﻿A new study of Nagrajomyces: with two new species proposed and taxonomic status inferred by phylogenetic methods

﻿Abstract Nagrajomyces (incertae sedis, Ascomycota) is a monotypic genus with a previously unknown systematic position. In this report, two new species are proposed, Nagrajomycesfusiformis and Nagrajomyceslaojunshanensis. These new taxa are proposed based on morphological characteristics evident via light microscopy and molecular data. Multi-locus phylogenetic analyses (ITS rDNA, nrLSU rDNA, RPB2, and TEF1-α) show that specimens recently collected in Yunnan Province, China are closely related to Gnomoniaceae. Both new species and known species were discovered repeatedly in their asexual developmental form exclusively on twigs of Rhododendron spp. (Ericaceae). This indicates a host specificity of Nagrajomyces spp. for species of Rhododendron.


Introduction
Gnomoniaceae is a distinct family of Diaporthales, established by Winter (1886). The traditional classification of species in Gnomoniaceae was mainly based on morphological features, such as the morphology of ascomata and ascospores as well as the position of necks (Barr 1978;Monod 1983). Sogonov et al. (2008) used phylogenetic analyses of molecular sequence data of several genes (TEF1-α, nrLSU, and RPB2) to revise the concepts of leaf-inhabiting genera, and discovered that several traditional genera in Gnomoniaceae are polyphyletic. Phylogenetic analyses indicate that host specificity can be used to circumscribe genera and species of Gnomoniaceae. Based on phylogenetic analyses and morphological characteristics, Senanayake et al. (2017) described new taxa and excluded some genera from Gnomoniaceae. Since then, additional genera have been introduced for species observed in sexual developmental stages, as well as those that have only been observed as pycnidial asexual morphs, and rarely for species known in both sexual and asexual forms (Senanayake et al. 2018;Crous et al. 2019;Minoshima et al. 2019;Yang et al. 2020).
Many species of Gnomoniaceae are important plant pathogens, such as Apiognomonia errabunda (Roberge ex Desm.) Höhn, which causes oak anthracnose (Sogonov et al. 2007), Gnomoniopsis fructicola (G. Arnaud) Sogonov, which causes strawberry stem rot (Maas 1998), and Ophiognomonia leptostyla (Fr.) Sogonov, which causes walnut anthracnose (Neely and Black 1976). Species of Gnomoniaceae can also have wide host ranges, with species on Fagaceae, Onagraceae, and Rosaceae being frequently infected. Species of Rhododendron (Ericaceae), which is the largest genus of woody plants in the northern hemisphere, are also known hosts of species of Gnomoniaceae (Monod 1983).
In the present study, two new species were discovered on twigs of Rhododendron spp. in Yunnan and assigned to the genus Nagrajomyces based on morphological characteristics, habitat, and host. Phylogenetic analysis revealed that the proposed Nagrajomyces species belong to Gnomoniaceae.

Specimen collections and isolation
Fieldwork for the discovery of fungi was conducted during June 2021 in Yunnan Province, China. Fresh pycnidia were repeatedly discovered and collected on twigs of Rhododendron spp. Twigs with conidiomata were packed in paper bags and transported to the laboratory for morphological tests. Conidiomata were cut off in the laboratory using a razor blade, wrapped in paper packets, disinfected with 75% ethanol for 10 s, then 10% sodium hypochlorite for 2 min 30 s, and rinsed with distilled water three times. After absorbing the water with sterile filter paper, the conidiomata were transferred to potato dextrose agar (PDA) plates (Jiang et al. 2021) then incubated at 25 °C to obtain cultures. Dry specimens were deposited at the China Forest Biodiversity Museum of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn) and the Herbarium of the College of Life Science, Capital Normal University (BJTC; http://smkxxy.cnu.edu.cn). Ex-type living cultures were deposited at the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/).

Morphological analysis
Conidiomata were photographed and cut by hand using a razor blade under a Nikon SMZ-1000 stereomicroscope (Japan). Morphological characteristics of conidiomata, conidiophores, and conidia were photographed and measured with an Olympus EX-51 upright microscope (Japan), and for each structure at least 20 measurements were made. Color values were taken from ColorHexa (https://www.colorhexa.com/).

DNA extraction, polymerase chain reaction amplification, and phylogeny
Genomic DNA was extracted from specimens and cultures via the M5 Plant Genomic DNA Kit (Mei5 Biotechnology Co., Ltd., China) in accordance with the manufacturer's instructions. Table 1 summarizes the primers used to obtain sequence data for ITS rDNA, nrLSU rDNA, RPB2, and TEF1-α, and the polymerase chain reaction (PCR) amplification protocols. PCR products were analyzed in 1% electrophoretic agarose gel with a 200-bp DNA ladder, purified, and sequenced by Beijing Zhongke Xilin Biotechnology Co., Ltd. (Beijing, China). SeqMan was used to align the sequences obtained by forward and reverse primers to obtain a consensus sequence. A partition homogeneity test was performed to determine the congruence of the four datasets (Farris et al. 1994). Sequences for phylogenetic analyses were selected based on Yang et al. (2020), supplemented by sequences of Apiosporopsis carpinea (Fr.) Mariani, Apiosporopsis sp., Juglanconis juglandina (Kunze) Voglmayr & Jaklitsch, Juglanconis oblonga (Berk.) Voglmayr & Jaklitsch, and Melanconis marginalis (Peck) Wehm. from Senanayake et al. (2018) used as outgroup taxa. All sequences used in this study are listed in Table 2. Subsequent alignments were generated with online MAFFT tools fRPB2-5F/fRPB2-7cR Liu et al. (1999) Liu et al. (1999 (Nylander 2004). Analyses of four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 100,000,000 generations, and other operational methods were applied used as described by Guo et al. (2021). The maximum parsimony (MP) tree was constructed using PAUP version 4.0 beta 10 (Swofford 2003) with 1000 random sequence additions, 1000 maxtrees were obtained, and bootstrap analysis was conducted based on 1000 replicates, with 10 replicates of random stepwise additions of taxa. For further details see Guo et al. (2021). Trees were viewed via Treeview (Page 1996).

Phylogenetic analysis
Multi-locus phylogenetic analyses of species of Gnomoniaceae (Diaporthales) include sequences of 51 ingroup taxa and sequences of an outgroup formed by Apiosporopsis carpinea, Apiosporopsis sp., Juglanconis juglandina, J. oblonga, and Melanconis marginalis (Fig. 1). The multi-locus dataset (ITS rDNA, LSU rDNA, RPB2 and TEF1-α) comprises 2875 characters, of which 945 are parsimony-informative, 200 are parsimony-uninformative and 1730 are constant. Maximum parsimony analysis of sequences resulted in one most parsimonious tree with a length (TL) of 3730 steps, a consistency index (CI) of 0.463, a retention index (RI) of 0.690, and a homoplasy index (HI) of 0.537. Bayesian and maximum likelihood trees exhibited topologies similar to this parsimony tree. The topology of the phylogenetic tree obtained in the current study was similar to the topology presented by Yang et al. (2020). Nineteen sequences of five specimens recently collected on Rhododendron spp. in China form a clade with high support values. This clade is sister to sequences of species of Siroccocus and Neognomoniopsis, but with poor support values. The newly discovered clade is divided into two small subclades labeled Nagrajomyces fusiformis and N. laojunshanensis. Diagnosis. This new species differs from N. dictyosporus and N. laojunshanensis by fusoid to elongate-fusoid conidia with pointed ends, usually 1-septate and smaller.
Culture characteristics. Cultures (ex-type CFCC 58177) on PDA 8 cm diameter after 1 month, with irregular margins, sparse aerial mycelium, colonies with whitish margins, with center turning black olive (#3b3c36) with increasing age. On MEA, 5.7 cm diameter after 1 month, with irregular margins, colonies with beaver (#9f8170) -colored margins, with center turning black olive (#3b3c36) with increasing age. Conidia not observed.

Discussion
Morphologically, the most distinctive features of the new species of Nagrajomyces are septate conidia with long, single, apical appendages. The presence of this structure distinguishes them from all anamorphic genera known to belong to Gnomoniaceae. Both new species proposed in the present study and the known species N. dictyosporus inhabit twigs of Rhododendron. In spite of the absence of molecular data for the type species of Nagrajomyces, these two new species are accommodated in Nagrajomyces based on significant morphological features (distinctive conidia) and identical ecology.
Many coelomycetous genera have conidia with appendages (Nag Raj 1993), and some of them share morphological characteristics with the new species proposed in this study. For example, species of Uniseta and Urohendersonia have septate conidia and a long apical appendage attached to each conidium. Uniseta is a monotypic genus typified by U. flagellifera (Ellis & Everh.) Ciccar. (Ciccarone 1947). Nag Raj (1974Raj ( , 1993  mentioned that U. flagellifera has a sexual morph called Cryptodiaporthe comptoniae (Schwein.) Barr (Barr 1991, syn. C. aubertii var. comptoniae (Schwein.) Wehm.) that is considered a synonym of Cryptodiaporthe aubertii (Westend.) Wehm. (Wehmeyer 1933). Conidia of this species are two-celled, hyaline, relatively inequilateral or curved, and bear a long flagellate appendage at one end (Wehmeyer 1933). Cryptodiaporthe acerina J. Reid & Cain and C. aubertii are included in the phylogenetic tree (Fig. 1) and located distant from the new species proposed herein. Furthermore, U. flagellifera differs from the new species proposed here by an asexual morph growing on branches of Comptonia asplenifolia damaged by fire (Ellis and Everhart 1889), while Nagrajomyces spp. develop on twigs of Rhododendron. Because of these differences, we consider the genus Uniseta to be separate from the genus Nagrajomyces. Spegazzini (1902) introduced Urohendersonia Speg. with Ur. platensis Speg. as the type species. Nag Raj (1993) listed only five species in this genus, including the type species. Urohendersonia spp. differ from Nagrajomyces spp. by having globose to subglobose conidiomata immersed in host tissues, and yellowish brown to brown conidia each with an extracellular gelatinous appendage, and their host species (Nag Raj 1993). Urohendersonia spp. occur on diverse host species and various substrates of host, such as on leaves of Erythrina sp., Manihot carthagenensis, Pongamia pinnnata, Stipa spartea, or in the rhizospheres of Acerva persica and Dactyloctenium aegyptium (Nag Raj 1993;Wijayawardene et al. 2016). Unfortunately, there are no molecular sequence data available for any species within those genera.
In the phylogenetic analysis presented herein, the two new species, N. fusiformis and N. laojunshanensis form a clade with high support values, which is separate from other species of Gnomoniaceae represented by sequence data in GenBank. These two new species described in this study fill gaps in the molecular data of Nagrajomyces and also enable the taxonomic status of the new species to be determined.
A total of 38 genera are currently included in the family Gnomoniaceae based on morphological and molecular analyses (Senanayake et al. 2018;Crous et al. 2019;Jiang et al. 2019;Minoshima et al. 2019;Yang et al. 2020). Sexual morphs have been described for all but four; Asteroma, Flavignomonia, Millerburtonia, and Sirococcus. Sirococcus spp. are closely related to the new species described herein, whereas phylogenetic data indicate that the other three genera are distantly related to Nagrajomyces spp. Asteroma spp. have cylindrical to fusiform, acicular or broadly fusiform conidia (Senanayake et al. 2018). Conidia of Flavignomonia are cylindrical to oblong , and conidia of Millerburtonia are filiform and aciculate (Ciferri 1951).
In addition to morphological characteristics and molecular sequence data, host ranges are often useful to delineate genera and species of Gnomoniaceae . Species of Gnomonia, for example, are generally associated with host plants in the Betulaceae family, mostly belonging to the subfamily Coryloideae ). The two new species identified in the present study, and the known species, all develop on twigs of Rhododendron spp. indicating that they are specialized with respect to this host. The differences in conidiomatal structure could be explained by differences in host epidermal features or maturity. Two species of Gnomoniaceae are known to inhabit Rhododendron spp. Plagiostoma rhododendri (Auersw.) Sogonov was reported on dry twigs and inflorescences of Rhododendron hirsutum L., and occasionally on dead leaves of R. ferrugineum L. (Monod, 1983). Only the sexual form of this species has been described, and phylogenetic analysis places it somewhat distant to species of Nagrajomyces (Fig. 1). The second species is Gnomonia sp., reported on rotten leaves of R. ferrugineum (Rehm 1906). This species lacks a specific morphological description.
Rhododendron is the largest genus of woody plants in the northern hemisphere, and its species diversity is highest in the Himalaya-Hengduan Mountains and Southeast Asia (Chamberlain et al. 1996;Shrestha et al. 2018). Considering the host preference of Gnomoniaceae species and the biodiversity of Rhododendron worldwide, additional Gnomoniaceae species are expected to exist on these plants.