Reassessment of Dyfrolomyces and Four New Species of Melomastia from Olive (Olea europaea) in Sichuan Province, China

Pleurotremataceae species are saprobes on decaying wood in terrestrial, mangrove, and freshwater habitats. The generic boundary of the family has traditionally been based on morphology. All genera of Pleurotremataceae have a high degree of morphological overlap, of which the generic circumscription of Melomastia and Dyfrolomyces has not been well resolved. Thus, the delimitation of genera has always been challenging. Melomastia traditionally differs from Dyfrolomyces in having 2-septate, oblong, with obtuse-ends ascospores. These main characteristics have been used to distinguish Melomastia from Dyfrolomyces for a long time. However, the above characteristics sometimes overlap among Dyfrolomyces and Melomastia species. Based on the morphology and multigene phylogeny with newly obtained data, we synonymized Dyfrolomyces under Melomastia following up-to-date results. Four novel species (i.e., Melomastia fusispora, M. oleae, M. sichuanensis and M. winteri) collected from the dead branches of Olea europaea L. in Chengdu Olive Base, Sichuan Province in China are introduced based on detailed morphological characterization and phylogenetic analyses of sequences based on nuclear ribosomal (LSU and SSU) and protein-coding gene (tef1-α). The 11 new combinations proposed are Melomastia aquatica (=Dyfrolomyces aquaticus), M. chromolaenae (=D. chromolaenae), M. distoseptata (=D. distoseptatus), M. mangrovei (=D. mangrovei), M. marinospora (=D. marinosporus), M. neothailandica (=D. neothailandicus), M. phetchaburiensis (=D. phetchaburiensis), M. sinensis (=D. sinensis), M. thailandica (=D. thailandica), M. thamplaensis (=D. thamplaensis) and M. tiomanensis (=D. tiomanensis).


Introduction
The family Pleurotremataceae was introduced by Watson [1] for the monotypic genus Pleurotrema Müll. Arg. with P. polysemum as the type of species. The family is characterized by a clypeus on the substrate, immersed ascomata and multi-septate ascospores, with or without a sheath in bitunicate/fissitunicate asci [1,2]. Barr assigned Pleurotremataceae to Xylariales and accepted five genera, viz. Daruvedia Dennis., Melomastia Nitschke ex Sacc., Phomatospora Sacc., Pleurotrema Müll. Arg. and Saccardoella Speg., based on their unitunicate and non-fissitunicate ascal characteristics [2]. Later, Daruvedia and Phomatospora were transferred to Pleosporales and Phomatosporales based on DNA sequence data, respectively [3,4]. Melomastia and Saccardoella were referred to Ascomycota genera incertae sedis [5,6], while Pleurotrema was retained in Pyrenulales [7]. The familial placement of Pleurotrema has been controversial due to the confusion over the nature of the asci, as they are neither typically unitunicate nor bitunicate [8,9]. Thus, Pleurotrema has been classified in the Xylariales [10], Pyrenulales [11] and Chaetosphaeriales [12]. The type of species P. polysemum was re-examined to confirm the placement of Pleurotrema [2,13]. The bitunicate, non-fissitunicate and not lichenized asci of Pleurotrema determined that it has morphological affinities to the genera Melomastia and Saccardoella. Based on the above evidence, Maharachchikumbura et al. [13] excluded Pleurotremataceae from Sordariomycetes and placed it in Dothideomycetes.
Pang et al. [20] established the family Dyfrolomycetaceae with the type of genus Dyfrolomyces to accommodate D. tiomanensis and accept three marine species transferred from Saccardoella (i.e., Saccardoella mangrovei, S. marinospora and S. rhizophorae). Subsequently, Dyfrolomycetaceae was raised as the order Dyfrolomycetales [21]. Maharachchikumbura et al. [13] synonymized Dyfrolomycetaceae under Pleurotremataceae based on morphological investigations. Dyfrolomyces is charactered by immersed, clypeate, globose ascomata, 8-spored, bitunicate, fissitunicate, cylindrical asci with relatively short pedicel, and overlapping uniseriate, fusiform, hyaline, multi-septate ascospore with or without a gelatinous sheath, which is very similar to Melomastia. However, the affiliation between these two genera is unclear due to the lack of sequence data of Melomastia species. With the description of Melomastia italica, Melomastia was revealed as closely related to Dyfrolomyces, and there was no significant support in the molecular phylogenies to differentiate Melomastia from Dyfrolomyces [18]. Presently, 11 Dyfrolomyces species have been accepted in the genus.
During a survey of micro-fungi on olive in Sichuan Province, China, we collected nine pleurotrema-like taxa from Olea europaea. Four new species, viz. Melomastia fusispora, M. oleae, M. sichuanensis and M. winteri were introduced. Moreover, we evaluated the morphology and phylogeny of accepted species in Dyfrolomyces and Melomastia, and synonymized Dyfrolomyces under Melomastia.

Isolation and Morphology
The dead branches of Olea europaea were collected from Chengdu Olive Base, Shuangliu District, Chengdu City, Sichuan Province, China, in 2020. The samples were transported to a laboratory in paper envelopes. Examination and observation of samples were carried out as detailed by Senanayake et al. [22]. Measurements were made with the Tarosoft (R) Image Framework program v. 0.9.7, following Liu et al. [23].
Single spore isolations were prepared following the method of Chomnunti et al. [24]. Germinating spores were transferred aseptically onto fresh potato dextrose agar (PDA) plates and incubated at room temperature in daylight. The strains isolated in this study were deposited at the China General Microbiological Culture Collection Center (CGMCC), Beijing, China and the University of Electronic Science and Technology Culture Collection (UESTCC), Chengdu, China. Herbarium specimens were deposited at Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (KUN-HKAS), Kunming, China and Herbarium, University of Electronic Science and Technology (HUEST), Chengdu, China.

DNA Extraction, PCR Amplification and Sequencing
Genomic DNA was extracted from 14d-old fungal colonies growing on PDA at 25 • C using the EZ geneTM fungal gDNA kit (GD2416) according to the manufacturer's instructions. Phylogenetic analyses were conducted using partial sequences of the large subunit rDNA (28S, LSU), small subunit rDNA (18S, SSU) and the translation elongation factor-1 alpha (tef1-α) using the primer pairs LR0R/LR5 [25], NS1/NS4 [26] and 983F/2218R [27], respectively. Polymerase chain reaction (PCR) amplifications were conducted in 25 µL reaction volumes. The PCR mixture consisted of 2× PCR MasterMix (12.5 µL) (Tsingke Co., China), genomic DNA (1 µL), each primer (1 µL) and ddH2O (9.5 µL). The PCR thermal cycle program for LSU, SSU and tef1-α amplification was performed using the following parameters: 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 50 s, elongation at 72 • C for 1 min and a final extension at 72 • C for 10 min, and finally kept at 4 • C in a thermal cycle. PCR amplification products were visualized on 1% agarose electrophoresis gels stained with GoldView. Purification and sequencing of PCR products were carried out at Beijing Tsingke Biological Engineering Technology and Services Co., Ltd. (Beijing, China).
Pang et al. [20] introduced Dyfrolomyces to accommodate species with immersed coriaceous ascomata with clypeated ostiolar neck, peridium composed of 2-zone, multicellular ascospores. The family Dyfrolomycetaceae was proposed based on the phylogenetic analyses of a multi-gene [20] to accommodate this single genus. Morphologically similar Melomastia species were not included in their analyses due to the limited availability of the sequence data, and thus the affiliation of these two genera has remained unclear.
Culture characteristics: Colonies on PDA reaching 15 mm diam. after 2 weeks at 25 • C. Cultures from above white, dense, circular, margin entire, papillate; reverse dark brown.
Culture characteristics: Colonies on PDA reaching 30 mm diam. after 4 weeks at 25 • C. Cultures from above, white, dense, circular, margin erose, umbonate, papillate with somewhat fluffy, pale orange at the center; reverse dark brown at the center, pale yellow at the edge.

Discussion
Species of Melomastia have a wide geographical distribution, e.g., Africa, China, Germany, Italy, Japan, Poland and the United States of America (California) [18], and are reported in marine, freshwater and terrestrial habitats [9,20,28]; they do not seem to have specific host preferences. Though a few species are known as saprophytes on mangrove wood (Avicennia marina, Kandelia, Rhizophora sp.), the rest are reported from various woody hosts (eg. Clematis sp., Chromolaena odorata and Vitis vinifera) [18]. This study provides the first report of Melomastia species reported from Olea europaea.
A well-resolved revision of the family Pleurotremataceae is challenging since the type species of the early established genera Melomastia and Pleurotrema do not possess molecular data. Norphanphoum et al. [18] proposed combining Dyfrolomyces and Pleurotrema under Melomastia based on similar phenotypic characteristics, and they suggested this taxonomic assumption needs to be reinforced via increasing the number of taxa with sequence data in each genus. In the present study, we have included the taxa representing all species of Pleurotremataceae (of which the sequences are available in GenBank) and nine pleurotrema-like taxa obtained from olive. Considering the morphological comparison ( Table 2) and multi-gene phylogenetic analysis (Figure 1), Melomastia and Dyfrolomyces are congeneric, and the epithet Melomastia should be adopted as it is the oldest name. Further studies with more collections of Pleurotremataceae, especially of Melomastia mastoidea and Pleurotrema polysemum are essential for a better understanding of the taxonomic relationship of this family.  The differences between the peridium of the ascomata and ascomatal position on the substrate probably play prominent roles in differentiating some fungal groups [56,57]. However, the results of this study suggest that it may also vary depending on the condition of habitats (terrestrial or aquatic environment) and substrates (herbaceous or woody plants), e.g., Melomastia produce fully immersed ascomata in an aquatic habitat (M. tiomanensis) and superficial or semi-erumpent ascomata in a terrestrial environment (e.g., M. fusispora, and M. sinensis) [45]; the peridium (perithecia) of the ascomata on woody plants is composed of 2-zone (e.g., M. oleae, M. sichuanensis), while those on herbaceous plants or semi-woody were 1-zone (e.g., M. chromolaenae, M. italica) [18,42]. Thus, the ascomatal position on the substrate and the zonation of the peridium of the ascomata have no taxonomic significance for generic delimitation in Pleurotremataceae, and could even change depending on the host and location.

Conclusions
This study comprises research investigating fungi associated with oil trees in Sichuan Province in China, and here we introduced four new species of Pleurotremataceae isolated from olive (Olea europaea). With detailed descriptions, Dyfrolomyces was synonymized under Melomastia based on molecular phylogeny and morphology. Olives are cultivated in many regions of the world with Mediterranean climates, such as Australia, Chile, Italy, Peru, South Africa and the USA (California and Oregon). This study represents the first discovery of Melomastia on the Olea europaea in China. Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.