, Papua New Guinea, and Trinidad [23,68]. Hypoxylon pilgerianum s. Ju & Rogers is similar to H. tibeticum in stromatal morphology, but it differs in having shorter [8.5–12 (–13.5) μm] and narrower ascospores [4–5 (–5.5) μm] [23]. Moreover, the phylogenetic analyses (Figure 1) showed that they are different species.
Dichotomous key to Hypoxylon species from China
1. Stromata on bamboo ............................................................................................................ 2
1. Stromata on dicot wood ...................................................................................................... 4
2. Most perispore indehiscent in 10% KOH .............................................. H. wuzhishanense
2. Perispore dehiscent in 10% KOH ....................................................................................... 3
3. KOH-extractable pigments ochreous (44), honey (64) or amber (47); ascospores 8.5–12 (–13.5) × 4– 5 (–5.5) µm ............................................................................. H. pilgerianum
3. KOH-extractable pigments orange (7); ascospores 9.8–13 × 5.1–6.9 µ....... H. tibeticum
4. Stromatal surface dark cyan blue or olivaceous ............................................................... 5
4. Stromatal surface other colors.............................................................................................. 6
5. Stromatal surface dark cyan blue; ascospores 11.5–13.5 × 5–6 µm..............H. cyanescens
5. Stromatal surface olivaceous or isabelline; ascospores 9–13 × (4–) 4.5–6 µm .........................................................................................................................H. musceum
6. Ascospores equilateral or nearly equilateral ......................................................................7
6. Ascospores inequilateral ......................................................................................................16
7. Ostioles higher than the stromatal surface ........................................................................8
7. Ostioles lower than the stromatal surface .........................................................................9
8. Stromata glomerate to pulvinate, with very conspicuous perithecial mounds; KOH-extractable pigments isabelline (65) or hazel (88) ...........................................H. croceum
8. Stromata pulvinate, with inconspicuous perithecial mounds; KOH-extractable
pigments brick (59) ........................................................................................H. parksianum
9. Perispore dehiscent in 10% KOH............................................................... H. hypomiltum
9. Perispore indehiscent in 10% KOH.....................................................................................10
10. Perithecia tubular to long tubular........................................................................................ 11
10. Perithecia obovoid .................................................................................................................13
11. Stromatal surface fulvous (43), rust (39), sinna (8), ochreous (44), or apricot (42); KOH-extractable pigments orange (7) ...............................................................H. cinnabarinum
11. Stromatal surface sepia (63) or chestnut (40) ......................................................................12
12. KOH-extractable pigments greenish yellow (16), dull green (70), or dark green (21);
ascospores 6.5–9.5 (–10) × 3–4.5 µm ..................................................................H. investiens
12. KOH-extractable pigments livid violet (79), violaceous gray (113), or violet slate (99);
ascospores (10.5–) 11–16 × (4.5–) 5–6.5 µm .................................................H. sclerophaeum
13. Without apparent KOH-extractable pigments or dilute grayish sepia ..........................14
13. With KOH-extractable pigments .........................................................................................15
14. Without apparent KOH-extractable pigments or dilute grayish sepia (106); ascospores
6.5–10 (–11) × (3–) 3.5–4 µm ......................................................................... H. dieckmannii
14. Without apparent KOH-extractable pigments; ascospores (9.5–) 10.5–11.5 (–12.5) ×
4.5–6 µm .......................................................................................................... H. yunnanense
15. KOH-extractable pigments olivaceous (48), greenish olivaceous (90), gray olivaceous
(127), or olivaceous gray (121); ascospores (11.5–) 12–15 (–16) × 5.5–7 µm ......................
.....................................................................................................................H. fuscopurpureum
15. KOH-extractable pigments hazel (88); ascospores 7–8.5 × 4–4.5 µm.......... H. gilbertsonii
16. Stromata hemispherical to spherical .................................................................................17
16. Stromata pulvinate to effused-pulvinate...........................................................................23
17. Ascospore length up to 20 µm ..........................................................................................18
17. Ascospore length less than 20 µm.....................................................................................19
18. Ascospores 18–28 × 6–10 µm........................................................................... H. apiculatum
18. Ascospores 8–20 × 4–8 µm....................................................................................... H. fuccum
19. Perithecia tubular .................................................................................................................20
19. Perithecia spherical to obovoid ..........................................................................................21
20. Stromata with orange red granules, with KOH-extractable pigments orange (7) or
scarlet (5); ascospores 13.5–18 (–19) × 7–8 (–8.5) µm............................. H. haematostroma
20. Stromata with dark reddish brown or blackish granules, with KOH-extractable
pigments olivaceous (48), greenish olivaceous (90), isabelline (65), or dull green (70);
ascospores 8.5–18.5 × 4.5–8 (–8.5) µm........................................................ H. placentiforme
21. KOH-extractable pigments amber (47) with greenish yellow (16) tone, or greenish
yellow (16) with citrine (13) tone; ascospores (8–) 9–12 (–13) × 4–6 µm...... H. perforatum
21. KOH-extractable pigments orange (7) ..............................................................................22
22. Ascospores (10.5–) 11–15 × 5–6.5 (–7) µm, with straight germ slit................H. fragiforme
22. Ascospores 7–9.5 (–10) × 3–4.5 µm, with slight sigmoid germ slit ............H. howeianum
23. Ostioles at the same level or higher than the stromatal surface ....................................24
23. Ostioles lower than the stromatal surface .........................................................................29
24. Perithecia tubular, ascospores 6–7.5 × 3–3.5 µm.................................... H. lienhwacheense
24. Perithecia spherical to obovoid............................................................................................25
25. KOH-extractable pigments orange (7), scarlet (5) or amber (47) .....................................26
25. KOH-extractable pigments with other colors ....................................................................28
26. Stromata with red or scarlet granules; ascospores 7.5–9.5 × 3.5–4.5 µm........... H. rutilum
26. Stromata with orange granules ...........................................................................................27
27. Ascospores 8–10 × 3.5–4.5 µm.................................................................................. H. laschii
27. Ascospores 9.9–12.8 × 4.6–7 µm....................................................................... H. medogense
28. KOH-extractable pigments hazel (88), sienna (8), cinnamon (62), fulvous (43), umber
(9), or ochreous (44); ascospores 9.5–15 (–16) × 4–6.5 (–7) µm ................... H. lenormandii
28. KOH-extractable pigments pale vinaceous (85) to livid vinaceous (83) and vinaceous
purple (101); ascospores 6.1–9.6 × 3.2–5 µm .................................................. H. hainanense
29. Without apparent KOH-extractable pigments; ascospores (12–) 13–16 × 5–6 µm ..........
................................................................................................................ H. kretzschmarioides
29. With KOH-extractable pigments ........................................................................................30
30. Most ascospore length less than 8 µm ..............................................................................31
30. Most ascospore length more than 8 µm ...........................................................................33
31. KOH-extractable pigments olivaceous gray (12), greenish olivaceous (90), or gray
olivaceous (107); ascospores 5.5–8 × 2.5–3.5 µm ........................................ H. brevisporum
31. KOH-extractable pigments orange.......................................................................................32
32. Perithecia obovoid to tubular; ascospores (4.5–) 5–7 × 2.5–3.5 µm ....................................
.............................................................................................. H. subgilcum var. microsporum
32. Perithecia spherical to ovoid; ascospores 6.5–8.5 × 4–5 µm .......................... H. hubeiense
33. Asci with apical apparatus highly reduced or lacking, not bluing in Melzer’s reagent. ...
...................................................................................................................................................34
33. Asci with apical apparatus bluing in Melzer’s reagent.....................................................39
34. KOH-extractable pigments orange tone..............................................................................35
34. KOH-extractable pigments other colors..............................................................................37
35. Ascospores with inconspicuous coil-like ornamentation, (9–) 9.5–12 × 5–6 µm................
..............................................................................................................................H. cercidicola
35. Ascospores with conspicuous coil-like ornamentation.....................................................36
36. KOH-extractable pigments orange (7), sienna (8), or amber (47); ascospores 9.2–15.6 × 5.5–7.5 µm, with spore-length straight germ slit........................................H. baihualingense
36. KOH-extractable pigments luteous (12); ascospores 12–14 × 5.5–6.5 (–7) µm....H. shearii
37. Ascospores with inconspicuous coil-like ornamentation, (11–) 12–16 × (5.5–) 6–7.5 µm....
..................................................................................................................................H. notatum
37. Ascospores with conspicuous coil-like ornamentation...................................................38
38. Ascospores 8–10.6 (–11.1) × 4.1–6.3 (–7.1) µm, with conspicuously straight spore-length
germ slit..................................................................................................H. chrysalidosporum
38. Ascospores 11–15.2 × 5.1–7 µm, with more sigmoid to less straight spore-length germ
slit............................................................................................................H. cyclobalanopsidis
39. Ascospores with conspicuous coil-like ornamentation....................................................40
39. Ascospores smooth or with inconspicuous coil-like ornamentation..............................44
40. Most perispore indehiscent in 10% KOH; ascospores 8.2–10.5 × 4.1–5.5 µm. ....................
....................................................................................................................................H. damuense
40. Perispore dehiscent in 10% KOH..........................................................................................41
41. Ascospores with straight germ slit.......................................................................................42
41. Ascospores with straight to slightly sigmoid germ slit......................................................43
42. Perithecia bilayer; ascospores 9.2–11.6 × 4–5.7 µm..................................H. deperithecium
42. Perithecia monolayer; ascospores 10.3–13.6 × (4.2–) 4.7–6.1 µm................H. jianfengense
43. KOH-extractable pigments orange (7) or scarlet (5); ascospores (9) 9.5–12 × 4.5–5 µm.......
.....................................................................................................................................H. retpela
43. KOH-extractable pigments isabelline (65) or amber (47); ascospores 9.5–13 (–14.5) ×
4.5–6.5 µm..................................................................................................................H. duranii
44. Ascospore length up to 15 µm ...........................................................................................45
44. Ascospore length less than 15 µm .....................................................................................46
45. Stromatal surface cinnamon (62), fulvous (43), apricot (42), sienna (8), rust (39), or bay
(6); ascospores (9–) 9.5–15 (–17.5) × 4–7 (–7.5) µm.......................................H. crocopeplum
45. Stromatal surface rust (39), sienna (8), fulvous (43), or bay (6); ascospores 15.5–22.9 (–
23.6) × 7.3–10.6 µm...................................................................................................H. larissae
46. Ascospores with sigmoid germ slit......................................................................................47
46. Ascospores with straight, straight or slightly sigmoid germ slit.....................................48
47. KOH-extractable pigments orange (7); ascospores (8–) 9–12 × 4–5.5 µm..........H. fendleri
47. KOH-extractable pigments vinaceous purple (101); ascospores 9.5–12.5 × 5–6 µm...........
.................................................................................................................................H. fuscoides
48. Ascospores with straight germ slit.......................................................................................49
48. Ascospores with straight to slightly sigmoid germ slit......................................................53
49. KOH-extractable pigments orange tone .............................................................................50
49. KOH-extractable pigments other colors .............................................................................51
50. Stromata with orange granules; ascospores (10–) 10.5–11.5 (–12.5) × 5–6.5 µm..............
.......................................................................................................................................H. dengii
50. Stromata with yellowish brown or brown granules; ascospores (8–) 9–12 × 4–5.5 µm.......
...........................................................................................................................H. rubiginosum
51. Perithecia obovoid to tubular; ascospores 8–11 × 3.5–4.5 µm ...........................H. trugodes
51. Perithecia spherical, ovoid to obovoid................................................................................52
52. Stromatal surface brown vinaceous; ascospores 11–13 × 5–6 µm ...H. vinosopulvinatum
52. Stromatal surface livid red and vinaceous; ascospores 10.9–14.6 × 4.8–6.4 µm ..H. zangii
53. KOH-extractable pigments orange ......................................................................................54
53. KOH-extractable pigments other colors .............................................................................55
54. Asci with apical apparatus bluing to faintly bluing in Melzer’s iodine reagent, 0.3–1 µm high × 1.5–2.2 µm broad; ascospores 7–11 × 3.5–5 µm....................................H. subgilvum
54. Asci with apical apparatus bluing in Melzer’s iodine ragent, 0.2–0.5 µm high × 1–1.5 µm broad; ascospores 8–9.5 (–11) × 4–5 µm.........................................................H. jecorinum
55. Perithecia tubular; ascospores 11–12.5 × 4.5–5 µm........................................H. lividicolor
55. Perithecia subglobose or obovoid to tubular......................................................................56
56. Perithecia obovoid to tubular; ascospores 8.5–13.5 × 4–6 µm...................H. anthochroum
56. Perithecia subglobose; ascospores 8.5–10 × 4.5–6 µm................................H. wujiangensis

4. Discussion

Currently, the genus Hypoxylon is still considered a paraphyletic group in Hypoxylaceae based on a single-region (ITS sequences) or multi-locus phylogeny involving both protein-coding and rDNA genes [33,70,71,72]. In this study, two species of Hypoxylon from Tibet of China, H. diperithecium and H. tibeticum, are proposed as new species based on morphological features and multi-gene (ITS-LSU-RPB2-TUB) phylogenetic analyses. Fifty-five species of Hypoxylon have been reported and described in China [19,35,36,66,73,74], and this study expanded the numbers of Hypoxylon species to 57 around China. However, studies in China are still few and the relationships amongst Hypoxylon species remain unresolved. Therefore, more comprehensive studies on the diversity, phylogeny, and evolution of the genus Hypoxylon depend on more collections and data from poorly sampled areas. With the in-depth investigation of Hypoxylon in Tibet, an increasing number of new species and new records will be discovered, and the species diversity will be richer.

Author Contributions

A.-H.Z., Z.-K.S. and H.-X.M. prepared the samples; Z.-K.S. made morphological examinations and performed molecular sequencing; J.-F.W. and H.-W.G. performed phylogenetic analyses; A.-H.Z. and H.-X.M. wrote the manuscript; H.-X.M. conceived and supervised the work. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed by the Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (No. 1630032022001), the National Natural Science Foundation of China (No. 31972848), the Collection, preservation, identification and evaluation of tropical crop germplasm resources in the remote tropical area (NONYNCBKFSXM2023-2025).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequences newly generated were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 16 October 2023); Table 1). All new taxa were deposited in MycoBank (https://www.mycobank.org/ (accessed on 18 October 2023); MycoBank identifiers follow new taxa).

Acknowledgments

We gratefully acknowledge Zhen Liu (Motuo Forestry and Grassland Administration) and Zhu-Nian Wang, Qing-long Wang, Hu-Biao Yang, Shi-Song Xu (Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences) for help during field collections. Special thanks to Xiao-Wei Qin (Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences) and Xiao-Peng Wu (Analysis and Testing Center, Chinese Academy of Tropical Agricultural Sciences) for assistance in micrographs produced by SEM.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. BI phylogenetic tree of the genus Hypoxylon inferred from multi-gene alignment of ITS-LSU-RPB2-TUB. ML bootstrap support (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95 are given at the nodes in this order. New species in this study are indicated in bold.
Figure 1. BI phylogenetic tree of the genus Hypoxylon inferred from multi-gene alignment of ITS-LSU-RPB2-TUB. ML bootstrap support (BS) ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.95 are given at the nodes in this order. New species in this study are indicated in bold.
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Figure 2. Hypoxylon diperithecium (holotype, FCATAS 4226). (a,b). Stromata; (c,d). Stroma in vertical section showing the perithecia and tissue below the perithecial layer; (e,o). Ascus in water; (f,g). Ascospore in water; (h). Ascospores and germ slit; (i,j). Ascospore in 10% KOH; (k). Ascospores under SEM; (l). KOH-extractable pigments; (m). Apical apparatus; (n). Ascus in Melzer’s reagent. Scale bars: (a) = 1 mm; (b–d) = 200 μm; (e–j,m–o) = 10 μm; (k) = 5 μm.
Figure 2. Hypoxylon diperithecium (holotype, FCATAS 4226). (a,b). Stromata; (c,d). Stroma in vertical section showing the perithecia and tissue below the perithecial layer; (e,o). Ascus in water; (f,g). Ascospore in water; (h). Ascospores and germ slit; (i,j). Ascospore in 10% KOH; (k). Ascospores under SEM; (l). KOH-extractable pigments; (m). Apical apparatus; (n). Ascus in Melzer’s reagent. Scale bars: (a) = 1 mm; (b–d) = 200 μm; (e–j,m–o) = 10 μm; (k) = 5 μm.
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Figure 3. Hypoxylon tibeticum (holotype, FCATAS 4226). (a,b). Stromata; (c). Stromatal surface; (d). Stroma in vertical section showing the perithecia and tissue below the perithecial layer; (e). KOH-extractable pigments; (f). Ascus in water; (g,h). Ascus and apical apparatus in Melzer’s reagent; (i–l). Ascus in water; (m–p). Ascospore in water; (q,r). Ascospores in 10% KOH; (s,t). Ascospores under SEM. Scale bars: (a,b,e) = 1 cm; (c,d) = 200 μm; (f) = 20 μm; (g–t) = 10 μm.
Figure 3. Hypoxylon tibeticum (holotype, FCATAS 4226). (a,b). Stromata; (c). Stromatal surface; (d). Stroma in vertical section showing the perithecia and tissue below the perithecial layer; (e). KOH-extractable pigments; (f). Ascus in water; (g,h). Ascus and apical apparatus in Melzer’s reagent; (i–l). Ascus in water; (m–p). Ascospore in water; (q,r). Ascospores in 10% KOH; (s,t). Ascospores under SEM. Scale bars: (a,b,e) = 1 cm; (c,d) = 200 μm; (f) = 20 μm; (g–t) = 10 μm.
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MDPI and ACS Style

Zhu, A.-H.; Song, Z.-K.; Wang, J.-F.; Guan, H.-W.; Ma, H.-X. Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China. Microorganisms 2024, 12, 72. https://doi.org/10.3390/microorganisms12010072

AMA Style

Zhu A-H, Song Z-K, Wang J-F, Guan H-W, Ma H-X. Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China. Microorganisms. 2024; 12(1):72. https://doi.org/10.3390/microorganisms12010072

Chicago/Turabian Style

Zhu, An-Hong, Zi-Kun Song, Jun-Fang Wang, Hao-Wen Guan, and Hai-Xia Ma. 2024. "Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China" Microorganisms 12, no. 1: 72. https://doi.org/10.3390/microorganisms12010072

APA Style

Zhu, A. -H., Song, Z. -K., Wang, J. -F., Guan, H. -W., & Ma, H. -X. (2024). Multi-Locus Phylogeny and Morphology Reveal Two New Species of Hypoxylon (Hypoxylaceae, Xylariales) from Motuo, China. Microorganisms, 12(1), 72. https://doi.org/10.3390/microorganisms12010072

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