Morphological Diversity and Preliminary DNA Barcoding of Xylaria (Xylariales) from Estación Científica San Francisco, Including Xylaria aenea as a New Record for Ecuador

Abstract

The genus Xylaria comprises numerous species, particularly prevalent in tropical ecosystems such as those of Ecuador. Despite its ecological importance, the taxonomy of the genus remains challenging, and much of its diversity in the Neotropics remains under-documented. This study provides a preliminary characterization of the Xylaria diversity at the Estación Científica San Francisco, an Andean biodiversity hotspot in Southern Ecuador. Through an integrated approach including detailed macro- and micro-morphological descriptions and nuclear ribosomal DNA (nrDNA ITS and LSU) phylogenetic analyses, 20 Xylaria specimens were examined. As a result, ten species were recognized: Xylaria adscendens, X. cf. anisopleura, X. apiculata, X. curta, X. enterogena, X. fissilis, X. globosa, X. aff. telfairii, X. tuberoides, and X. aenea, the latter representing a new record for Ecuador. The phylogenetic analysis presented here serves as a preliminary systematic positioning of these specimens within the genus rather than a comprehensive global reconstruction. While these ribosomal markers provided preliminary insights into species relationships, partial incongruence with morphospecies highlights the evolutionary complexity of certain lineages and underscores the need for future multilocus studies. Furthermore, four additional phylotypes found in their anamorphic state are documented, suggesting that local diversity exceeds current records. By providing detailed morphological documentation supported by preliminary barcode data from a poorly sampled region, this study contributes vital information to the global understanding of Xylaria and underscores the importance of Southern Ecuador as a reservoir of fungal diversity.

1. Introduction

Xylaria Hill ex Schrank is the largest genus within the family Xylariaceae Tul. & C. Tul., with 901 epithets deposited in Mycobank (accessed: 8 July 2024). Ecologically, species of Xylaria are recognized as decomposers of organic matter [1,2], particularly lignin- and cellulose-rich wood [3,4,5]. Morphologically, the genus is characterized by having perithecia embedded in sometimes branched stromata, which are commonly carbonaceous, cracked, and rough. In their immature stage, the stromata typically produce white conidia along their upper portions. In some species, such as those within the Xylaria polymorpha complex [6], including X. anisopleura, X. bulbosa, X. feejeensis, X. longipes, X. obovate, X. polymorpha, X. schweinitzii, and X. tuberiformis, these conidial structures may later be replaced by perithecia during stromatal maturation [6,7,8]. Microscopically, Xylaria have unitunicate and inoperculate cylindrical asci with an amyloid apical apparatus, generally with eight ascospores in each. The ascospores are typically pigmented, ranging from pale brown to black, ellipsoid-inequilateral in shape, and typically having a hyaline germ slit [7,8].

The genus exhibits a high degree of intraspecific variability and interspecific similarity, which makes it difficult to identify taxonomically informative characters [9]. This has contributed to significant confusion in historical taxonomy [10,11]. Due to this morphological complexity, species identification is most effectively achieved using integrated morphological and molecular datasets. In this approach, morphological data are validated through phylogenetic analysis using a combination of genetic markers. Essential markers include the barcode marker nrDNA ITS-5.8S and LSU [12,13,14,15,16,17,18]. Other useful markers include α-actin, β-tubulin, and RNA polymerase subunit II [19,20].

Species of Xylaria have a global distribution, often reported as saprotrophs in tropical and subtropical regions with varying levels of endemism [21,22,23]. This significant group of fungi remains understudied, particularly in South America. Notable studies are limited to a few countries, including Argentina [24,25,26], Colombia [27], Brazil [28,29,30], and Venezuela [31]. Although Ecuador is a mega-diverse country, there are few relevant studies on fungi across its different ecosystems [32,33,34].

To address this knowledge gap, we conducted a mycological survey of Xylaria species at the Estación Científica San Francisco, a site located within the Andean biodiversity hotspot [35]. Currently, mycorrhizal fungi are the only group of fungi from this site that have been studied in depth, using both molecular [36,37,38] and morphological methods [39]. No collection-based studies on macrofungi have been conducted at the Estación Científica San Francisco to date. Therefore, this study aims to provide an important contribution to the knowledge of fungal diversity in Ecuador by performing a morphological and molecular characterization of Xylaria specimens collected from this forest reserve in southern Ecuador.

2. Materials and Methods

2.1. Collecting and Site Description

Sampling of Xylaria specimens was carried out at Estación Científica San Francisco, a tropical mountain forest reserve bordering Podocarpus National Park (PNP) in Zamora Chinchipe province. Detailed information about the forest is given in [40]. The ascomata were collected between April 2014 and October 2015 from the paths named AT and T2, at elevations of approximately 1900 and 2500 m a.s.l. respectively. Vouchered specimens were deposited in the fungarium of Universidad Técnica Particular de Loja’s Herbarium [HUTPL(F)] with accession codes provided in

Table 1. Specimens of Xylaria studied, and species defined by morphological and molecular data.

No.	Fungarium Code	Determination	ITS-5.8S and LSU D1/D2 (Accession Numbers)
1	HUTPL(F)-604	Xylaria adscedens *	OR086092 only ITS-5.8S
2	HUTPL(F)-1448	Xylaria adscedens	OR088119
3	HUTPL(F)-1543	Xylaria aenea	OR088132
4	HUTPL(F)-1458	Xylaria cf. anisopleura	OR088123
5	HUTPL(F)-581	Xylaria apiculata	OR088126
6	HUTPL(F)-704	Xylaria apiculata	OR088127
7	HUTPL(F)-1532	Xylaria curta	OR088133
8	HUTPL(F)-1437	Xylaria enterogena	OR088131
9	HUTPL(F)-1436	Xylaria fissilis	OR088122
10	HUTPL(F)-1514	Xylaria fissilis	OR088121
11	HUTPL(F)-1542	Xylaria fissilis	OR088120
12	HUTPL(F)-663	Xylaria globose *	OR086093 only ITS-5.8S
13	HUTPL(F)-1497	Xylaria globosa	OR086091 only ITS-5.8S
14	HUTPL(F)-1083	Xylaria aff. telfairii	OR088130
15	HUTPL(F)-1559	Xylaria tuberorides	OR088134
16	HUTPL(F)-603	Xylaria sp. *	OR086094 only ITS-5.8S
17	HUTPL(F)-921	Xylaria sp. *	OR088128
18	HUTPL(F)-1077	Xylaria sp. *	OR088124
19	HUTPL(F)-1100	Xylaria sp. *	OR088125
20	HUTPL(F)-1533	Xylaria sp. ^	Sequence not available

* Anamorph state; assigned to phylogenetic species based on molecular data derived from nrITS-5.8S and LSU D1/D2 sequences. ^ Anamorph state without DNA information.

.

2.2. Morphological Diagnosis

We recorded the observable characteristics of the ascomata, including color, texture, and measurements of stipe and stroma length and width. To examine internal structures, we made freehand horizontal and transversal sections of each stroma to observe the color of the perithecia and internal tissues. For microscopic analysis, fresh and dried samples of perithecial contents were stained with Melzer’s reagent to test for an amyloid reaction. Other reagents, such as 1% Phloxine, 1% Methyl blue, and 3% Potassium hydroxide (KOH), were also used as needed. All sections were examined at 1000x magnification using an Olympus BX51 light microscope (Olympus Corporation, Tokyo, Japan).

The length and width of various microscopic structures were measured using iWork EX image analysis software (version 2.0), which was integrated with a Lanoptik-MDX503 digital microscopy camera (Lanoptik Ltd., Fuzhou, China). We took at least 25 measurements for perithecia and asci, and at least 30 measurements for ascospores. Ascospore measurements are presented as a range (min–) with the mean (–max) and the Q-value (average spore length/width ratio) [41]. We classified ascospore shapes primarily according to [42].

Species were identified using scientific literature and standard taxonomic keys, which are cited in the corresponding species descriptions. The term “cf.” (confer) indicates that specimens are morphologically similar to a specific species but have an uncertain phylogenetic relationship at the species level. The term “aff.” (affinis) indicates that a described species resembles its holotype but is considered distinct and potentially undescribed.

2.3. PCR Amplification and Sequencing of DNA

PCR was performed using the Phire^® Plant Direct PCR Kit (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s instructions. A small piece of stroma, approximately 1 mm³ in size and containing perithecia when possible, was placed into 20 μL of Dilution Buffer. Of this solution, 2 μL were used as the PCR template. The total PCR reaction volume was 20 μL, including 0.4 μL (25 pmol) of each primer and 0.4 μL of bovine serum albumin (BSA 10%) as an additive. For amplification, we used a nested PCR approach. The first reaction utilized the universal primers ITS1-F [43] and LR5 [44]. A subsequent, nested PCR was then performed using the primers ITS1 [45] and NL4 [46]. The PCR conditions for both reactions were as follows: an initial denaturation at 98 °C for 5 s, followed by 40 cycles. Each cycle consisted of denaturation at 98 °C for 10 s, annealing at 55 °C for 20 s, and extension at 72 °C for 30 s. A final extension at 72 °C for 10 min was performed to complete the reaction. PCR products were visualized on a 1% (w/v) agarose gel stained with 1X GelRed™ Safe Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA).

All positive PCR products (15–18 μL) were purified by adding an equal volume (1:1 ratio) of PEG (20% Polyethylene glycol 8000/2.5 M NaCl), followed by incubation at 37 °C for 15 min. The mixture was then centrifuged at 16,500 RCF for 15 min. The resulting pellets were rinsed with 80% cold ethanol and dried for 45 min at room temperature under a sterile flow hood. Finally, the dried pellets were re-suspended in 60 μL of deionized water. The purified PCR products, at a final concentration of 20–40 ng/μL, were sent to Macrogen (Seoul, Republic of Korea) for sequencing.

2.4. Phylogenetic Analyses

Sequence chromatograms were verified using Codon Code Aligner 5.1.4 (CodonCode Corporation, Centerville, MA, USA). The resulting sequences were aligned with reference sequences from the NCBI GenBank (https://www.ncbi.nlm.nih.gov) and UNITE (https://unite.ut.ee) databases (accessed: 10 January 2026). Sequences showing at least 95% similarity, as well as those containing taxonomic information such as type material or species names, were incorporated into the analysis (Table 2).

All nrITS-5.8S and LSU D1/D2 sequences were aligned using the G-INS-i strategy in MAFFT v.7 [47]. The ITS-5.8S region of the sequences was defined and extracted using ITSx v.1.0.11 [48], while the LSU D1/D2 region was empirically defined by its length within the alignment against sequences with known gene notation (e.g., AY373291, AJ313443, AY373294).

Phylogenetic trees were constructed separately for each region (ITS-5.8S and LSU D1/D2). Maximum Likelihood (ML) phylogenetic trees were built using MEGA 11 [49] with 1000 bootstrap replicates [50] and the GTR+G+I DNA substitution model [51]. Bayesian Posterior Probability (BPP) analysis was performed using the GTR+I+G substitution model [52]. Two independent runs of four Markov chains each were run for 4 million generations with random starting trees. Trees were sampled every 100 generations, and a 50% majority-rule consensus tree was generated from the final 24,000 trees. Convergence and effective sample sizes (ESS) were assessed with Tracer v1.5 [53].

The trees presented represent the best-scoring topologies inferred from the ML analysis. Node support values are indicated as ML bootstrap percentages (BS) and Bayesian posterior probabilities (BPP) from the MrBayes analysis. The sequences generated in this study are available in GenBank under accession numbers OR086091–OR086094 and OR088119–OR088134.

Table 2. Xylaria sequences included in the phylogenetic analysis, with accession numbers and associated published sources.

Fungarium or Strain Code	Determination	ITS-5.8S (Accession Numbers)	Source	References
Strain 570 (HAST, JF)	Xylaria adscedens	GU300101	From fruitbody	[20]
Strain 1114	Xylaria adscedens	KP133298	From fruitbody	[33]
Strain 865 (JDR)	Xylaria adscedens	GU322432	From fruitbody	[20]
Strain PR7	Xylaria globosa	AY909007	From fruitbody	[13]
Strain INBio:261A	Xylaria adscedens	KR534704	Endophyte in Smilax panamensis	[54]
Strain E63	Xylaria multiplex	JQ814313	Endophyte in Hevea brasiliensis	[55]
Strain MP748	Xylaria brevipes	KJ154952	From fruitbody	Vietnam (Unpublished)
Voucher 15223	Xylaria curta	JF908804	Fruitbody	[56]
Strain 938	Xylaria anisopleura	KP133317	From fruitbody	[33]
Strain 1074	Xylaria anisopleura	KP133318	From fruitbody	[33]
Strain 1089	Xylaria apiculata	KP133325	From fruitbody	[33]
Strain 1062	Xylaria apiculata	KP133335	From fruitbody	[33]
Strain 1090	Xylaria apiculata	KP133337	From fruitbody	[33]
Strain 94080508 (HAST)	Xylaria venosula	EF026149	From “twigs”	[57]
Strain SCAU-F-161	Xylaria apiculata	KF881786	Endophyte in Melia toosendan	[58]
Strin 89041211 (HAST)	Xylaria arbuscula	GU300090	From “bark”	[20]
Strain 205 (WSP:205)	Xylaria bambusicola	NR_153200	From Holotype	[20]
Strain 494 (HAST, JF)	Xylaria curta	GU322444	From fruitbody	[20]
Strain 92092022 (HAST)	Xylaria curta	GU322443	From fruitbody	[20]
QCAM4545	Xylaria sp.	MG768837	Fruitbody	[59]
Strain 897	Xylaria curta	KP133352	From fruitbody	[33]
Strain R1	Xylaria curta	KP133356	From fruitbody	[33]
Strain Vega421	Xylariaceae sp.	EU009999	Endophyte in Coffea arabica	Colombia (Unpublished)
Strain C227	Xylaria curta	MK304420	Endophyte in Ageratina adenophora	[60]
BCC 1067	Xylaria sp.	DQ139271	Fruitbody	[61]
Strain 1027	Xylaria cuneata	KP133351	From fruitbody	[33]
Strain 917	Xylaria enterogena	KP133371	From fruitbody	[33]
Strain 1049	Xylaria enterogena	KP133372	From fruitbody	[33]
Strain 785 (HAST, JF)	Xylaria enterogena	GU324736	From fruitbody	[20]
Strain 987	Xylaria enterogena	KP133368	From fruitbody	[33]
Strain 1053	Xylaria aff. comosa	KP133304	From fruitbody	[33]
Robert L. Gilbertson Mycological Herbarium 1371	Xylaria sp.	KT289542	Endolichenic in Peltigera neopolydactyla	[62]
Robert L. Gilbertson Mycological Herbarium 1364	Xylaria sp.	KT289541	Endolichenic in Peltigera neopolydactyla	[62]
E13415E	Xylaria sp.	KF466899	Endophyte in Costus laevis	Ecuador (Unpublished)
Strain 367 (HAST, JF)	Xylaria fissilis	GU300073	From fruitbody	[20]
Strain 775 (HAST, JF)	Xylaria globosa	GU324735	From fruitbody	[20]
Strain 933	Xylaria globosa	KP133428	From fruitbody	[33]
Strain 979	Xylaria globosa	KP133429	From fruitbody	[33]
Strain 995	Xylaria globosa	KP133345	From fruitbody	[33]
Strain 976	Xylaria globosa	KP133426	From fruitbody	[33]
INBio:2303C	Xylaria schweinitzii	KR534679	Endophyte in plants	[54]
UOC MINNP MK33b	Xylaria schweinitzii	KT037020	From fruitbody	Sri Lankan (Unpublished)
Strain 421 (HAST, JF)	Xylaria telfairii	GU324737	From fruitbody	[20]
Strain 90081901 (HAST)	Xylaria telfairii	GU324738	From fruitbody	[20]
TSJ845	Xylaria telfairii	KF937370	Fruitbody	[63]
Strain 987	Xylaria telfairii	KP133368	From fruitbody	[33]
GAB193	Xylaria longipes	KY250409	Fruitbody	Gabon (Unpublished)
Strain CBS 14873	Xylaria longipes	AY909013	From fruitbody	[13]
Strain R22	Xylaria tuberorides	KP133545	From fruitbody	[33]
Strain 475 (HAST, JF)	Xylaria tuberorides	GU300074	From fruitbody	[20]
Hao & Guo & Han 060203	Xylaria xanthinovelutina	MH425284	From fruitbody	China (Unpublished)
Strain 553 (HAST, JF)	Xylaria ianthinovelutina	GU322441	From fruitbody	[20]
Strain 189 (JDR)	Xylaria culleniae	GU322442	From fruitbody	[20]
Strain C24	Xylaria ianthinovelutina	JQ936302	Endophyte in soybean	[64]
Strain 945	Xylaria aff. comosa	KP133303	From fruitbody	[33]
Strain R0	Xylaria aff. comosa	KP133308	From fruitbody	[33]
Strain 901	Xylaria aff. comosa	KP133307	From fruitbody	[33]
Strain 860 (JDR)	Xylaria cubensis	GU991523	From fruitbody	[20]
NR-2006-D65	Xylaria sp.	DQ480358	Endophyte in Garcinia plants	[65]
STRI:ICBG-Panama:TK985	Fungal endophyte	KF436104	Endophyte in Saccharum sp.	[66]
Strain F1124	Xylariaceae sp.	KU747729	Endophyte in Campyloneurum serpentinum	[67]
Strain ATCC 58729	Parahypoxylon papillatum	NR_155153	from epitype of Hypoxylon papillatum	[68]
Strain CHTAE15	Xylaria curta	JF773598	Endophyte in Taxus globosa	[69]
STRI:ICBG-Panama:TK985	Fungal endophyte	KF436104	Endophyte in Saccharum sp.	[66]
Strain CBS 385.35	Xylaria mali	MH867225	From fruitbody	[70]
Strain CBS 162 22	Xylaria polymorpha	MH866242	From fruitbody	[70]
Strain BCC 22966	Xylaria papulis	AB376811	Endophyte on wood	[71]
Strain DSM 110363	Xylaria multiplex	MN833802	Deciduous deadwood	[72]
Strain C4015B2SNA2CC460	Xylaria hypoxylon	KP143687	From marine sponge	[73]
Strain FL0490	Xylaria venustula	JQ760209	Endolichenic in Cladonia didyma	[74]
Strain BCC 1085	Xylaria coccophora	AB376688	Endophyte/saprobe on wood	[71]
Strain FL0512	Xylaria sp.	JQ760231	Endophyte in Usnea mutabilis	[74]
Strain 931	Xylaria sp.	KP133516	From fruitbody	[33]
Strain BCC 17352	Xylaria anisopleura	AB376732	Endophyte/saprobe on wood	[71]
Strain 1m_VC1	Xylaria cf. microceras	KT250979	From fruitbody	[62]
MFLUCC 11-0606	Xylaria bambusicola	KU863148	Fruitbody	[67]
Strain BCC 1086	Xylaria juruensis	AB376689	Endophyte/saprobe on wood	[71]
Strain FL1283	Xylaria arbuscula	JQ760898	Endolichenic in Cladonia leporina	[74]
Strain CBS 126416	Xylaria arbuscula	MH875561	From fruitbody	[70]
Strain BCC 1083	Xylaria juruensis	AB376687	Endophyte/saprobe on wood	[71]
Strain B1A0816P30CC497	Xylariaceae sp.	KP306978	From marine sponge	[73]
STRI:ICBG-Panama:TK36	Fungal endophyte	KF435419	Endophyte in Cordia lasiocalyx	[66]
Strain BCC 1151	Xylaria curta	AB376702	Endophyte/saprobe on wood	[71]
Strain BCC 1115	Xylaria feejeensis	AB376696	Endophyte/saprobe on wood	[71]
Strain CBS 128357	Xylaria enteroleuca	MH876349	From fruitbody	[70]
Strain CHTAR111	Xylaria cubensis	GU048579	Endophyte in Taxus globosa	[69]
Strain NC1163	Xylaria cf. heliscus	JQ761807	Endophyte in Tsuga canadensis	[74]
Strain FL1767 (ARIZ)	Xylaria cf. heliscus	KU683899	Endophyte in Quercus inopina	[67]
Strain FL1161	Xylaria cf. heliscus	JQ760778	Endophyte in Parmotrema rampoddense	[74]
Strain B1a0283EM2CC388	Xylaria sp.	KP306964	From marine sponge	[73]
TSJ845	Xylaria telfairii	KJ130993	Fruitbody	Unpublished
Strain FL1777 (ARIZ)	Xylaria sp.	KU683903	Endophyte in Quercus inopina	[67]
Strain F1825	Xylaria sp.	KU747819	Endophyte in fern frond	[67]
Strain BCC 18361	Xylaria tuberorides	AB376736	Endophyte/saprobe on wood	[71]
Strain FL0632 (ARIZ)	Xylaria cubensis	JQ760340	Endolichenic in Parmotrema tinctorum	[74]
Strain NC1232	Xylaria cubensis	JQ761869	Endophyte in Hypnum sp.	[74]
Strain F0358	Xylariaceae sp.	KU747599	Endophyte in Phlebodium pseudoaureum	[67]
Strain ATCC 58729	Parahypoxylon papillatum	NG_066379	from epitype of Hypoxylon papillatum	[68]

Table 2. Xylaria sequences included in the phylogenetic analysis, with accession numbers and associated published sources.

Fungarium or Strain Code	Determination	ITS-5.8S (Accession Numbers)	Source	References
Strain 570 (HAST, JF)	Xylaria adscedens	GU300101	From fruitbody	[20]
Strain 1114	Xylaria adscedens	KP133298	From fruitbody	[33]
Strain 865 (JDR)	Xylaria adscedens	GU322432	From fruitbody	[20]
Strain PR7	Xylaria globosa	AY909007	From fruitbody	[13]
Strain INBio:261A	Xylaria adscedens	KR534704	Endophyte in Smilax panamensis	[54]
Strain E63	Xylaria multiplex	JQ814313	Endophyte in Hevea brasiliensis	[55]
Strain MP748	Xylaria brevipes	KJ154952	From fruitbody	Vietnam (Unpublished)
Voucher 15223	Xylaria curta	JF908804	Fruitbody	[56]
Strain 938	Xylaria anisopleura	KP133317	From fruitbody	[33]
Strain 1074	Xylaria anisopleura	KP133318	From fruitbody	[33]
Strain 1089	Xylaria apiculata	KP133325	From fruitbody	[33]
Strain 1062	Xylaria apiculata	KP133335	From fruitbody	[33]
Strain 1090	Xylaria apiculata	KP133337	From fruitbody	[33]
Strain 94080508 (HAST)	Xylaria venosula	EF026149	From “twigs”	[57]
Strain SCAU-F-161	Xylaria apiculata	KF881786	Endophyte in Melia toosendan	[58]
Strin 89041211 (HAST)	Xylaria arbuscula	GU300090	From “bark”	[20]
Strain 205 (WSP:205)	Xylaria bambusicola	NR_153200	From Holotype	[20]
Strain 494 (HAST, JF)	Xylaria curta	GU322444	From fruitbody	[20]
Strain 92092022 (HAST)	Xylaria curta	GU322443	From fruitbody	[20]
QCAM4545	Xylaria sp.	MG768837	Fruitbody	[59]
Strain 897	Xylaria curta	KP133352	From fruitbody	[33]
Strain R1	Xylaria curta	KP133356	From fruitbody	[33]
Strain Vega421	Xylariaceae sp.	EU009999	Endophyte in Coffea arabica	Colombia (Unpublished)
Strain C227	Xylaria curta	MK304420	Endophyte in Ageratina adenophora	[60]
BCC 1067	Xylaria sp.	DQ139271	Fruitbody	[61]
Strain 1027	Xylaria cuneata	KP133351	From fruitbody	[33]
Strain 917	Xylaria enterogena	KP133371	From fruitbody	[33]
Strain 1049	Xylaria enterogena	KP133372	From fruitbody	[33]
Strain 785 (HAST, JF)	Xylaria enterogena	GU324736	From fruitbody	[20]
Strain 987	Xylaria enterogena	KP133368	From fruitbody	[33]
Strain 1053	Xylaria aff. comosa	KP133304	From fruitbody	[33]
Robert L. Gilbertson Mycological Herbarium 1371	Xylaria sp.	KT289542	Endolichenic in Peltigera neopolydactyla	[62]
Robert L. Gilbertson Mycological Herbarium 1364	Xylaria sp.	KT289541	Endolichenic in Peltigera neopolydactyla	[62]
E13415E	Xylaria sp.	KF466899	Endophyte in Costus laevis	Ecuador (Unpublished)
Strain 367 (HAST, JF)	Xylaria fissilis	GU300073	From fruitbody	[20]
Strain 775 (HAST, JF)	Xylaria globosa	GU324735	From fruitbody	[20]
Strain 933	Xylaria globosa	KP133428	From fruitbody	[33]
Strain 979	Xylaria globosa	KP133429	From fruitbody	[33]
Strain 995	Xylaria globosa	KP133345	From fruitbody	[33]
Strain 976	Xylaria globosa	KP133426	From fruitbody	[33]
INBio:2303C	Xylaria schweinitzii	KR534679	Endophyte in plants	[54]
UOC MINNP MK33b	Xylaria schweinitzii	KT037020	From fruitbody	Sri Lankan (Unpublished)
Strain 421 (HAST, JF)	Xylaria telfairii	GU324737	From fruitbody	[20]
Strain 90081901 (HAST)	Xylaria telfairii	GU324738	From fruitbody	[20]
TSJ845	Xylaria telfairii	KF937370	Fruitbody	[63]
Strain 987	Xylaria telfairii	KP133368	From fruitbody	[33]
GAB193	Xylaria longipes	KY250409	Fruitbody	Gabon (Unpublished)
Strain CBS 14873	Xylaria longipes	AY909013	From fruitbody	[13]
Strain R22	Xylaria tuberorides	KP133545	From fruitbody	[33]
Strain 475 (HAST, JF)	Xylaria tuberorides	GU300074	From fruitbody	[20]
Hao & Guo & Han 060203	Xylaria xanthinovelutina	MH425284	From fruitbody	China (Unpublished)
Strain 553 (HAST, JF)	Xylaria ianthinovelutina	GU322441	From fruitbody	[20]
Strain 189 (JDR)	Xylaria culleniae	GU322442	From fruitbody	[20]
Strain C24	Xylaria ianthinovelutina	JQ936302	Endophyte in soybean	[64]
Strain 945	Xylaria aff. comosa	KP133303	From fruitbody	[33]
Strain R0	Xylaria aff. comosa	KP133308	From fruitbody	[33]
Strain 901	Xylaria aff. comosa	KP133307	From fruitbody	[33]
Strain 860 (JDR)	Xylaria cubensis	GU991523	From fruitbody	[20]
NR-2006-D65	Xylaria sp.	DQ480358	Endophyte in Garcinia plants	[65]
STRI:ICBG-Panama:TK985	Fungal endophyte	KF436104	Endophyte in Saccharum sp.	[66]
Strain F1124	Xylariaceae sp.	KU747729	Endophyte in Campyloneurum serpentinum	[67]
Strain ATCC 58729	Parahypoxylon papillatum	NR_155153	from epitype of Hypoxylon papillatum	[68]
Strain CHTAE15	Xylaria curta	JF773598	Endophyte in Taxus globosa	[69]
STRI:ICBG-Panama:TK985	Fungal endophyte	KF436104	Endophyte in Saccharum sp.	[66]
Strain CBS 385.35	Xylaria mali	MH867225	From fruitbody	[70]
Strain CBS 162 22	Xylaria polymorpha	MH866242	From fruitbody	[70]
Strain BCC 22966	Xylaria papulis	AB376811	Endophyte on wood	[71]
Strain DSM 110363	Xylaria multiplex	MN833802	Deciduous deadwood	[72]
Strain C4015B2SNA2CC460	Xylaria hypoxylon	KP143687	From marine sponge	[73]
Strain FL0490	Xylaria venustula	JQ760209	Endolichenic in Cladonia didyma	[74]
Strain BCC 1085	Xylaria coccophora	AB376688	Endophyte/saprobe on wood	[71]
Strain FL0512	Xylaria sp.	JQ760231	Endophyte in Usnea mutabilis	[74]
Strain 931	Xylaria sp.	KP133516	From fruitbody	[33]
Strain BCC 17352	Xylaria anisopleura	AB376732	Endophyte/saprobe on wood	[71]
Strain 1m_VC1	Xylaria cf. microceras	KT250979	From fruitbody	[62]
MFLUCC 11-0606	Xylaria bambusicola	KU863148	Fruitbody	[67]
Strain BCC 1086	Xylaria juruensis	AB376689	Endophyte/saprobe on wood	[71]
Strain FL1283	Xylaria arbuscula	JQ760898	Endolichenic in Cladonia leporina	[74]
Strain CBS 126416	Xylaria arbuscula	MH875561	From fruitbody	[70]
Strain BCC 1083	Xylaria juruensis	AB376687	Endophyte/saprobe on wood	[71]
Strain B1A0816P30CC497	Xylariaceae sp.	KP306978	From marine sponge	[73]
STRI:ICBG-Panama:TK36	Fungal endophyte	KF435419	Endophyte in Cordia lasiocalyx	[66]
Strain BCC 1151	Xylaria curta	AB376702	Endophyte/saprobe on wood	[71]
Strain BCC 1115	Xylaria feejeensis	AB376696	Endophyte/saprobe on wood	[71]
Strain CBS 128357	Xylaria enteroleuca	MH876349	From fruitbody	[70]
Strain CHTAR111	Xylaria cubensis	GU048579	Endophyte in Taxus globosa	[69]
Strain NC1163	Xylaria cf. heliscus	JQ761807	Endophyte in Tsuga canadensis	[74]
Strain FL1767 (ARIZ)	Xylaria cf. heliscus	KU683899	Endophyte in Quercus inopina	[67]
Strain FL1161	Xylaria cf. heliscus	JQ760778	Endophyte in Parmotrema rampoddense	[74]
Strain B1a0283EM2CC388	Xylaria sp.	KP306964	From marine sponge	[73]
TSJ845	Xylaria telfairii	KJ130993	Fruitbody	Unpublished
Strain FL1777 (ARIZ)	Xylaria sp.	KU683903	Endophyte in Quercus inopina	[67]
Strain F1825	Xylaria sp.	KU747819	Endophyte in fern frond	[67]
Strain BCC 18361	Xylaria tuberorides	AB376736	Endophyte/saprobe on wood	[71]
Strain FL0632 (ARIZ)	Xylaria cubensis	JQ760340	Endolichenic in Parmotrema tinctorum	[74]
Strain NC1232	Xylaria cubensis	JQ761869	Endophyte in Hypnum sp.	[74]
Strain F0358	Xylariaceae sp.	KU747599	Endophyte in Phlebodium pseudoaureum	[67]
Strain ATCC 58729	Parahypoxylon papillatum	NG_066379	from epitype of Hypoxylon papillatum	[68]

3. Results

3.1. Phylogeny

After DNA amplification, sequences for the ITS-5.8S region and the partial LSU D1/D2 region were obtained from 15 specimens. Four additional specimens yielded only the ITS-5.8S region, and one specimen, HUTPL(F)-1533, failed to amplify (Table 1).

The phylogenetic trees (Figure 1 and Figure S1) inferred using the ITS-5.8S and partial LSU DNA regions, showed partial agreement between some morphologically and phylogenetically defined species. This was evident in taxa like Xylaria cf. anisopleura and Xylaria aff. telfairii (Table 1; Figure 1 and Figure S1), which belong to species complexes with overlapping morphological traits [19].

The ITS-5.8S sequence for Xylaria adscendens showed high similarity to published sequences of the same species (GU322432; KP133298). However, the LSU sequence did not cluster with X. adscendens but instead grouped with a likely misidentified sequence of Xylaria curta (JF773598; Table 2, Figure S1), which was isolated as an endophyte from Taxus globosa.

The ITS-5.8S phylogenetic tree allowed the placement of sequences from several anamorphic specimens into species: HUTPL(F)-1448 as Xylaria adscedens; HUTPL(F)-704 as Xylaria apiculata; HUTPL(F)-1436 as Xylaria fissilis and HUTPL(F)-663 as Xylaria globosa. However, other sequences from anamorphic specimens were only closely related to species-level identifications [i.e., HUTPL(F)-603 related to Xylaria cuneata KP133351; HUTPL(F)-921 close together Xylaria aff. comosa KP133308; HUTPL(F)-1077 and HUTPL(F)-1100 related to Xylaria cubensis GU991523]. The remaining sequences from anamorphic specimens [HUTPL(F)-603, HUTPL(F)-921, HUTPL(F)-1077, HUTPL(F)-1100] clustered separately and are provisionally designated as Xylaria spp. Additionally, the sequence from the Xylaria aenea morphospecies did not cluster with any described species (Table 2, Figure 1 and Figure S1), supporting the notion that no reference sequences for this taxon are currently available.

3.2. Morphology

Among the 20 Xylaria collections analyzed, 11 teleomorph specimens were identified to species level using an integrative approach combining morphological and molecular analyses. These analyzed specimens corresponded to 10 species of Xylaria: Xylaria adscendens, X. aenea, X. cf. anisopleura, X. apiculata, X. curta, X. enterogena, X. fissilis, X. globosa, X. aff. telfairii, and X. tuberoides. Nine collections were found only in the anamorph state. Four [HUTPL(F)-1448, HUTPL(F)-604, HUTPL(F)-663, HUTPL(F)-1436] were assigned by molecular methods to species level (Table 1), and the other five [HUTPL(F)-603, HUTPL(F)-921, HUTPL(F)-1077, HUTPL(F)-1100, HUTPL(F)-1533] were not classified into any described species (Table 1).

3.3. Taxonomy

Xylaria adscendens Fr., Linnaea 5: 537 (1830)

MycoBank No: 190364

Figure 2A–G

Synonymy: ≡Xylaria adscendens (Fr.) Fr., Nova Acta Regiae Societatis Scientiarum Upsaliensis 1: 128 (1851); ≡Xylosphaera adscendens (Fr.) Dennis, Kew Bulletin 13 (1): 102 (1958); ≡Xylosphaera hypoxylon subsp. adscendens (Fr.) Dennis, Bulletin du Jardin Botanique de l’État à Bruxelles 31: 124 (1961); ≡Xylaria hypoxylon subsp. adscendens (Fr.) D. Hawksw., Transactions of the British Mycological Society 61 (1): 199 (1973); Sensu Mycobank Database [3].

Description. Stromata usually solitary and occasionally forming small groups, cylindrical and slightly apiculate without branches, externally black, rough to cracked, and the entostromata white to cream, 32–50 × 3–5 mm with stipe of 8–22 × 1–2 mm. In the immature asexual state, the stromata exhibit a slightly creamy white coloration at the upper part. Perithecia totally immersed into the stromata, black with ostioles slightly papillate. Asci unitunicate, inoperculate, and cylindrical with eight ascopores, 75–84 × 4–5 μm.

Apical apparatus amyloid, 2–3 × 1–2 μm, tubular, parallel, and slightly flattened at the upper apical part. Ascospores uniseriate, guttulate, with a straight spore-length germ slit [(10–) 11 (–12) × (4–) 4.5 (–5) μm], elliptical and inequilateral or oblique with Q value 2.4 μm, dark brown colored. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path AT (1900 m.a.s.l.), growing on decaying wood, 19 May 2015, A. Chamba EM-166, HUTPL(F)-1448; same locality, path AT (1900 m.a.s.l.), growing on decaying wood, 3 April 2014, D. Cruz IR-109, HUTPL(F)-604 (asexual state only).

Remark. Our specimen HUTPL(F)-1448 (Figure 2), characterized by ellipsoid and inequilateral ascospores, exhibits a general morphological resemblance to several specimens identified as Xylaria adscendens. Notably, it aligns with descriptions from tropical South American forests, such as Brazilian collections with ascospores dimensions of 9–14 × 4–5 μm [75] or (9–)11–14.5(–15) × 3–5 μm [30]. Comparable measurements have also been reported from cloud forests in Mexico for ascospores of X. adscendens with (9–) 10.5–13(–14) × 4.5–5 μm [76], and Papua Nueva Guinea 11–14 × 4.5 μm [77,78].

The specimen HUTPL(F)-1448 described here is consistent with Xylaria adscendens, differing from the branched stromata of X. hypoxylon [75] and the massive, fused stromata of X. multiplex [76,77], while sharing the pointed apex and long, striped peeling layer characteristic of the X. hypoxylon aggregate [20]. Based on its morphology and phylogenetic placement with other ITS-5.8S sequences identified as X. adscendens from ascomata-derived strains, we retain the identification of specimen HUTPL(F)-1448 and HUTPL(F)-604 as Xylaria adscendens.

Xylaria aenea Mont., Annales des Sciences Naturelles Botanique 3: 100 (1855).

MycoBank No: 535638

Figure 3A–H

Synonymy: ≡Xylaria aenea Mont. (1855); ≡Xylosphaera aenea (Mont.) Dennis, Kew Bulletin 13 (1): 102 (1958); Sensu Mycobank Database (http://www.mycobank.org/).

Description. Stromata solitary, unbranched, clavate and rounded at the top, with fluted or large wrinkled black surface, but white to cream internally, 99 × 8 mm with stipe of 47 × 4 mm. Perithecia totally immersed in the stromata, black with discoid ostioles slightly papillate. Asci with eight partially biseriate ascospores 133–169 × 5–11.5 μm. Apical apparatus amyloid to Melzer’s reaction, 4–5 × 3–4 μm, tubular, parallel, and slightly urn-shaped, with a narrow neck and a broad opening. Ascospores biseriate, with two to three internal oil drops, partial ventral germ slit [(30–)33.5(–37) × (5–)6(–7) μm], elliptical, inequilateral or partially falcate with Q value 5.6, and dark brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 25 August 2015, D. Cruz EM-593, HUTPL(F)-1543.

Remark.

Our specimen HUTPL(F)-1543 (Figure 3) matches with Xylaria cf. aenea as interpreted by Rogers [31] and epitypified by Ju and Hsieh [79], particularly in the shape and size range of both stromata “cylindrical or clavate, unbranched, rounded and fertile at apex, on a short to long stipe, up to 7 cm in total length × 0.4–2.1 cm broad, 2.5–5.8 cm long at fertile parts, 0.7–5.0 cm long at stipes; surface lacking perithecial mounds” and ascospores “(26.5–)28.5–34(–37.5) × (4.5–)5–6(–8) µm brown to dark brown, unicellular, falcate, with broadly rounded ends, bearing a tiny cellular appendage on one end”. Rogers [31] examined and compared specimens originally described from Venezuela by Dennis [80], subsequently, Ju and Hsieh [79] studied the same material examined by Dennis and proposed an epitypification for Xylaria aenea (VENEZUELA. Aragua, Colonia Tovar, Fendler A. 253, as X. aenea by Dennis, R.W.G. [K(M) 169675 ex Berk. herb.] EPITYPE). The concept of Xylaria aenea based on the epitype aligns with our specimen in terms of ascospore shape and dimensions. However, it clearly differs in stromatal morphology, presenting shorter, more compact stromata with a finely cracked, bronze-colored surface. In contrast, our specimen develops larger stromata, reaching up to 99 mm in length and 8 mm in width, with a prominently fluted or wrinkled surface, whereas the epitype measures only 70 mm in length and 4–21 mm in width.

Based on the strong morphological correspondence in ascospore characteristics, we assign our specimen HUTPL(F)-1543 to Xylaria aenea. The observed differences in stromatal size and surface texture are interpreted as intraspecific variation, likely influenced by environmental conditions.

To date, no reference sequences for Xylaria aenea exist in GenBank or other databases. However, the ITS-5.8S sequence of our specimen matches the unidentified “Xylaria sp.” reported by Thomas [33], and shows close similarity to sequences of X. enterogenea, suggesting a possible genetic relationship.

Xylaria cf. anisopleura (Mont.) Fr., Sylloge generum specierumque plantarum cryptogamarum: 204 (1851)

MycoBank No: 190626

Figure 4A–F

Synonymy: ≡Hypoxylon anisopleuron Mont., Annales des Sciences Naturelles Botanique 13: 348 (1840); ≡Xylosphaera anisopleura (Mont.) Dennis, Kew Bulletin 13 (1): 102 (1958); Sensu Mycobank Database (http://www.mycobank.org/).

Description. Stromata gregarious, unbranched, subglobose to clavate, rounded and moriform surface to the top, commonly black to strong brown, with white entostromata limited at the stromata base by one black line, 9–12 × 3–4 mm with stipe of 2–4 × 0.7–1 mm. Perithecia totally immersed into the stromata, black with umbilicated ostioles. Asci cylindrical with eight ascospores, 142–164 × 9–12 μm. Apical apparatus amyloid to Melzer’s reaction, 4–5 × 3–4 μm, tubular slightly curved, parallel, with flattened at the upper apical part. Ascospores uniseriate, with one or two internal oil drops, partial spiral to oblique ventral germ slit [(19–)23(–28) × (7–)9(–13) μm], elliptical, inequilateral with Q value 2.5, and dark brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 02 June 2015, A. Chamba EM-506, HUTPL(F)-1458.

Remark. The specimen HUTPL(F)-1458 (Figure 4) with small and their partial spiral to oblique ventral germ slit in the ascospores correspond to Xylaria anisopleura applying the taxonomic keys [30,81]. Additionally, our specimen is morphologically like many descriptions of X. anisopleura from different tropical forest [30,75,78,81,82,83]. Our X. cf. anisopleura (HUTPL(F)-1458) is different to the specimen HUTPL(F)-1497 presented here as Xylaria globosa, specially by the shorter stromata and the partial spiral to oblique ventral germ slit.

However, Rogers [31] considered to X. anisopleura part of a complex group closely related to morphospecies such as X. globosa, which was later treated as synonym by Van der Gucht [75] and Hamme and Trinidad [29], as well as to X. polymorpha and X. scruposa according to Dennis [80] and Cruz [84]. Due to the high degree of morphological overlap between X. anisopleura and X. globosa, Fournier [85] also treated both taxa as synonymous.

However, Dennis [80] suggests Xylaria anisopleura differs from X. polymorpha mainly by its smaller stromata, and Cruz [84] suggest it differs from X. scruposa by its larger ascospores [13–21 × 4.5–7 µm in X. scruposa], and in its distinctly moriform stromata.

Xylaria apiculata Cooke, Grevillea 8 (46): 66 (1879)

MycoBank No: 190405

Figure 5A–G

Synonymy: ≡Xylosphaera apiculata (Cooke) Dennis, Kew Bulletin 13 (1): 102 (1958); Sensu Mycobank Database (http://www.mycobank.org/).

Description. Stromata grouped or solitary, unbranched, cylindrical, smooth when young and becoming vertically striping so called “zebra-stripping” coming splits with perithecial swelling, blackish color, with a sterile apex (without perithecia), and white to cream entostromata, 11–23 × 2–3 mm with stipe of 5–10 × 0.4–1 mm, slightly tomentose. Perithecia totally immersed into the stromata, present in the whole fertile area, black with umbilicated barely visible ostioles. Asci stipitate at the base and continue cylindrical with eight ascospores, 90–110 × 6–10 μm. Apical apparatus amyloid to Melzer’s reaction, 9–11 × 5–8 μm, tubular, parallel, and slightly flattened at the upper apical part. Ascospores uniseriate, with one or two internal oil drops, partial ventral germ slit [(14–)16(–18) × (6–)7(–8) μm], elliptical, inequilateral with Q value 2.3, and light brown to dark brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path AT (1900 m.a.s.l.), growing on decaying wood, 3 April 2014, A. Chamba IR-85, HUTPL(F)-581; same locality, path AT (1900 m.a.s.l.), growing on decaying wood, 3 July 2014, A. Chamba IR-211, HUTPL(F)-704.

Remark. Xylaria apiculata is distinguished by short stromata less than 3 mm and surface texture reassembling “zebra-stripping” typically in this species, as well as ascospores measurements. The ascospores of X. apiculata have been reported by other authors as measuring 16–18.5(19) × (4)5–6.5 μm [75] and 16–21 × 6–7.5 μm [80]. This species differs from closely related taxa such as Xylaria venosula, which has significantly larger ascospores measuring 21–25 × 7.5–8 μm [80] and has been considered a synonym of Xylaria xylaroides by Hladki and Romero [26].

Phylogenetically, the ITS-5.8S sequences from specimens HUTPL(F)-581 and HUTPL(F)-704 clustered with sequences identified as X. apiculata from the Ecuadorian cloud forest [33]. Interestingly, one sequence labeled as X. venosula also grouped within this clade, possibly due to misidentification, as the two species are morphologically distinct.

Additionally, LSU data show that our X. apiculata specimens are closely related to X. arbuscula. However, key morphological differences—particularly in stromatal architecture and ascospore dimensions support their distinction as separate species (see X. arbuscula description).

Xylaria curta Fr., Nova Acta Regiae Societatis Scientiarum Upsaliensis 1: 126 (1851)

MycoBank No: 179336

Figure 6A–G

Synonymy: ≡Xylosphaera curta (Fr.) Dennis, Kew Bulletin 13 (1): 103 (1958); Sensu Mycobank Database (http://www.mycobank.org/).

Description. Stromata 13–24 × 3–6 mm, with stipe of 8–11 × 1–3 mm, clustered in groups of two to six fruit bodies, cylindrical, rough, rounded to the apex, blackish in color, but sometimes with external scales conferring brownish color; white to cream entostromata. Perithecia globose, totally immersed in the stromata, black with papillate ostioles. Asci cylindrical with eight ascospores, 65–77 × 4–4.8 μm. Apical apparatus amyloid to Melzer’s reaction, 2–3 × 1–2 μm, tubular, parallel, and narrow at the base, becoming slightly flattened toward the apical portion. Ascospores uniseriate, guttulate central, with ventral straight germ slit commonly covering the whole spore (9–)10(–11) × (4–)4.5(–5) μm, elliptical, inequilateral with Q value 2.2, and brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 25 August 2016, D. Cruz EM-582, HUTPL(F)-1532.

Remark. The specimen [HUTPL(F)-1532] (Figure 6), based on its morphology, fits within the species Xylaria curta according to the taxonomic key provided by Dennis [80]. However, the size of the stroma appears to be highly variable (ranging from 1.8 cm to 5 cm), as also reported in specimens of X. curta from Mexico, Brazil, Costa Rica, France, and Venezuela [30,75,83].

Hsieh [20] placed Xylaria curta within the X. corniformis aggregate, which also includes X. feejeensis, X. montagnei, and X. plebeja, all characterized by similar stromatal textures finely cracked or wrinkled and small, ellipsoid ascospores. Our specimen of X. curta resembles X. feejeensis in ascospore size (8–12 × 4–5 µm), but differs by having stouter, often sessile and clustered stromata, with a white to cream immature surface that is distinctly cracked into persistent angular or rounded scales between the ostiolar papillae.

Phylogenetically, the ITS-5.8S and LSU sequences of specimen HUTPL(F)-1532 cluster with other X. curta sequences (AB376702, GU322444, KP133352, KP133356) derived from axenic cultures of ascomata, as reported by Thomas [33], Hsieh [20], and Okane [71]. These analyses indicate that X. curta forms a distinct clade, separate from closely related taxa, supporting its recognition as an independent species within the complex. Morphologically, our specimen differs from X. montagnei and X. feejeensis by its shorter stromata, and from X. plebeja by having broader ascospores.

Xylaria enterogena Mont., Syll. gen. sp. crypt. (Paris): 203 (1856)

MycoBank No: 250347

Figure 7A–E

Description. Stromata solitary, unbranched, clavate with rounded apex, yellowish cream in color, 63 × 4–6 mm with stipe 16 × 3 mm, with a smooth and hard surface; entostromata wet and white when fresh, darkening and becoming hollow in age. Perithecia immersed into the stromata, with ostiolar black rings. Asci cylindrical with eight ascospores, 131–142 × 6–11 μm. Apical apparatus amyloid to Melzer’s reaction, 5–6 × 3–5 μm, tubular slightly curved, parallel, with flattened at the upper apical part. Ascospores uniseriate, with one or two guttules, partial ventral germ slit sometimes oblique to one apex (18–)21(–24) × (5–)7(–9) μm, elliptical, inequilateral with Q value 3, and brown color. Paraphyses absent (Figure 7).

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 19 May 2016, A. Chamba EM-485, HUTPL(F)-1437.

Remark. Xylaria enterogena is usually confusing with Xylaria telfairii (Berk.) Sacc., specially by the ascospores shape and measurements. Rogers et al. (1988) [31] report to X. enterogena with ascospores (13–)17.5–25 × 6–7.5 μm and X. telfairii with ascospores (15–)16–21 × 6–7.5 μm. Based on similar ascospore dimensions, these species were suggested as conspecific by Dennis (1956) [80], who considered that X. enterogena to be a young state of X. telfairii.

The specimen HUTPL(F)-1437 (Figure 7) has characteristics close to the description of X. aff. enterogena (sensu [81]) from Mexico, which has 68 mm stromata length and ascospores measurements (19–)20–23(–25) × 8–9.5(–10). Here, the specimen HUTPL(F)-1437 differ to the specimen HUTPL(F)-1083 for the stromata 95 × 6–14 mm (fertile portion 38 mm length), with long brown, and smooth stipe 57 × 4 mm, but similar ascospores shape and measurements (18–)21(–25) × (5–)7(–9) μm, and molecular placement (Figure 7). Molecular data show a close relationship between our specimen HUTPL(F)-1437 Xylaria enterogenea and X. telfairii. This affinity is further supported by recent analyses by Forin [19], which include the sequence [HUTPL(F)-1437, OR088131] within a distinct clade referred to as the Xylaria enterogenea complex, positioned close to but separate from the X. telfairii clade.

Xylaria fissilis Ces. Atti dell’Accademia di Scienze Fisiche e Matematiche Napoli 8 (3): 16 (1879)

MycoBank No: 145665

Figure 8A–G

Description. Stromata gregarious mostly with two lateral branches, cylindrical, black color, slightly cracked, flattened flabelliform, pointing towards the apex, with blackish entostromata, internal 17–18 × 1–4 mm, with stipe 3.5–11 × 1–2.5 mm. Perithecia mostly immersed into the stromata, black with papillate ostioles. Asci cylindrical with eight ascospores, 66–96 × 4–6 μm. Apical apparatus amyloid to Melzer’s reaction, 2–4 × 2–3 μm, tubular, parallel, and slightly flattened at the upper apical part. Ascospores uniseriate, with germ slit, almost full spore length (10–)13(–14) × 4(–6) μm, navicular to elliptical, inequilateral with Q value 3.2 and brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path AT (1900 m.a.s.l.), growing on decaying wood, 21 July 2016, A. Chamba EM-564, HUTPL(F)-1514; same locality, path T2 (2500 m.a.s.l.), growing on decaying wood, 25 August 2015, D. Cruz EM-592, HUTPL(F)-1542; same locality, path AT (1900 m.a.s.l.), growing on decaying wood, 19 May 2015, A. Chamba EM-484, HUTPL(F)-1436.

Remark. When follow the taxonomic key given by Hladki and Romero [24,26] for Xylaria spp. from Argentina our description for the specimens revised here fit into the Xylaria fissilis specially by the ascospores measurements 14.5–17.5 × 4–7 μm. Trierveiler Pereira [30] mention that X. nigromedullosa presents similar dark brown to black entostromata as described for X. fissilis and X. luxurians (Rehm) Lloyd. However, our specimens, identified as Xylaria fissilis, differs from X. luxurians Dennis [80] and X. nigromedullosa Trierveiler Pereira [30] in their stromatic features which are slightly cracked, flattened, and flabelliform with an apex-oriented growth as well as in ascospore size 21–24 × 8–9 and 7–9.5 × 4–5 µm respectively and the presence of a germ slit extending nearly the entire length of the ascospores.

Xylaria globosa (Spreng.) Mont. (1855)

MycoBank No: 292028

Figure 9A–G

Synonymy: ≡Hypoxylon globosum (Spreng. ex Fr.) Fr., Nova Acta Regiae Societatis Scientiarum Upsaliensis 1: 130 (1851); Sensu Mycobank Database (http://www.mycobank.org/).

Description. Stromata usually solitary or sometimes grouped, cylindrical to obclavate, rounded to the apex, blackish color, roughly and slightly cracked, with white to cream entostromata, 16–29 × 4–8 mm with stipe sometimes difficult to distinguish from the stromata 5–14 × 1–2 mm. In asexual state present red exudates on young stromata. Perithecia totally immersed into the stromata, black with papillate ostioles. Asci cylindrical with eight ascospores, 146–210 × 8–14 μm. Apical apparatus amyloid to Melzer’s reaction, 7–10 × 5–7 μm, tubular slightly curved, parallel, with flattened at the upper apical part. Ascospores uniseriate, guttulate commonly with one to two oil drops, with partial spiral ventral germ slit [(25–)28(–33) × (8–)9(–11) μm], elliptical, inequilateral with Q value 3, and strong brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 7 July 2016, A. Chamba EM-545, HUTPL(F)-1497; same locality, path AT (1900 m.a.s.l.), growing on decaying wood, 5 June 2014, A. Chamba IR-170, HUTPL(F)-663 (asexual state).

Remark. Xylaria globosa is distinguished by its large elliptical ascospores, as observed in specimen HUTPL(F)-1497 (Figure 9). This specimen fit well within the concept of X. globosa when applying the taxonomic key for Xylaria species from Argentina proposed by Hladki and Romero [26] specially by the ascospores size 22–30 × 8–9.5 μm. The other specimen HUTPL(F)-663 is determined as X. globosa because their asexual state presents a red exudate, a morphological character typical for this species (Figure S1).

Phylogenetically, our ITS-5.8S sequences cluster with reference sequences of Xylaria globosa (e.g., GU324735, KP133429), forming a well-supported clade consistent with that reported by Forin [19], and clearly separated from the clade containing Xylaria cf. anisopleura [HUTPL(F)-1458]. Although X. globosa has been previously synonymized with X. anisopleura due to morphological variability, the ascospores of our specimens [(25–)28(–33) × (8–)9(–11) μm] closely match the measurements reported by Hladki and Romero [26], supporting their identification as X. globosa. Furthermore, Forin [19] identified Xylaria aurantiorubroguttata as the sister group to X. globosa, but distinguishable by morphological traits such as slightly shorter ascospores (~23.9 × 8 μm) with a non-sigmoid, oblique germ slit.

Xylaria aff. telfairii (Berk.) Sacc., Sylloge Fungorum 1: 320 (1882)

MycoBank No: 191825

Figure 10A–F

Synonymy: ≡Sphaeria telfairii Berk., Annals and Magazine of Natural History 3: 397 (1839) [MB#165107] ≡Xylosphaera telfairii (Berk.) Dennis, Kew Bulletin 13 (1): 106 (1958); Sensu Mycobank Database (http://www.mycobank.org/).

Description. Stromata solitary unbranched, cylindrical to slightly clavate, and rounded apex, cream to brownish, smooth and hard surface, with cream entostromata, without ventral hole, 95 × 6–14 mm (fertile portion 38 mm length), with long black smooth stipe 57 × 4 mm. Perithecia immersed into the stroma, black with umbilicate ostioles. Asci cylindrical and thin at the basal part with eight ascospores, (125–)142–153 × 6–11 μm. Apical apparatus amyloid to Melzer’s reaction, 5–6 × 3–5 μm, tubular slightly curved, parallel, with flattened at the upper apical part. Ascospores uniseriate, with one or two internal oil drops, partial ventral germ slit sometimes oblique to one apex (18–)21(–25) × (6–)7(–9) μm, elliptical, inequilateral with Q value 3, and brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica of San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 05 March 2015, A. Chamba EM-166, HUTPL(F)-1083.

Remark. Morphologically, our specimen HUTPL(F)-1083 is closely related to Xylaria enterogena and X. telfairii by having cream to brownish stromata (Figure 10), and similar shape and measurements of ascospores. The ascospores measurements (18–)21(–25) × (5–)7(–9) μm in the specimen HUTPL(F)-1083 are into the range of the ascospores measurements found in X. enterogena (15–)16–21 × 6–7.5 μm [31], X. telfairii (13–)17.5–25 × 6–7.5 μm [31], and X. telfairii (15–)16–22(–25) × 6–8 μm [80]. However, the entostromata in the specimen HUTPL(F)-1083 was not hollow, probably due to the perithecia maturity state [80].

The specimens HUTPL(F)-1083 and the specimen HUTPL(F)-1437 are different in color, size (fertile portion 38 mm and stipe 57 mm length in HUTPL(F)-1083, against 49 mm and 16 mm in HUTPL(F)-1437 respectively), and shape of the stroma, but identical in shape of the ascospores (Figure 10).

Based on morphology (Figure 10) the specimen HUTPL(F)-1083 is considered similar most of the microscopical shapes and measurements of Xylaria telfairii available in Dennis [80], and Rogers [31].

The morphological and phylogenetic evidence is not conclusive to confidently identify the specimen HUTPL(F)-1083 as Xylaria telfairii. Therefore, we refer to it as Xylaria aff. telfairii. Although Forin [19] includes our sequence within the X. telfairii clade, it also shows a close relationship to the X. enterogenea clade, highlighting the need for further analysis.

Xylaria tuberoides Rehm, Hedwigia 40: 146 (1901)

MycoBank No: 181321

Figure 11A–G

Description. Stromata solitary, globose to subglobose, black, and hard surface, with white to cream entostromata, 12–16 × 8–11 mm with stipe 3–4 × 2–4 mm. Perithecia immersed into the stromata, black with slightly papillate ostioles. Asci cylindrical with eight ascospores, 156–203 × 7–12 μm. Apical apparatus amyloid to Melzer’s reaction, 5–6 × 3–5 μm, tubular, parallel, and slightly urn-shaped, with a narrow neck and a broad opening. Ascospores uniseriate, with two or three internal oil drops, partial ventral germ slit slightly clear (24–)28(–32) × (6–)7(–11) μm, elliptical, inequilateral with Q value 3.9, and brown color. Paraphyses absent.

Examined specimens. SOUTH AMERICA: ECUADOR. Zamora Chinchipe, el Tambo, Estación Científica San Francisco, path T2 (2500 m.a.s.l.), growing on decaying wood, 3 September 2015, J.S. Eguiguren EM-609, HUTPL(F)-1559.

Remark. Xylaria tuberoides is morphologically described with globose and clavate stromata, close to our specimen [HUTPL(F)-1559] (Figure 11) which is globose to subglobose, however the morphological variability in the fruit bodies within the same species of Xylaria is common, especially in different development states. Regarding the microscopic structures, our specimen [HUTPL(F)-1559], with elliptical ascospores elliptical, inequilateral ascospores [(24–)28(–32) × (6–)7(–11) μm], is similar to the ascospores described for Xylaria tuberoides by Cruz (2015) [84] 24–29 × 7–9 μm, both featuring a partial ventral germ slit—slightly visible in our specimen (Figure 11) and inconspicuous in the original description. Additionally, both share a subglobose, stromatal shape.

Phylogenetically, the ITS-5.8S sequence of specimen HUTPL(F)-1559 (Xylaria tuberoides) forms a well-supported clade (Figure 1) together with other sequences identified as X. tuberoides, including KP133545 [33], GU300074 [20], and AB376736 partial LSU (Figure S1) [71]). Based on this phylogenetic congruence, we maintain the identification of our specimen as Xylaria tuberoides.

4. Discussion

Fungal species diversity is high in tropical forests, such as the Estación Científica San Francisco in the Andes, a recognized biodiversity hotspot [35,86]. It is likely that this tropical rainforest harbors thousands of fungal species, as has already been reported by molecular fungal diversity studies from orchid roots [87,88] including ascomycetes. This high richness is consistent with patterns observed in other megadiverse regions, such as the tropical and mixed forests of Guizhou, Yunnan, and Guangxi in China, where Xylariales exhibit high diversity and endemism while fulfilling key ecological roles as decomposers of decaying wood and fallen branches [89]. In this study, 15 of 20 xylaroid specimens (Table 1) were assigned to ten morphological distinct species Xylaria adscendens, X. cf. anisopleura, X. apiculata, X. curta, X. enterogena, X. fissilis, X. globosa, X. aff. telfairii, X. tuberoides, and X. aenea, the latter representing a new record for Ecuador. While the species X. cf. anisopleura, X. aff. telfairii, and X. aenea are partially supported by our molecular data, the phylogenetic analysis presented here (Figure 1 and Figure S1) serves as a preliminary systematic positioning of these specimens within the genus rather than a global reconstruction. This local approach complements broader multigene studies (e.g., [19,20,89]) by providing detailed morphological and molecular data from a Neotropical locality often underrepresented in international databases. Despite the scarcity of reference sequences for the LSU region (Figure S1) which precluded a more refined phylogeny, the ITS-5.8S marker proved more informative (Figure 1), as discussed below.

Correspondence between morphological and molecular data.

Xylaria adscendens [specimen HUTPL(F)-1448] matches well with several published descriptions of the species based on ascospores morphology and measurements [30,75,76,77,78]. Additionally, our ITS-5.8S sequences are cluster with published sequences (Table 2) GU322432 by Hsieh [20] and KP133298 from Ecuadorian cloud forest reported by Thomas [33], both identified as X. adscendens (Figure 1). However, our LSU sequence is cluster together with one sequence identified as Xylaria curta (JF773598; Figure S1), isolated as an endophyte from Taxus globosa [90] that probably represents a misnamed sequence, which is unfortunately common [91].

The Xylaria aenea [HUTPL(F)-1543] is proposed here as a new record for Ecuador, as no previous validated reports of this species have been found in the available literature. For example, Lodge [92] reported numerous Xylaria species for cloud and high montane forest in six Neotropical countries (Belize, Ecuador, the Guianas, Mexico, Puerto Rico, and Venezuela), but X. aenea was not included for Ecuador. Similarly, Thomas [33] did not describe X. aenea in their study, though their unidentified “Xylaria sp. strain 931” matches our specimen at the ITS locus (KP133516). Furthermore, our ITS-5.8S sequence for X. aenea (Figure 1 and Figure S1) did not cluster with any sequences of well-described species available in the GenBank database or in published studies (Table 2), suggesting that reference sequences for this species are currently lacking.

The continuous study of biodiversity hotspots is essential for uncovering global fungal richness. This is exemplified by recent research in Asia, where intensive surveys documented Xylaria frustulosa, X. glebulosa, and X. longissima as new records for the Chinese mainland, significantly expanding the known geographical distribution of these taxa [93]. Our discovery of X. aenea in the Andean foothills similarly contributes to bridging these distributional gaps in the Neotropics. However, molecular evidence indicates a close affinity with X. enterogenea. According to Ju and Hsieh [79], some specimens of X. enterogenea were probably misclassified as X. aenea because of overlapping morphological features.

Xylaria cf. anisopleura [specimen HUTPL(F)-1458] is closely related to the sequence KP133317, obtained from strain 938 (Ecuadorian cloud forest) identified as Xylaria anisopleura [33]. Comparisons based on the LSU D1/D2 region show that the specimen clusters with sequence AB376732, derived from an axenic culture of an ascocarp identified as X. anisopleura [71]. Given the inconclusive evidence—specifically, the morphology (Figure 4) and the ITS-5.8S and LSU D1/D2 sequences (Figure 1 and Figure S1)—specimen HUTPL(F)-1458 is tentatively identified as Xylaria cf. anisopleura. In a recent study by Forin [19], this clade is treated as part of the X. anisopleura complex.

The species Xylaria anisopleura and X. globosa based on their highly variable morphology are difficult to differentiate, as extensively discussed by Fournier [85]. However, our specimens HUTPL(F)-1497 and HUTPL(F)-663 are considered as X. globosa due to their asexual state exhibiting a red exudate, a typical morphological character for this species. Moreover, phylogenetic analysis of the ITS-5.8S region (Figure 1) shows that our sequences cluster with other identified as X. globosa (e.g., KP133429; [33]) and are genetically distinct from Xylaria cf. anisopleura [HUTPL(F)-1458]. These results are consistent with those reported by Forin [19], who recovered both species in separate clades based on ITS-5.8S and multilocus analyses (ITS, ACT, TUB2, and RPB2).

Specimens [HUTPL(F)-581 and HUTPL(F)-704] are proposed as Xylaria apiculata based on morphological characteristics (see remark) and the phylogenetic ITS-5.8 sequences placement (Figure 1). These specimens clustered with two sequences (accession number KP133325 and KP133335) from ascomata collected in the Ecuadorian cloud forest, previously identified as X. apiculata [33]. However, one ITS-5.8S sequence (EF026149) obtained from stroma identified as X. venosula and GU300090 identified as X. arbuscula also grouped within the same clade as X. apiculata. This suggests that the two species might be synonymous, as proposed by Dennis [80]. On the other hand, when compared to our X. apiculata LSU sequences (Figure S1), one sequence (JQ760898) identified as X. arbuscula by U’Ren [74] appears closely related. However, morphologically, X. arbuscula differs from X. apiculata primarily in its shorter, branched stromata and smaller ascospores (typically less than 16 × 6 μm), sensu Dennis [80]. These species appear to be genetically and morphologically related, as noted by Hsieh [20] who proposed the Xylaria arbuscula aggregate. This group includes X. arbuscula, X. arbuscula var. plenofissura, X. bambusicola, X. striata, and X. venosula, all sharing common characteristics such as a “pointed apex on cylindrical stromata and a striped, persistent peeling layer on the stromatal surface”.

Based on morphology (see remark) and phylogenetic placement (ITS-5.8S and LSU), specimen HUTPL(F)-1532 is identified as Xylaria curta. This identification is supported by the similarity of our specimen to other X. curta sequences, such as KP133352 and MG768837, obtained from axenic cultures in the Ecuadorian cloud forest by Thomas [33], as well as the fruitbody QCAM4545 reported by Guevara [59] (Table 2). In the LSU phylogeny, a closely related sequence identified as Xylaria feejeensis (AB376696) clusters within the X. curta clade, which may indicate a close genetic relationship or a possible misidentification. Both species are considered part of the X. corniformis aggregate and share morphological features such as a “finely cracked outer stromatal layer, wrinkled surface, and ascospores mostly 8–16 μm in length” [19].

In this study, we identified specimen HUTPL(F)-1437 as Xylaria enterogena based on morphological characteristics and ITS-5.8 sequence analysis. Our sequence clusters with other sequences (KP133370, KP133371, and KP133372) from strains isolated from ascomata collected in the Ecuadorian cloud forest and identified as X. enterogena by Thomas [33]. While X. enterogena HUTPL(F)-1437 shares similar ascospore shape and measurements with Xylaria aff. telfairii HUTPL(F)-1083, these two species are genetically distinct.

The specimen HUTPL(F)-1083 is suggested as Xylaria aff. telfairii due to their macro and microscopical similarity to Xylaria telfairii reported by Dennis [80], and Rogers [31]. Genetically, our sequence of Xylaria aff. telfairii is closely related to other sequences (KP133541, KP937370) identified as Xylaria telfairii from ascomata collected in the Ecuadorian cloud forest by Thomas [33]. However, the genetic difference among these sequences exceeds 3% based on pairwise distance of the ITS-5.8S region. This level of divergence is also evident in the ITS-5.8S phylogenetic tree presented by Forin [19], where the sequence from HUTPL(F)-1083 is nevertheless retained under the name Xylaria telfairii. Both species, Xylaria enterogena and X. telfairii had been discussed as related species that could overlap the shape and measurements of ascospores [31,80].

Specimens HUTPL(F)-1514, HUTPL(F)-1542, and HUTPL(F)-1436 are proposed as Xylaria fissilis based on morphological similarities and ITS-5.8S sequence clustering with KP133404, a sequence from an Xylaria fissilis isolate collected in the Ecuadorian cloud forest by Thomas [33].

Xylaria tuberoides, specimen HUTPL(F)-1559, is supported by the phylogeny (Figure 1). Sequences from HUTPL(F)-1559 cluster with KP133545 [isolated from ascomata in the Ecuadorian cloud forest by Thomas [33], GU300074 (from ascomata, Hsieh [20]), and AB376736 partial LSU (Figure S1) from axenic culture, by Okane [71].

This study provides a baseline of morpho-molecularly characterized species, contributing significantly to our understanding of fungal diversity and their ecological roles. All Xylaria spp. encountered in this study were saprotrophic on decaying wood, confirming their well-established role as decomposers of organic matter [1,2,94], thereby contributing to nutrient recycling in the forest.

It is likely that many Xylaria species represent cryptic lineages, a phenomenon increasingly documented across the fungal kingdom [95]. Consequently, further research on Xylaria species, including examination of herbarium and type specimens, is necessary to comprehend the diversity within this genus, as outlined by Chen [23]. Future studies should incorporate additional molecular markers, such as beta-tubulin [16,20] to analyze intra- and inter-specific variability in depth, thereby establishing a robust barcode gap for accurate species delineation.

In this study, the observed incongruence between morphology and nrDNA ITS-5.8S and LSU sequences likely reflects the high intragenomic polymorphism and paralogous copies characteristic of Xylaria [20]. As demonstrated in other groups such as Golovinomyces [96], these polymorphisms often lead to sequence overlaps between distinct species, complicating identification via ribosomal markers alone. This biological complexity explains the partial resolution of our nrDNA ITS-5.8S and LSU data and reinforces the necessity of adopting multi-locus frameworks in future research. By integrating these molecular findings with exhaustive morphological evidence, our results establish a taxonomic baseline that facilitates the inclusion of Neotropical specimens into broader evolutionary frameworks, connecting local inventories with global multigene phylogenies [19,20,89,93].

Adhering to the guidelines of the “Outline of Fungi and fungus-like taxa” [97], which seeks to stabilize fungal classification and prevent incorrect taxonomic inferences, our study places these species within the Xylariaceae (Xylariales). This family remains one of the most diverse within the Sordariomycetes, with Xylaria being a notably speciose genus that accounts for a significant portion of the order’s diversity, currently estimated to exceed 500 species globally [97]. The high diversity found in this preliminary survey—comprising 10 species and 4 additional phylotypes—reflects the global pattern of "speciose genera" where intensive local sampling continues to reveal hidden richness, especially in under-documented Neotropical regions. To date, scientific records from the Estación Científica San Francisco have been limited to specific groups, including approximately 75 mycorrhizal fungi [37] and three morphologically distinct Tulasnella species [39,98], making this contribution a vital expansion of the area’s known mycobiota.