﻿New species of Hydnotrya (Ascomycota, Pezizomycetes) from southwestern China with notes on morphological characteristics of 17 species of Hydnotrya

﻿Abstract 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.

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.

Materials and methods
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).

Phylogenetic analysis
The ML and Bayesian analyses of the 50 ITS sequences, are shown in Fig. 1 with associated bootstrap supports for branches.
In the phylogenetic tree, the 46 ITS sequences from Hydnotrya ascomata revealed the phylogenetic relationship of 14 species: Clade 1 includes 5 sequences of H. bailii from Europe.Clade 2 includes 2 sequences of H. brunneospora from China.Clade 3 includes 4 sequences of H. tulasnei from Europe.Clade 4 includes 3 sequences of H. puberula from China.Clade 5 includes 2 sequences of H. badia from China.Clade 6 includes 2 sequences of H. nigricans from China.Clade 7 includes 6 sequences of H. cerebriformis from Germany, China, and Mexico; two other distinct clades were revealed, one comprising Eurasian specimens, and the other comprising specimens from Mexico, which is probably because these specimens, respectively, are from Holarctic and Neotropical regions.Clade 8 includes 3 sequences of H. variiformis from the USA.Clade 9 includes 2 sequences of H. cubispora from the UK and USA.Clade 10 includes 3 sequences of H. michaelis from Europe.Clade 11 includes 3 sequences of new species, H. oblongispora from China.Clade 12 includes 3 sequences of Hydnotrya sp. from the USA.They may be new species from North America that have not yet been reported.Clade 13 includes 6 sequences of H. laojunshanensis from China.When the latter was reported, only one specimen was found, and many more were collected over the past few years, so new DNA sequences of H. laojunshanensis were added.Clade 14 includes 2 sequences of a new species, H. zayuensis from China.The phylogenetic analysis shows that the new species are distinct from other Hydnotrya species.In addition to the ITS sequences used in this phylogenetic analysis, the LSU sequences were amplified from the newly supplemented specimens in this study and uploaded to NCBI for future study.
Based on the ITS locus, two major monophyletic lineages are presented, showing a strong sister relationship (BS=100%; PP = 1.0).They are Clade A (including Clade 1-9) and Clade B (include Clade 10-14) respectively.The species included in these two phylogenetic morphologically share commonalities and uniqueness.Description.Ascomata irregularly globose, 1.0-2.5 cm in diameter when fresh, smooth, sometimes gently folded inward, surface light khaki (4C5) to reddish brown (8D8); nearly single-chambered with a primary apical opening up to 0.2-0.8cm in diameter, sometimes the opening is just an almost closed seam, white fluffy inside cavity.Elastic and crisp.No special smell was noticed.
Ecology and distribution.Hypogeous, solitary, or in groups in soil, under A. forrestii mixed with shrubs of Rhododendron spp., fruiting from late summer to early autumn.Known only from Yunnan Province, China.

Hydnotrya zayuensis
Ecology and distribution.Hypogeous, solitary in the humus under Abies sp.mixed with shrubs of Rhododendron spp.Fruiting in summer, from July to September.Known only from Zayu, Tibet, China.
Notes.Morphologically, H. zayuensis is similar to H. laojunshanensis.However, H. zayuensis has much smaller ascospores, and a thinner peridium, as well as lighter colored ascomata.Molecular analysis showed that H. zayuensis is distinct from H. laojunshanensis and other species of Hydnotrya.Description.Ascomata irregularly globose, 1.0-3.0cm in diameter when fresh, brownish orange (6C8), smooth, mostly single-chambered with a primary apical opening to 0.1-0.5 cm in diameter, the opening rarely narrowing into a slit, sometimes folded forming few channels, lined with white fluffy hymenium.Elastic to crisp.No special smell was noticed.
Notes.When the species was described in 2013 by Li et al., only one collection from Mt. Laojun in Yunnan Province, China, was reported.More specimens of H. laojunshanensis have been found at other places in Yunnan since then.We discovered that this species had not only simple chambered ascomata but also folded, chambered ascomata.This species has large, rectangular ascospores (including thickened exsporium) with a rough surface differentiating from other species in Hydnotrya.

Discussion
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).
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. cerebriformi 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).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.

Hydnotrya cerebriformis
Harkn.1899     This study 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;Laessøe 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.

Figure 1 .
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.

Plate 3 .
Hydnotrya laojunshanensis A young sarcomata cut in half B mature ascomata with one cut in half C infolded and chambered ascoma D section of hymenium in 5% KOH E a peridium section in 5% KOH F ascospores released from asci in 5% KOH G-I ascospores under SEM (I.SEM of a single ascospore cut in half).Scale bars: 1 cm (A, B); 50 μm (D, E); 20 μm (F); 5 μm (G-I).
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.

Table 1 .
Taxa information and GenBank accession numbers of the sequences used in this study.The newly generated sequences are in bold.

Taxonomy Hydnotrya oblongispora L. Li & S.H. Li, sp. nov.
Diagnosis.Differs from other species in the genus Hydnotrya by its nearly single-chambered ascomata and long ellipsoidal ascospores.

Table 2 .
List of main characteristics of Hydnotrya.