Micromelanconis kaihuiae gen. et sp. nov., a new diaporthalean fungus from Chinese chestnut branches in southern China

Abstract Melanconis-like fungi are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae. A new Melanconis-like genus of Pseudomelanconidaceae was discovered on branches of Chinese chestnut (Castanea mollissima) in southern China, which was confirmed by both morphology and phylogenetic analysis of combined ITS, LSU, tef1a and rpb2 sequences. The new genus Micromelanconis is characterized by two types of conidia from natural substrate and manual media of PDA, respectively. Conidia from Chinese chestnut branches are pale brown, ellipsoid, multiguttulate, aseptate with hyaline sheath. While conidia from PDA plates are pale brown, long dumbbell-shaped, narrow at the middle and wide at both ends, multiguttulate, aseptate, and also with hyaline sheath. All Pseudomelanconidaceae species were only reported on tree branches in China until now. More interesting taxa may be discovered if detailed surveys on tree-inhabiting fungi are carried out in East Asia in the future.

Melanconis-like taxa are distributed in several families of Diaporthales, mainly Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae, which are four morphologically similar clades in distinct phylogenetic clades within this order ). Species of these four families are usually discovered on branches of Betulaceae, Juglandaceae and Fagaceae, but they are not strong pathogens (Wehmeyer 1937;Du et al. 2017;Voglmayr et al. 2019).
In the present study, investigations were conducted in Castanea mollissima plantations in Hunan Province of south China. Two Melanconis-like specimens were collected on a cultivated chestnut tree. The aim of the present study was to identify the fresh collections and confirm their phylogenetic positions.

Collection, examination and isolation
The fresh specimens of diseased and dead chestnut branches were collected in a Castanea mollissima plantation in Hunan Province of south China. Morphological characteristics of the conidiomata were determined under a Nikon AZ100 dissecting stereomicroscope. More than 20 fruiting bodies were sectioned, and 50 conidia were selected randomly for measurement using a Leica compound microscope (LM, DM 2500). Isolates were obtained by removing a mucoid conidial mass from conidiomata, spreading the suspension onto the surface of 1.8% potato dextrose agar (PDA), and incubated at 25 °C for up to 24 h. Single germinating conidium was removed and plated onto fresh PDA plates. Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were recorded, including the colony color and conidiomata structures. Specimens were deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures were maintained in the China Forestry Culture Collection Centre (CFCC).

DNA extraction, PCR amplification and phylogenetic analyses
Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). DNA was estimated by electrophoresis in 1% agarose gel and the quality was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Four partial loci, including the 5.8S nuclear ribosomal DNA gene with the two flanking internally tran-scribed spacer (ITS) regions, the large subunit of the nrDNA (LSU), and the translation elongation factor 1-alpha (tef1a) and DNA-directed RNA polymerase II second largest subunit (rpb2) genes, were amplified by the following primer pairs: ITS1 and ITS4 for ITS (White et al. 1990), LR0R and LR5 for LSU (Vilgalys and Hester 1990), EF1-728F and EF2 for tef1a (O'Donnell et al. 1998;Carbone and Kohn 1999), and RPB2-5F and fRPB2-7cR for rpb2 (Liu et al. 1999). The polymerase chain reaction (PCR) conditions were as follows: an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 50 s at 48 °C (ITS, LSU) or 54 °C (tef1a) or 55 °C (rpb2), and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. PCR products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).
For phylogenetic reconstruction, newly-generated sequences of ITS, LSU, tef1a and rpb2 were initially subjected to BLAST search (BLASTn) in NCBI website (https:// www.ncbi.nlm.nih.gov). Then species and their sequences from recently published articles were selected and listed in Table 1 (Crous et al. 2012b;Alvarez et al. 2016;Senanayake et al. 2017;Braun et al. 2018;Fan et al. 2018a;Jiang et al. 2020a;Wang et al. 2020). The sequence alignments of the four individual loci (ITS, LSU, tef1a and rpb2) were conducted using MAFFT 7 (http://mafft.cbrc.jp/alignment/server/index.html), manually edited in MEGA 7.0.21, and then assembled as a dataset of ITS-LSU-tef1a-rpb2 to infer the phylogenetic placement of our new isolates.
ML and Bayesian analysis were implemented on the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014) and MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003), respectively. For ML analyses, a GTR+GAMMA substitution model with 1000 bootstrap iterations was set. MrModeltest 2.3 was used to estimate the best nucleotide substitution model settings for each gene. Bayesian inference (BI) was performed based on the DNA data set from the results of the MrModeltest, using a Markov chain Monte Carlo (MCMC) algorithm in MrBayes 3.1.2. Two MCMC chains were run from random trees for 1000 million generations and stopped when the average standard deviation of split frequencies fell below 0.01. Trees were saved each 1000 generations. The first 25% of trees were discarded as the burn-in phase of each analysis, and the Bayesian posterior probabilities (BPPs) were calculated from the remaining trees. Phylogenetic trees were viewed with FigTree v.1.3.1 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxon have been deposited in GenBank (Table 1).

Results
The ITS, LSU, tef1a and rpb2, and combined data matrices contained 624, 867, 513, 865, and 2869 characters with gaps, respectively. The alignment comprised 92 strains, with Nakataea oryzae (CBS 243.76) and Pyricularia grisea (Ina168) from Magna- porthales as outgroup taxa. The ML analysis yielded a tree with a ln likelihood value of -45806.266577 and the following model parameters: alpha = 0.298226, Π(A) = 0.241173, Π(C) = 0.258552, Π(G) = 0.275145, and Π(T) = 0.225130. For BI analyses, the general time reversible model, additionally assuming a proportion of invariant sites with gamma-distributed substitution rates of the remaining sites (GTR+I+G), was determined to be the best for the ITS, LSU, and tef1a loci by MrModeltest, whereas the most appropriate model for the rpb2 locus was the Tamura-Nei model, additionally assuming a proportion of invariant sites with gamma-distributed substitution rates of the remaining sites (TrN+I+G). The phylogeny resulting from the RAxML maximum likelihood analysis of the combined gene sequence data is shown in Fig. 1. Overall, the topologies obtained from the different phylogenetic analyses were similar, and the best scoring RAxML tree is illustrated here. The bootstrap support values above 50% of maximum likelihood analysis (ML) and Bayesian posterior probability scores (≥0.90) are noted at the nodes. The Diaporthales separates into 32 clades, representing 32 families, and the new isolates were clustered with a well-supported clade (ML/BI = 100/1) in Pseudomelanconidaceae. The two new isolates were different from any known genera in Pseudomelanconidaceae, and represented a new genus (Fig. 1). Ex-type strains are marked by an asterisk (*) and the strains from this study are in bold.  Description. Sexual morph: not observed. Asexual morph: Conidiomata acervular, conspicuous, immersed in host bark to erumpent, covered by brown to blackish exuding conidial masses at maturity. Central column beneath the disc more or less conical. Conidiophores unbranched, aseptate, cylindrical, pale brown, smooth-walled. Conidiogenous cells annellidic, occasionally with distinct annellations and collarettes.
Notes. Micromelanconis is the third genus after Neopseudomelanconis and Pseudomelanconis in the family Pseudomelanconidaceae (Fig. 1). Micromelanconis is united in this family based on the Melanconis-like conidiomata, and pale brown conidia with conspicuous hyaline sheath. Micromelanconis produces two types of conidia from natural branches and manual media respectively, which differs from Neopseudomelanconis and Pseudomelanconis Jiang et al. 2018a). Additionally, Neopseudomelanconis is characterized by its septate conidia (Jiang et al. 2018a). Etymology. Named after Kaihui Yang, a Chinese heroine; Kaihui is also the name of the town where holotype was collected.
Key to Pseudomelanconidaceae genera and species

Discussion
Diaporthales is a well-studied order based on integrated approaches of morphology and phylogeny in recent years (Castlebury et al. 2002;Rossman et al. 2007;Voglmayr and Jaklitsch 2014;Alvarez et al. 2016;Senanayake et al. 2017Senanayake et al. , 2018Voglmayr et al. 2017;Braun et al. 2018;Fan et al. 2018a;Jiang et al. 2020a). Thirty-two accepted families are monophyletic and supported by morphological characters; four of them contain Melanconis-like fungi, namely Juglanconidaceae, Melanconidaceae, Melanconiellaceae and Pseudomelanconidaceae ). The Melanconis-like fungi were similar in their asexual morph, but well-separated in the phylogeny and their hosts (Voglmayr et al. 2012(Voglmayr et al. , 2017Fan et al. 2018a, b;Jaklitsch and Voglmayr 2020). In the present study, a new genus and species were clustered in the family Pseudomelanconidaceae (Fig. 1), and differed from the other Melanconis-like genera by its long dumbbell-shaped conidia formed on PDA plates. Hosts are useful taxonomic information in some families of Diaporthales, such as Coryneaceae, Cryphonectriaceae, Erythrogloeaceae and Gnomoniaceae (Voglmayr et al. 2012;Jaklitsch and Voglmayr 2019;Roux et al. 2020;Wang et al. 2020;Yang et al. 2020). Hosts are important to separate Melanconis-like genera, Juglanconis inhabit Juglans and Pterocarya of Juglandaceae, Melanconiella and Melanconis occur only on the plant family Betulaceae (Voglmayr et al. 2012(Voglmayr et al. , 2017Fan et al. 2018b;Jaklitsch and Voglmayr 2020). Melanconis species are discovered only on Alnus and Betula, while Melanconiella occurs in the subfamily Coryloideae with the exception of M. betulae and M. decorahensis on Betula (Voglmayr et al. 2012;Du et al. 2017;Fan et al. 2018a). Species of Pseudomelanconidaceae inhabit Carya of Juglandaceae, and Castanea of Fagaceae Jiang et al. 2021). More interesting Melanconis-like may be revealed by more detailed surveys on tree-inhabiting fungi in the future.