Molecular and morphological evidence reveal a new genus and species in Auriculariales from tropical China

Abstract Grammatus labyrinthinus gen. et sp. nov. is proposed based on DNA sequences data and morphological characteristics. It is known so far from southern, tropical China. The new species is characterised by an annual, resupinate basidiocarp with a shallow, subporoid hymenophore, a hymenium restricted to the bottom of the tubes, a dimitic hyphal system, presence of encrusted skeletocystidia and dendrohyphidia, longitudinally septate basidia and smooth, oblong-ellipsoid to cylindrical, acyanophilous basidiospores. Phylogenetic analyses based on ITS + nLSU DNA sequences data indicate that G. labyrinthinus belongs to Auriculariaceae in which it has an isolated position. Phylogenetic inferences show G. labyrinthinus to be related to Heteroradulum. However, the ITS sequences similarity between G. labyrinthinus and H. kmetii, the type species of Heteroradulum, were 89.84% and support the establishment of the new genus. Inversely, Heteroradulum semis clustered with G. labyrinthinus with strong support and it is transferred to Grammatus.


Introduction
Auriculariales was established by Schroeter (1889) and originally accommodated species which, with transversely septate basidia, is also known as auricularioid basidia. Based on the micromorphology and ultra-structure of the septal pore and the spindle pole body, Bandoni (1984) redefined Auriculariales, in order to accommodate all heterobasidiomycetes with continuous parenthesomes, transversely or longitudinally septate basidia and hyphal haploid stages. Currently, one family, Auriculariaceae is accepted and 198 species are distributed into 32 genera of Auriculariales (Kirk et al. 2008).
Molecular phylogeny had been widely used to investigate phylogenetic relationships amongst the genera and species in Auriculariales (Swann and Taylor 1993, Berres et al. 1995, Weiß and Oberwinkler 2001, Kirschner and Chen 2004, Wells et al. 2004, Kirschner et al. 2010, 2012, Zhou and Dai 2013, Sotome et al. 2014, Malysheva and Spirin 2017. These phylogenetic contributions provided a general overview of Auriculariales and show strong support at the species level, but so far, not all the deeper nodes received high support from molecular evidence. China is very rich in wood-decaying fungi and extensive studies on species diversity, taxonomy, ecology and phylogeny of wood-decaying fungi have been carried out recently (Yuan and Dai 2008;Dai , 2011Dai , 2012Yuan et al. 2015). During a continuous survey of wood-decaying Basidiomycetes in the Yunnan Province, tropical China, a species of Auriculariaceae was collected but could not be confidently identified to any known species. The morphology characteristics suggested they represent an undescribed genus in Auriculariaceae. The aim of this paper is to clarify the taxonomic status of the genus and to describe new taxa.

Materials and methods
Morphological studies. Specimens are deposited at the herbarium of Institute of Applied Ecology, Chinese Academy of Sciences (IFP). Microscopic procedures follow Yuan and Qin (2018). The microscopic studies were made from sections mounted in Cotton Blue (CB): 0.1 mg aniline blue dissolved in 60 g pure lactic acid; CB+/-= cyanophilous / acyanophilous. Amyloid and dextrinoid reactions were tested in Melzer's reagent (IKI): 1.5 g KI (potassium iodide), 0.5 g I (crystalline iodine), 22 g chloral hydrate, aq. dest. 20 ml; IKI-= neither amyloid nor dextrinoid reaction. KOH (5%) was used as a mounting reagent. Sections were studied at magnifications up to 1000× using a Nikon Eclipse E600 microscope and phase contrast illumination and dimensions were estimated subjectively with an accuracy of 0.1 µm. For spore measurements, the apiculus was excluded. In presenting the variation of spore size, 5% of the measurements at each end of the range are given in parentheses. The following abbreviations are used in the text: L = mean spore length, W = mean spore width, Q = range of length/width ratios for studied specimens and n = total number of spores measured from a given number of specimens. Special colour terms are from Petersen (1996).
Molecular procedures and phylogenetic analyses. The fungal taxa and strains used in this study are listed in Table 1. Phire Plant Direct PCR Kit (Finnzymes, Finland) procedure was used to extract total genomic DNA from the fruiting body and for the polymerase chain reaction (PCR). PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide (Stöger et al. 2006). DNA sequencing was performed at Beijing Genomics Institute (BGI). All newly generated sequences have been submitted to GenBank and are listed in Table 1.
Nuclear ribosomal RNA genes were used to determine the phylogenetic position of the new species. The internal transcribed spacer (ITS) regions were amplified with the primers ITS4 and ITS5 and the partial nLSU regions were amplified with primers LR7 and LR0R (White et al. 1990). The most similar sequences were searched for in GenBank NCBI (http://www.ncbi.nlm.gov) using the BLAST option and downloaded (Table 1). Sequences were aligned using ClustalX (Thompson et al. 1997) and the alignment was deposited in TreeBASE (http://treebase.org/treebase-web/) (submission ID: 22496). Identity/similarity between two sequences was calculated using the BioEdit v. 7.2.6 (Hall 2005). Maximum parsimony (MP), Maximum likelihood (ML) and Bayesian inference were applied to the ITS + LSU dataset. All characters were weighted and gaps were treated as missing data. Maximum parsimony analysis (PAUP* version 4.0b10) was used (Swofford 2002). Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Max-trees were set to 5000 and no-increase, branches of zero length were collapsed and all parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1,000 replicates (Felsenstein 1985). Maximum likelihood (ML) analysis was performed in RAxML v8.2.4 with GTR + I + G model (Stamatakis 2014). The best tree was obtained by executing 100 rapid bootstrap inferences and, thereafter, a thorough search for the most likely tree using one distinct model/data partition with joint branch length optimisation (Stamatakis et al. 2008). Bayesian analysis with the latest version of MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003) implementing the Markov Chain Monte Carlo (MCMC) technique. The best-fit models (GTR + I + G) were selected by hLRT in MrModeltest 2.3 (Nylander 2004). Four simultaneous Markov chains were run starting from random trees and keeping one tree every 100 th generation until the average standard deviation of split frequencies was below 0.01. The value of burn-in was set to discard 25% of trees when calculating the posterior probabilities. Bayesian posterior probabilities were obtained from the 50% majority rule consensus of the trees kept.

Phylogenetic analyses
The combined ITS + nLSU sequence dataset includes the new species and other related species in Auriculariales. Sistotrema brinkmannii was used as outgroup (Malysheva and Spirin 2017). The data matrix comprised 1413 base pairs with 818 constant characters, 206 parsimony-uninformative variable characters and 389 parsimony informative positions. Maximum parsimony analysis was performed and a strict consensus tree was obtained from the 2 equally most parsimonious trees. The same dataset and alignment was analysed using RAxML v8.2.4 and MrBayes 3.2.6 with the best-fit model (GTR + I + G) selected by MrModeltest 2.3 and a similar topology was generated and the maximum likelihood tree is shown in Fig. 1. Bayesian analysis ran 4 million generations and resulted in average standard deviation of split frequencies = 0.008219. In the phylogenetic tree, two sampled specimens of Grammatus labyrinthinus group together with full support and form a monophyletic lineage with Heteroradulum semis with strong support (97 % in ML, 100 % in MP and 1.00 BPP). The new taxon belongs to Auriculariales in which it has an isolated position. Diagnoses. Basidiocarps annual, resupinate; hymenium restricted to the base of the tubes. Hymenophoral surface irregularly irpicoid to subporoid, then labyrinthine to sinuous. Subiculum very thin. Tubes shallow. Hyphal system dimitic; generative hyphae bearing clamp connections; skeletal hyphae IKI-, CB+. Skeletocystidia clavate, the upper part heavily encrusted. Dendrohyphidia present, thin-to slightly thickwalled. Basidia subglobose, longitudinally septate. Basidiospores oblong-ellipsoid to cylindrical, hyaline, thin-walled, smooth, IKI-, CB-.
Etymology. labyrinthinus (Lat.): refers to labyrinthine hymenophore. Basidiocarps annual, resupinate, coriaceous, without special odour or taste when fresh, corky when dry, up to 15 cm long, 3 cm wide and 0.2 mm thick. Hymenophoral surface cream to pale buff when fresh, cinnamon-buff to yellowish-brown upon drying, firstly irregularly irpicoid to subporoid, the separate plates grow laterally and then develop into labyrinthine to sinuous pores, mostly 4-5 per mm, dissepiments thin; sterile margin up to 0.2 mm wide, pale yellow. Subiculum very thin (ca. 0.1 Figure 1. Maximum likelihood tree illustrating the phylogeny of Grammatus labyrinthinus and related taxa in Auriculariales, based on the combined ITS + nLSU sequence dataset. Branches are labelled with maximum likelihood bootstrap higher than 50%, parsimony bootstrap proportions higher than 50% and Bayesian posterior probabilities more than 0.95. mm thick), cream to pale buff. Tubes corky, concolorous with pore surface, shallow, up to 130 µm deep, tube walls 120-200 µm thick. Hymenium restricted to the base of the tubes. Hyphal structure. Hyphal system dimitic; generative hyphae bearing clamp connections, skeletal hyphae IKI-, CB+; tissue unchanged in KOH.

Discussion
Anatomically, the longitudinally septate basidia of Grammatus labyrinthinus point toward affinities with Auriculariales, which is confirmed by molecular data. The new taxa are phylogenetically closely related to Heteroradulum. Heteroradulum kmetii, type of the genus, has perennial, effused-reflexed and pinkish or reddish basidiocarps with hymenial surface first smooth then with irregularly arranged, sharpened outgrowths (Malysheva and Spirin 2017), in which feature, it differs from G. labyrinthinus. The similarity between the ITS sequences of G. labyrinthinus and H. kmetii are of 89.84%. The unique morphological characteristics and molecular sequence analyses both support the establishment of the new genus.
Heteroradulum semis was originally found and described from high elevation temperate north-eastern China. It is characterised by resupinate, leathery basidiocarps covered by blunt-pointed spines, a dimitic hyphal structure with clamped generative hyphae, encrusted tramal skeletocystidia, simple or sparsely branched dendrohyphidia, longitudinally septate basidia and broadly cylindrical to narrowly obovate basidiospores (Malysheva and Spirin 2017). The sterile and blunt-pointed outgrowths on the hymenial surface of H. semis identify the irregularly irpicoid and separate plates of G. labyrinthinus when young, but the irregularly irpicoid and separate plates of G. labyrinthinus would form the labyrinthine or subporoid structure when old and can be distinguished from the former species. Phylogenetic analyses confirm that H. semis clustered with G. labyrinthinus with strong support. So it is transferred to Grammatus and a new combination, G. semis is proposed.
There are 216 genera and more than 1800 species of wood-inhabiting fungi in Polyporales (Kirk et al 2008) with high diversity of hymenophore structure from smooth, hydnoid, lamellate and poroid. Amongst the poroid taxa, Grammothele Berk. & M.A. Curtis, Hymenogramme Mont. & Berk., Porogramme (Pat.) Pat. and Theleporus Fr. are characterised by a hymenium restricted to the bottom of the pores, which differentiate them from the other typical polypores. These genera are characterised by non-septate basidia and are members of Polyporales. In comparison, Auriculariales are relatively poor in genera and species, but still, their hymenophore structures are diverse. Grammatus labyrinthinus is the representative of poroid species with a hymenium restricted to the bottom in Auriculariales. It is another instance of morphological convergent evolution across the order.