﻿Two new species of Craterellus (Cantharellales, Hydnaceae) with veined hymenophore from north-eastern China

﻿Abstract In this contribution to the genus Craterellus in northern China, two new species are introduced: Craterellusconnatus and C.striatus. These species and C.atrobrunneolus, initially described in south-western China, are highly similar and closely related. The species delimitation is molecularly supported by multigene phylogenetic analysis of the nr LSU and tef-1α region. Craterellusconnatus is characterised by its medium-sized basidiomata, greyish-brown and smooth pileus with an off-white margin, the hymenophore with a strongly anastomosing vein, turning khaki upon drying, connate stipe, broad ellipsoid to ellipsoid basidiospores (6.1–7.8 × 4.8–5.9 μm), slender basidia with (2)4–6 sterigmata and the absence of clamp connection. Craterellusstriatus is characterised by its small-sized basidiomata, fibrillose, greyish-brown to yellowish-brown, fully perforated pileus with a brown fringe, the hymenophore with a forking vein, the stipe inflated at the base, broad ellipsoid to ellipsoid basidiospores (6.8–8.0 × 5.1–6.0 μm), 2–6 spored basidia, encrusted hyphae and the absence of clamp connection. Detailed macroscopic and microscopic descriptions, accompanied by illustrations and a taxonomic discussion, are presented. A key to the Chinese Craterellus species is also provided.


Introduction
Craterellus Pers., typified with C. cornucopioides (L.) Pers. (Persoon 1825), belongs to Hydnaceae of Cantharellales (Hibbett et al. 2014). It is well represented in northern temperate zones and occurs in the tropics (Dahlman et al. 2000). Species in this genus are characterised by funnel-shaped fruiting bodies, hollow stipes, usually dark grey to black or yellow pileus (Corner 1966). Craterellus is an important genus of wild edible mushrooms and is renowned for its high economic, medicinal and ecological values (Pilz 2003). Approximately 162 records of Craterellus (including infraspecific taxa) have been found in Index Fungorum (http://www.indexfungorum.org). However, most of these species have been transferred to other genera, based on a phylogenetic analysis of the large nuclear subunit (nr LSU) and the internal transcribed spacer region (ITS), including Cantharellus Adans. ex Fr., Gomphus Pers. and Polyozellus Murrill etc. (Dahlman et al. 2000;Contu et al. 2009;Olariaga et al. 2009;Wilson et al. 2012;Montoya et al. 2021). Nearly 100 names have been considered legitimate in MycoBank (https://www.mycobank.org/) to date.
In China, species diversity, taxonomy and phylogeny of macrofungi have been investigated in recent years, many new species having been discovered (Zhong et al. 2018;Cui et al. 2019;Sun et al. 2020;Zhang et al. 2020a;Cao et al. 2021a, b;Liu et al. 2021Liu et al. , 2022aJi et al. 2022;Sun et al. 2022). Craterellus is one of the important fungal genera of macrofungi, 14 species of Craterellus having been recorded in China: C. albidus Chun Y. Deng, M. Zhang & J. Zhang, C. atratus (Corner) Yomyart, Watling, Phosri, Piap. & Sihan., C. atrobrunneolus T. Cao (Bi 1994;Mao 1998;Dai et al. 2010;Gu et al. 2012;Li et al. 2015;Zhong et al. 2018;Zhang et al. 2020a;Cao et al. 2021a, b). Nevertheless, due to their less attractive appearance, which can be confused with the environment, it is challenging to find Craterellus species in the field, causing them to be overlooked and thus remain undescribed. In addition, some species of Craterellus, especially those with the non-perforated pileus, are easily confused with other morphologically similar taxa, for example,, some species of Clitocybe (Fr.) Staude, Gomphus or Polyozellus, making it never easily distinguished from others in fieldwork.
During a recent survey of macrofungi in northern China, we discovered some exciting and novel species of Craterellus. In this contribution, the subsequent morphological and molecular analyses of the transcription elongation factor 1-alpha (tef-1α) and nr LSU sequences represented two new species, C. connatus G. P. Zhao, J. J. Hu, B. Zhang & Y. Li and C. striatus G. P. Zhao, J. J. Hu, B. Zhang & Y. Li which are described and illustrated herein. A key of Craterellus in China is provided as well.

Vouchers and morphological analyses
This paper is principally based on materials collected by the senior author and collaborators over the past four years in northern China and specimens have been deposited at the Mycological Herbarium of Jilin Agricultural University (HMJAU).
Photographs and descriptions of macroscopic morphological characteristics were made from fresh materials in the field. The colours correspond to the "Flora of British fungi: colour identification chart" (Royal Botanic Garden 1969). Collections were dried in an oven at 45 °C and rehydrated with 95% alcohol. All microscopic observations and measurements were made in ammoniacal Congo red with a 5% aqueous potassium hydroxide (KOH) solution to improve tissue dissociation and matrix dissolution. Melzer's Reagent was also used in the study. Measurements of basidiospores cite length and length/width ratio (Q) in this format: (minimum-) mean minus standard deviation-mean value-mean plus standard deviation (-maximum measured), Q m = average Q of all basidiospores measured ± sample standard deviation; spore measurements are based on 20 spores. Microscopic features were examined with the aid of the ZEISS Axio Lab A1.

DNA extraction, amplification and sequencing
Genomic DNA was extracted from dried or fresh material stored in desiccant silica gel. The extraction method followed the nuclear Plant Genomic DNA Kit (Kangwei Century Biotechnology Company Limited, Beijing, China). The amplified segments were the nr LSU and the tef-1α regions. The nr LSU region was amplified using LR0R and LR5 (Vilgalys and Hester 1990). The tef-1α region was amplified using tefF and tefR (Morehouse et al. 2003). Polymerase chain reaction (PCR) amplifications were performed in a total volume of 25 μl containing 1 μl template DNA, 1 μl of each primer, 10.5 μl distilled water and 12.5 μl PCR mix (2× Es Taq MasterMix, CWBIO, China). PCR amplification conditions followed Vilgalys and Hester (1990) for the nr LSU region and Morehouse et al. (2003) for the tef-1α region. The PCR products were subjected to electrophoresis on 1% agarose gel. Sequencing was performed by Sangon Biotech (Shanghai, China) using the same primer pairs used for the PCR.

Phylogenetic analyses
This study is based on around 25 specimens, including two outgroups (Hydnum ellipsosporum Ostrow & Beenken and Cantharellus cibarius Fr.) (Bijeesh et al. 2018;Zhong et al. 2018;Zhang et al. 2020a;Cao et al. 2021b). Sequences of the nr LSU and tef-1α regions were newly produced in this study and downloaded from GenBank (http://www.ncbi.nlm.nih.gov/). Detailed sample information is provided in Table 1. Sequences were assembled and edited using the software package Sequencher 5.4.6 (Gene Codes Corp., USA). Alignment of sequence data was performed with MUSCLE in MEGA 7.0.21 (Kumar et al. 2016). For Bayesian Inference (BI) analyses, the most suitable substitution model for each gene partition was calculated with ModelFinder (Kalyaanamoorthy et al. 2017) in PhyloSuite 1.2.2. (Zhang et al. 2020b). BI analyses were performed using MrBayes in PhyloSuite 1.2.2 (Zhang et al. 2020b), implementing the Markov Chain Monte Carlo (MCMC) technique. Four simultaneous Markov chains were run from random trees, keeping one tree every 200 th generation until the average standard deviation of split frequencies was below 0.01. The Maximum Likelihood analysis was performed using RAxML-HPC2 on XSEDE 8.2.12 (Stamatakis 2014) in the CIP-RES Science Gateway portal (https://www.phylo.org/portal2/tools.action) with the GTR-GAMMA model and searching for the most likely tree with 1000 heuristic replicates. The bootstrap support (BS) of ≥ 50% in the ML tree and BPP of ≥ 0.75 indicated statistical significance. The phylogenetic trees were visualised using FigTree 1.4.23 (Andrew 2016).

Phylogeny
Seven new DNA sequences (3 nr LSU, 4 tef-1α) were produced for this study. After removing introns and low-homology regions, the final combined alignment of these two genes totalled 2,027 characters (nr LSU: 1,024 characters; tef-1α: 1,003 characters). The best models for the BI analysis of the concatenated dataset were SYM+I+G4 for nr LSU and SYM+G4 for tef-1α. The most likely tree inferred by ML analysis of the combined dataset exhibited a quite similarly supported topology as the Bayesian majority-rule consensus tree with an average standard deviation of split frequencies = 0.005143. The most likely tree, based on 1000 searches, is depicted in Fig. 1  Etymology. Connatus: referring to several stipes grown together from the base upwards.
Diagnosis. Differs from other Craterellus species by its greyish-brown and smooth pileus with an off-white margin, hymenophore with a strongly anastomosing vein and the colouration turning khaki upon drying.
Description. Fruiting body 12-45 mm high. Pileus 3-21 mm in diam., 1-10 mm tall, plane-convex firstly, soon infundibuliform and usually perforated continuous to the base of the stipe; margin inrolled when young, then expanding outwards, old becoming incurved, broadly wavy; surface blackish-brown, cream near the margin at first, gradually lighter to the centre with age, yellowish-brown finally, turning pale greyish-brown when drying, covered with brown fringe and fibrillose scales. Hymenophore decurrent, consisting of longitudinal ridges (<1 mm) with prominent forking, cross vein and off-white. Stipe 11-35 × 1-6 mm, cylindrical, inflated at the base, up to 10 mm in diam., hollow, central, smooth, blackish-brown. Text leathery, dirty white. Odour light. Taste unknown. Spore print not obtained.
Habitat. Scattered to gregarious on the ground in a coniferous and angiosperm mixed forest.

Discussion
In our study, two new species formed a sister relationship with Craterellus atrobrunneolus. These three species show close morphological and phylogenetic similarities with each other. All species share the brown pileus, grey hymenophore, hollow stipe, narrow basidia and absence of clamps. Craterellus atrobrunneolus was initially described in south-western China. It is characterised by a dark brown to brownish-grey colouration, convex to plano-convex pileus with shallow depression, but not perforated centre, smooth to slightly folded hymenophore, absence of clamp connections in all tissues, narrow basidia with 2-6 sterigmata and broad ellipsoid to subglobose basidiospores (Cao et al. 2021b). Craterellus connatus is recognised in the field by the medium-sized, nearly fleshy basidiomata with the greyish-brown, fully perforated pileus, an off-white margin, strongly anastomosed, veined hymenophore and hollow stipe. Microscopically, it possesses broad ellipsoid to ellipsoid basidiospores (Q m = 1.32 ± 0.09), slender basidia with (2)4-6 sterigmata and an absence of clamp connections. Craterellus striatus is characterised by its small-sized basidiomata, blackish-brown pileus that turns yellowish-brown upon drying and is covered with brown fringe and spinous scales and off-white hymenophore consisting of longitudinal ridges (<1 mm) with prominent forking. It has a hollow and brown stipe and broad ellipsoid to ellipsoid basidiospores, 2-6 spored basidia, encrusted hyphae and the absence of the clamp connection. Craterellus atrobrunneolus differs by its dark brown to almost black throughout, convex but not perforated pileus, while C. connatus possesses greyish-brown pileus with an off-white margin and C. striatus possesses yellowish-brown, perforated pileus with brown fringe. Microscopically, C. atrobrunneolus possesses broad ellipsoid to subglobose basidiospores, while C. connatus and C. striatus possess broad ellipsoid to ellipsoid basidiospores. In addition, C. atrobrunneolus have 2-6 spored basidia, while C. connatus have mostly 4-6 spored basidia. Craterellus striatus have encrusted hyphae, while C. atrobrunneolus did not. Although the two new species have similar microscopic characteristics (spores and basidia), they are separated by their colouration of dried pileus and hymenophore and the configuration of hymenophore. Craterellus striatus is smaller (both pileus and stipe) than C. connatus. Further, C. striatus has a hymenophore composed of the forked longitudinal ridge and non-discolouring upon drying, whereas C. connatus has an anastomosing veined hymenophore, which turns khaki upon drying. Morphology and phylogenetic analyses indicated that the two new species in this study are not conspecific.
However, Craterellus atrobrunneolus was not included in any subgenus in Cao et al. (2021a). According to our study, C. atrobrunneolus, C. conatus and C. striatus have formed an isolated branch. Further research is needed to obtain a more precise infrageneric classification. Additionally, this study suggests that China, especially northeastern China, has considerable fungal diversity and possibly many endemic species.