More specimens of Hydnotrya have been collected from southwestern China in recent years. Morphological and molecular analyses showed that they belonged to three species of Hydnotrya, of which two are new to science, H. oblongispora and H. zayuensis. The third one was H. laojunshanensis, previously reported in 2013. The new species are described, and their relationship to other species of Hydnotrya is discussed. H. laojunshanensis is re-described in more detail. The main morphological characters of 17 species of Hydnotrya are compared and a key to them is provided as well.

Discinaceae, hypogeous fungi, ITS, morphological diversity, taxonomy

Hydnotrya Berk. & Broome is a genus of hypogeous fungi belonging to Pezizomycetes, Ascomycota. It was placed in the family Helvellaceae by Spooner (1992) and Abbott and Currah (1997) but based on the recent molecular analyses it has been shifted into the family Discinaceae (O’Donnell et al. 1997; Hansen and Pfister 2006; Tedersoo et al. 2006; Læssøe and Hansen 2007; Wang et al. 2023). Their ascomata are hollow to convoluted with simple or folded chambers, even nearly solid, lined with recognizable hymenium. Hydnotrya species usually forms a symbiotic relationship with both conifer and broadleaf trees and are distributed throughout the northern hemisphere (Trappe 1975; Spooner 1992; Trappe and Castellano 2000; Stielow et al. 2010; Xu et al. 2018; Slavova et al. 2021). There are 22 names listed in the Index Fungorum online database (http://www.indexfungorum.org/Names/Names.asp). However, among them, the species H. jurana Quél. and H. carnea (Corda) Zobel was synonymized with H. tulasnei (Berk.) Berk. & Broome (Gilkey 1954; Trappe 1969), H. ploettneriana (Henn.) Hawker, H. yukonensis Gilkey and H. dysodes Kirschstein with H. michaelis (E. Fisch.) Trappe (Soehner 1942; Trappe 1975), and H. convoluta (McAlpine) McLennan was renamed as Peziza jactata Burds. & Korf (Burdsall-Jr 1968), H. ellipsospora Gilkey combined as P. ellipsospora (Gilkey) Trappe (Trappe 1979). To date, there are 15 accepted species remaining in the genus Hydnotrya.

To date, nine Hydnotrya species have been reported in China: H. cerebriformis in Shanxi and Xinjiang, H. cubispora in Tibet, H. michaelis, H. tulasnei and H. brunneospora in Jilin (Tao and Liu 1989; Zhang 1991; Xu 2000; Xu et al. 2018), H. laojunshanensis and H. badia in Yunnan (Li et al. 2013), H. nigricans in Sichuan, H. puberula in Yunnan and Jilin (Xu et al. 2018).

Over the past two years, more Hydnotrya specimens have been collected in southwest China. Based on the morphological and molecular analyses, two new species were detected and described: H. oblongispora and H. zayuensis. Their relationships with other known Hydnotrya species are discussed and a more detailed supplementary description is given to another species H. laojunshanensis, previously found in Yunnan. Additionally, the main morphological characteristics of 15 species of Hydnotrya are listed and a key to the species of the genus is provided.

The specimens were collected from Yunnan and Tibet, China. The type and other studied specimens were deposited at the Biological Science Museum of Dali University (BMDLU) and HKAS (Herbarium of Kunming Institute of Botany, Academy Sinica), China.

Descriptions of microscopic and macroscopic characters were based on specimens (BMDLU L20069, L20067, L21197, L21211, L21212, L21215, L21217, L22024, L22027, and HKAS95802) following the methods of Kumar et al. (2017) and Truong et al. (2017). The sections were made with a razorblade by hand, mounted in a 5% KOH solution or water, and then stained with a cotton blue or lactophenol solution. The sections were observed under an Olympus BH-2 microscope. Key colors were obtained from Kornerup and Wanscher (1978).

Total genomic DNA was extracted from the specimen using the OMEGA Plant Genomic DNA Kit. The internal transcribed spacer (ITS) rDNA region was amplified with PCR primers ITS1F and ITS4 (White et al. 1990; Gardes and Bruns 1993; Truong et al. 2017). The large subunit nuclear ribosomal DNA (LSU) region was amplified with the PCR primers LROR and LR5 (Vilgalys and Hester 1990). PCR reactions were performed on a BIO-RAD C1000TM instrument. Thermal cycles with the following settings: initial denaturation for 5 min at 94 °C, followed by 32 cycles of 40 s denaturation at 94 °C, annealing at 56 °C for 40 s for ITS, and 52 °C for 30 s for LSU, extension for 1 min at 72 °C, and final extension at 72 °C for 10 min. The PCR products were verified on 1% agarose electrophoresis gels stained with ethidium bromide. The purification and sequencing of the PCR products was conducted by Sangon Biotech Limited Company (Shanghai, China).

ITS was used for the analysis of Hydnotrya species diversity in this study because ITS appears as a useful locus for the delimitation of Hydnotrya species. 46 ITS sequences from NCBI and this study representing 14 species of Hydnotrya (Table 1), including Gyromitra infula (Schaeff.) Quél. and Gyromitra esculenta Pers. ex Fr. as outgroups (Fig. 1). All Hydnotrya ITS sequences were extracted with an ascoma. Sequences of Hydnotrya species generated in this study were submitted to the GenBank database. We first used the Basic Local Alignment Search Tool for the GenBank database to recheck whether the newly generated sequences were amplified DNA from contaminant or not and examine clusters with closely related sequences. DNA sequences were retrieved and assembled using SeqMan. Sequence alignments were aligned using MAFFT version 7 (Katoh and Standley 2013), ITS gene was analyzed using BioEdit v. 7 (Hall 2007) Maximum Likelihood (ML) analysis was performed using RAxML-HPC2 v. 8.2.12 (Stamatakis 2014) as implemented on the Cipres portal (Miller et al. 2011), with the GTR+G+I model and 1,000 rapid bootstrap (BS) replicates for all genes. A reciprocal 70% bootstrap support approach was used to check for conflicts between the tree topologies from individual genes. As the topology of the ML tree and the Bayesian tree are similar, the ITS1, ITS2, and 5.8s sequences were combined using SequenceMatrix (Vaidya et al. 2011), partitioned phylogenetic analyses. For Bayesian Inference (BI), the best substitution model for each partition was determined by MrModeltest 2.2 (Nylander et al. 2004). The result suggested that ITS1: JC+I, 5.8S: GTR+G+I, ITS2: K80+I+G. Bayesian analysis was performed using MrBayes ver. 3.2.7a (Ronquist et al. 2011) on the Cipres (Miller et al. 2011), four parallel runs, were performed for 10 million generations sampling every 100^th generation for the single gene trees. Parameter convergence > 200 was verified in Tracer v. 1.7 (Rambaut et al. 2018). The phylogenetic clade was strongly supported if the bootstrap support value (BS) was ≥ 70% and/or a posterior probability (PP) <0.01.

Figure 1. 

Phylogeny derived from a maximum likelihood (ML) analysis of the nrDNA-ITS sequences from Hydnotrya species, using Gyromitra esculenta and G. infula as outgroup. Values next to nodes reflect, maximum likelihood bootstrap support values (BS), left, and Bayesian posterior probabilities (PP), right. Names of novel species and samples with newly generated sequences in bold. Symbols by taxon names indicate specific fruiting body types, the arrangement of the ascospores in the ascus and ascospore appearance.

To date, 17 species of Hydnotrya (including these two new species) are accepted worldwide (Kirk et al. 2008; Stielow et al. 2010; Li et al.2013; Xu et al. 2018). The main macroscopic and microscopic characters of these species are provided and discussed based on available literature (Table 2).

Table 2.

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List of main characteristics of Hydnotrya.

Species	Ascomata	Gleba	Ascospore		Asci	Host Plants	Distribution	References
Hydnotrya badia L. Fan, Y.W. Wang & Y.Y. Xu 2018	Irregularly subglobose, 7–15 × 14–19 mm diam., surface even, brown to earth brown.	Gleba solid, with numerous variably compacted canals and chambers (usually without empty space).		Roughly globose, 25–40 μm in diam. including ornamentation, 17.5–27.5 μm in diam. excluding ornamentation, red-brown to reddish, thickened exisporium with regular large protuberances	Asci broadly clavate to somewhat saccate, sessile, or narrowed at the base into a short stalk, 125–172.5 × 65–75 μm, randomly immersed in paraphyses, 8-spored, spores mostly biseriate.	Pinus sp.	Huize, China Asia 2000–2900m	Yu et al. 2018
Hydnotrya bailii Soehner 1959	Irregularly subglobose, 10–20(–25) mm diam., dark brown, with deep furrows often with multiple lobes, with one or many openings at the apex, with pleasant aromatic smell.	Gleba solid, dark brown, strongly convoluted cavities.		Globose, (27.5-) 30–34 (–37.5) μm in diam, brown reddish, exosporium thickening with blistered warts	Asci cylindrical, 250–300×30–40 μm, 8-spored, spores mostly uniseriate.	Picea abies	Europe	Stielow et al. 2010
Hydnotrya brunneospora L. Fan, Y.W. Wang & Y.Y. Xu 2018	Irregularly globose, 20–23 mm diam., dark brown when dry, surface smooth.	Gleba solid, scattered with some small, isolated, and irregularly shaped chambers.		Roughly globose, 26.25–46.25 μm diam., brown to golden brown at maturity (never reddish), exosporium thickening with small protuberances.	Asci cylindrical to clavate, narrowed at the base into a short stalk, 162.5–237.5 × 30–47.5 μm, randomly immersed in paraphyses, 8-spored, spores mostly uniseriate.	Betula platyphylla	Jilin, China Asia	Yu et al. 2018
Hydnotrya cerebriformis Harkn. 1899	Irregular spherical, lobulated, 10–35 × 10–20 mm diam., reddish-brown, cerebriform, with cavities that communicate with the gleba.	Gleba with labyrinthine chambers composed of invagination and fusion from the walls of the ascoma.		Globose ascospores 20–25μm diam. (x = 22.85 μm), excluding ornamentation, amber-brown, walls 1μm wide. Finely warty ornamentation, warts up to 4μm long.	Asci cylindrical, 175–200 × 25–35 μm, 8-spored, spores mostly uniseriate.	Pinus sp. Abies sp.	Europe North America 3100–4000m	Harkness 1899 Abbott and Currah 1997 Piña-Páez et al. 2017
Hydnotrya confusa Spooner 1992	Ovoid or irregular, size from ca. 20 × 20 × 15 cm up to 40 × 40 × 20 mm, greyish-brown or red-brown, with a primary apical opening and sometimes some smaller secondary openings.	Gleba hollow, with single chambered but mostly cerebriform folded.		Ovoid or very broad ellipsoid, 38–50 × 28–32 µm, golden brown, exosporium much thickened, vertically grooved, forming irregular warts.	Asci cylindrical, 290–320 × 38–43 μm, 8-spored, spores mostly uniseriate. clavate at immature, with irregular or biseriate, cylindric at maturity, strictly uniseriate.	Picea sp.	Europe 361m	Spooner 1992 Bemmann and Bandini 2011
Hydnotrya cubispora (E.A. Bessey & B.E. Thomps.) Gilkey 1939	Irregularly globose, 5–10 mm diam., Isabella color, with somewhat cerebriform folds radiating distinctly from central opening	Gleba with cavity simple, but somewhat irregular due to surface lobing.		Cubical, 47–50 × 23–32µm, including thickening exosporium, brownish, with maturity.	Asci cylindrical, 100–120 μm long, 8-spored, spores mostly uniseriate.	Coniferous forest	Europe North America	Gilkey 1939 Bryan 2003 K(M)189248
Hydnotrya inordinata Trappe & Castellano, 2000	Irregular globose, 8–30mm diam., dark red-brown, convolute and infolded ptychothecia with one or a few openings from the interior	Gleba complex, of infolded tramal plates forming canals and chambers 0.5–3mm broad.		Globose to ellipsoid, 20–30×20–28μm excluding ornamentation, brown-yellow, with aggregated , irregular flexuous spines	Asci cylindrical, ±300 × 25–33 μm, (6–) 8-spored, spores mostly uniseriate.	Abies amabilis Tsuga mertensiana	North America 1800m	Trappe and Castellano 2000
Hydnotrya laojunshanensis Lin Li, D.Q. Zhou & Y.C. Zhao 2013	Irregularly globose, 10–30 mm diam., brownish orange, smooth, mostly single-chambered with a primary apical opening, rare the opening narrowing into a slit, sometimes folded forming a few channels, lined with white fluffy hymenium. No special smell.	Gleba hollow, single-chambered, sometimes infolded and chambered, lined with hymenium with orange asci and whitish to yellowish paraphyses.		Ellipsoid without thickened exosporium, rectangle (including exosporium), (42.5–) 50.0–57.2(–60.3) × (27.5–)30.4–36.9(–38.2) µm, reddish orange, thickening exosporium with rough surface.	Asci cylindrical, 331.5–390.5 × 25.5–35.5 µm, 8-spored, spores strictly uniseriate	Abies spp.	Yunnan, China Asia 3500–3800m	Li et al. 2013 This study
Hydnotrya michaelis (E. Fisch.) Trappe 1975	Irregular or subspherical, up to 60 mm across, with rounded opening, wrinkled, lobulate, with numerous invaginations, odor very strong, somewhat pungent, rather persistent.	Gleba labyrinthoid, with large, sinuous cavities, separated by folded inwards portions of ascoma wall.		Broadly ellipsoid, (21.2–)24.9–29.6(–32.2) ×(18.8–)19.8–22.4 (–24.9) µm; ornamentation excluded), honey-yellow, exosporium thickened, with conspicuous, irregular, often interconnected warts	Asci cylindrical, 200–220 × 30–35 µm, 8-spored, spores strictly uniseriate	Pinaceae	Europe North America	Trappe 1975 Slavova et al. 2021
Hydnotrya nigricans L. Fan, Y.W. Wang & Y.Y. Xu 2018	Irregular globose, 13 × 9mm, black brown to blackish	Gleba solid, brown, red to dark reddish, with some irregularly shaped and isolated small chambers lined with pale whitish hymenium.		Irregularly globose, 25.0–37.5 μm in diam., red brown, exosporium unevenly thickened, and usually of trigonal outline in cross section	Asci broadly clavate to saccate, sessile or narrowed at the base into a short stalk, 87.5–190 × 25–62.5 μm, scattered between paraphyses in a hymenium,8-spored,with spores mostly biseriate.	Pinus sp.	Sichuan, China Asia	Yu et al. 2018
Hydnotrya oblongispora sp. nov.	Irregularly globose, 10–25mm in diam. when fresh, light khaki to reddish brown, smooth, mostly single-chambered with a primary apical opening up to 02–08 mm in diam., sometimes infolded.	Gleba hollow, single-chambered lined with milky white hymenium, hymenium surface fluffy.		Long-ellipsoid (20.0–) 26.5–39.0 × (9.5–) 11.0–21.5 μm, golden brown, thickened exporium with pitted surface.	Asci cylindrical, 102.5–138.5 × 13.0–25.5 µm, narrowed at the base into a long stalk (20–30 μm), 8-spored, spores strictly uniseriate	Abies forrestii	Yunnan, China Asia 3500–4000m	This study
Hydnotrya puberula L. Fan, Y.W. Wang & Y.Y. Xu 2018	Irregularly subglobose, 11–20 × 8–19 mm, brown to dark brown, sometimes with purple tints when fresh, much convoluted with deep furrows, ascoma surface tomentulose	Gleba solid, compact, dark brown to purple reddish at maturity, with numerous small chambers.		Roughly globose, 22.5–42.5 μm in diam., red brown to reddish, exosporium unevenly thickened by irregularly large protuberances.	Asci clavate to saccate, 125–190 × 55–80 μm, sessile or with a short stalk, borne among palisade-like paraphyses in the hymenium, 8-spored, with spores mostly biseriate.	Pinus sp.	Yunnan, China Asia	Yu et al. 2018
Hydnotrya soehneri Svrček, 1955	Irregularly subglobose, tuberous, 10–40 mm wide, reddish and reddish-gray to reddish brown, odor light fragrance	Gleba solid, whitish to yellowish gray, at maturity is colored reddish-brown corridors (from mature spores).	Spherical, 25 – 36 (– 42), red brown, exosporium thickened, coarsely warty.		Asci mostly cylindrical to saccate, 150–300 × 35–70 µm, 8-spored, mostly incompletely arranged biseriate.	Mixed woods	Europe	Svrček 1955
Hydnotrya subnix Trappe & Castellano, 2000	Irregular subglobose, 50–65mm in diam, dark red-brown, glabrous to minutely roughened. Odor and taste strongly of spicy garlic.	Gleba variable, deeply convoluted and infolded lacking openings from the interior, forming canals and locules 1–10mm broad.		Globose to rarely ellipsoid, 23–30μm in diam. excluding ornamentation, brown, coarsely warty.	Asci mostly cylindrical, 300–340 × 25–40 µm, 8-spored, mostly incompletely arranged uniseriate	Abies amabilis	North America 950m	Trappe and Castellano 2000
Hydnotrya tulasnei (Berk.) Berk. & Broome, 1846	Irregularly spherical or lobed, sometimes with inward folds, 20–70 mm diam., ochre-reddish to brick red	Gleba solid, later yellow brown, with labyrinthic chambers.		Globose, 20–30 (–33) μm diam. (including ornamentation), ochre-reddish, with conspicuous, irregular warts.	Asci broadly clavate or cylindrical, 175–210 × 30–62.5 µm, (4–) 8-spored, spores biseriate.	Coniferous forest	Europe North America 1600m	Dimitrova and Gyosheva 2008 Stielow et al. 2010
Hydnotrya variiformis Gilkey, 1947	Globose to subglobose to flattened, somewhat depressed, 7–40 mm broad, cinnamon-buff to cream-buff	Gleba variable, from a simple cavity to extremely lobed with numerous small chambers the interior, usually opening to the exterior at one or more points.		Ellipsoid, 24–28×36–36 μm, yellow-brown, thickened exosporium wall, surface appearing punctate and with small irregular nodules	Asci 240–280 × 24 µm, 8-spored, clavate at immaturity, spores incompletely biseriate; cylindrical at maturity, spores strictly uniseriate.	Coniferous forest	North America 1200–2400m	Gilkey 1947 Abbott and Currah 1997 Beug et al. 2014
Hydnotrya zayuensis sp. nov.	Irregularly globose, 15–20 mm in diameter when fresh, smooth, gentle inward folds, surface cinnamon. Mostly single-chambered with a primary apical opening, the opening is just an almost closed seam, white fluffy inside cavity. Elastic and crisp. No special smell.	Gleba hollow, single-chambered with a primary apical opening, sometimes the opening is just an almost closed seam.		Ellipsoid, (17–)20–30.5 × 15.5–18.0 μm, (including thickened exosporium), golden yellow, surface rough, looking like crumbly.	Asci cylindrical, 118.5–130.5×15.0–22.5 µm, 8-spored, spores strictly uniseriate	Abies sp.	Zayu, China Asia 3770m	This study

The ascospore morphology is highly variable among different species in Hydnotrya, which is useful for distinguishing species. Abbott and Currah (1997) once divided the genus Hydnotrya into two subgenera: subg. Hydnotrya and Cerebriformae, according to the characters of their ornamentation. The subg. Hydnotrya had four species of H. tulasnei, H. michaelis, H. cubispora, and H. variiformis showing ascospores with rounded or irregular warts. The subg. Cerebriformae has only one species of H. cerebriformis differs from Subg. Hydnotrya in ascospores with short, rounded aculei. However, the current phylogenetic analysis showed that ascospore characteristics were not reliable for differentiating species of Hydnotrya into these subgenera (Fig. 1).

Based on ITS analyses, 14 species of Hydnotrya are divided into two lineages, A and B. The species in the clade A mostly have nearly solid gleba (6 out of 9) and globose, warty ascospores, either uniseriately or biseriately arranged in asci. The clade A is divided into two subclades: the subclade Aa (clade 1–6) and Ab(clade 7–9). The species in the subclade Aa have solid ascomata. Two groups can be distinguished: the group 1 (clade 1 and 2) and group 2 (clade 3–6), both found in China and Europe. The group 1 contains two species with ascospores uniseriately arranged in asci; the group 2 contains four species with ascospores biseriately arranged in asci. Species in the subclade Ab are distributed in China, Europe, and America, and have hollow ascomata and ascospores uniseriately arranged in asci. The species in the clade B has hollow to chambered gleba and ellipsoidal ascospores (without thickened exosporium), biseriately arranged in asci. The clade B is divided into two groups: Ba and Bb. The group Ba (clade 10 and 11) contains 2 species distributed in China and Europe, with ellipsoidal ascospores, with a pitted surface. The group Bb (clade 13 and 14) contains two species, only found in China, with rectangular and ellipsoidal ascospores (with thickened exosporium), with a rough surface. (Fig. 1).

Based on the morphological and molecular phylogenetic analyses there seems to be a trend in morphological traits among the species within the genus Hydnotrya, that is, the gleba evolved from being hollow or chambered to nearly solid; the ascus becoming shorter and wider, with ascospores arranged from uniseriate to biseriate; ascospores from ellipsoidal to globose, with an ornamentation from smooth to rough as well. This evolutionary trend in the genus Hydnotrya is probably related to their hypogeous habits, that is, if the gleba has more chambers, the ascoma will hold more ascospores, and so there are more chances of ascospores to be dispersed by animals that eat them (Hawker 1955; Ławrynowicz 1990; Læssøe and Hansen 2007; Bonito et al. 2013). All of this improves their survival and reproduction. Of course, more collections would be needed for comprehensive morphological and molecular analyses to provide more evidence to support this hypothesis.

In China, 9 species were recorded before this study (Xu et al. 2018). In this paper, two new species are described. 11 species are now known in China, among which 7 species are distributed in southwest China.

Thanks to Mr. Shucheng He for his help in specimen collection. Thanks to the Fungal Diversity Conservation and Utilization Team in Northwest Yunnan for providing the research platform and team members for their help.