Taxonomic circumscription of melanconis-like fungi causing canker disease in China

Abstract Melanconis-like species comprise latent fungal pathogens with a wide range of woody hosts. Taxonomy of these pathogens is difficult due to their uninformative descriptions and similar asexual morphology. Based on molecular phylogenies, many species of this group were placed in various families of Diaporthales. In this study, eight species of melanconis-like fungi were isolated from Betulaalbosinensis, B.platyphylla (Betulaceae), Cornuscontroversa (Cornaceae), Corylusmandshurica (Betulaceae) and Juglansregia (Juglandaceae) in China. These species were phylogenetically placed in three families of Diaporhthales, i.e. Juglanconisjuglandina, J.oblonga (Juglanconidaceae), Melanconiellabetulicolasp. nov., M.corylinasp. nov. (Melanconiellaceae), Melanconisbetulae, Ms.itoana, Ms.stilbostoma (Melanconidaceae) and one new genus, Sheathospora (Melanconiellaceae). Sheathospora is proposed to accommodate Melanconiellacornuta with conical and discrete pycnidia with aseptate, hyaline, cylindrical to ellipsoidal conidia with distinct hyaline sheath on branches of Cornuscontroversa. Combined analyses of ITS, LSU, CAL, RPB2 and TEF1-α sequence data were used to construct the molecular phylogeny. Additionally, we provided separate phylogenetic trees for three families (Juglanconidaceae, Melanconidaceae and Melanconiellaceae) to show the species distribution of melanconis-like fungi in China.


Introduction
Melanconium (Diaporthales) was introduced by Link (1809) from dead branches of Fagus with M. atrum Link as the generic type. Corda (1837) extended this genus to 28 species. Subsequently, the genera Melanconis Tul. & C. Tul. and Melanconiella Sacc. were described as sexual morphs of Melanconium (Wehmeyer 1937(Wehmeyer , 1941. Sutton (1980) summarised more than 200 binomials that have been described in Melanconium, whereas no generic revision is available due to the uninformative descriptions and illustrations, few morphological characteristics, misplacement or poor condition of original specimens and lacking of ex-type cultures. In the Index Fungorum (2018), there are more than 235 species epithets of Melanconium with an estimated 50 species epithets by Kirk et al. (2008). Thus Melanconium species has serious obstacles for appropriate interpretation and is phylogenetically distributed throughout the Diaporthales, especially in Juglanconidaceae, Melanconidaceae and Melanconiellaceae. Although the genus Melanconium may be synonymous with Melanconis and would therefore have priority, the true identity of the generic type, M. atrum, is obscure and it was recommended to protect Melanconis over Melanconium (Rossman et al. 2015).
During trips to collect forest pathogens that cause canker or dieback diseases in China, several melanconis-like taxa associated with various disease symptoms were collected in Beijing, Gansu, Heilongjian, Jilin, Ningxia, Shaanxi and Tibet Provinces. As the higher-level phylogeny of many genera within the melanconis-like taxa remains largely unresolved in China, this project was initiated to address this issue. In this paper, we identified eight melanconis-like species residing in three families of Diaporthales; recognised three genera within Melanoconiellaceae; and described two new species in Melanconiella as well as one new genus to accommodate Melanconiella cornuta.

Isolation
Fresh specimens of melanconis-like fungi were collected from infected branches of seven hosts during collection trips in China (Table 1). A total of 47 isolates were established by removing a mucoid spore mass from ascomata or conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) and incubating at 25 °C for up to 24 h. Single germinating conidia/ascospores were removed and plated on to fresh PDA plates. Specimens and isolates were deposited in the Key Laboratory for Silviculture and Conservation of the Ministry of Education in the Beijing Forestry University (BJFU) and the working Collection of X.L. Fan (CF) housed at the BJFU. Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological studies
Species identification was based on morphological features of the ascomata or conidiomata produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Cross-sections were prepared by hand using a double-edge blade under a dissecting microscope. More than 10 conidiomata/ascomata, 10 asci and/or 50 conidia/ascospores were measured to calculate the mean size and standard deviation (SD). Microscopic photographs were captured with a Nikon Eclipse 80i microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination and the Nikon software NIS-Elements D Package v. 3.00. Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004). Colony diameters were measured and the colony colours described after 3 weeks according to the colour charts of Rayner (1970).

Phylogenetic analyses
DNA sequences generated by each primer combination were used to obtain consensus sequences using SeqMan v. 7.1.0 in the DNASTAR Lasergene Core Suite software package (DNASTAR Inc., Madison, WI, USA). Reference sequences were selected based on ex-type or ex-epitype sequences available from relevant published literature (Voglmayr et al. 2012, Fan et al. 2016, Du et al. 2017, Senanayake et al. 2017) (Table 1). All sequences were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/ alignment/server/index.html) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for maximum parsimony (MP) analysis (Swofford 2003), MrBayes v. 3.1.2 for Bayesian Inference (BI) analysis (Ronquist and Huelsenbeck 2003) and PhyML v. 7.2.8 for Maximum Likelihood (ML) analysis (Guindon et al. 2010). The first analyses were performed on the combined multi-gene dataset (ITS, LSU, RPB2, TEF1-α) to compare isolates of Diaporthales species to ex-type sequence data from recent studies (Table 1). A partition homogeneity test (PHT) with heuristic search and 1 000 search replicates was performed using PAUP to test for incongruence amongst the ITS, LSU, RPB2 and TEF1-α sequence datasets in reconstructing phylogenetic trees. Maximum parsimony (MP) analysis was run using 1 000 heuristic search replicates with randomadditions of sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5 000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). Maximum likelihood (ML) analysis was performed with a GTR site substitution model, including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated with a bootstrapping (BS) method of 1 000 replicates.
MrModeltest v. 2.3 was used to estimate the best nucleotide substitution model settings for each gene (Posada and Crandall 1998). Bayesian inference (BI) was performed based on the DNA dataset from the results of the MrModeltest, using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). Two MCMC chains were run from random trees for 1 000 M generations and stopped when the average standard deviation of split frequencies fell below 0.01. Trees were saved each 1 000 generations. The first 25% of trees were discarded as the burn-in phase of each analysis and the posterior probabilities (BPP) were calculated from the remaining trees (Rannala and Yang 1996).
In addition to the above analyses, we provided separate phylogenetic trees for Juglanconidaceae, Melanconidaceae and Melanconiellaceae, based on various gene regions (see below) and the same analyses parameters as given above. Phylograms were edited using FigTree v. 1.3.1 (Rambaut and Drummond 2010). Novel sequences generated in the current study were deposited in GenBank (Table 1). The aligned matrices used for phylogenetic analyses and the resulting trees can be found in TreeBASE (www. treebase.org; accession number: S23477).

Phylogenetic analyses
The combined matrix of ITS, LSU, RPB2 and TEF1-α of Diaporthales included 209 ingroup and two outgroup taxa, comprising 3 269 characters including gaps (776 characters for ITS, 517 for LSU, 1107 for RPB2 and 869 for TEF1-α) in the aligned matrix. Of these, 1 417 characters were constant, 192 variable characters were parsimony-uninformative and 1 660 characters were parsimony informative. The MP analysis resulted in 100 most parsimonious trees (TL = 10 370, CI = 0.341, RI = 0.806, RC = 0.275) and the first tree is shown as Fig. 1. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Branches with significant Bayesian posterior probability (≥ 0.95) in Bayesian analyses were thickened in the phylogenetic tree. The phylogram based on four genes resolved 28 known lineages, representing 26 known families and two incertae sedis genera Diaporthella and Phaeoappendispora due to lack of sequence data on their types. The current 47 melanconislike isolates are herein placed within Juglanconidaceae, Melanconidaceae and Melanconiellaceae in Diaporthales (Fig. 1). A phylogenetic tree of each family or genus was constructed separately based on different DNA datasets. Tree topologies of all genera computed from the MP, ML and Bayesian analyses were similar for the individual gene region and in the combined dataset.
For the single genus Juglanconis (Juglanconidaceae), a combined ITS, LSU, CAL and RPB2 matrix of 23 ingroup accessions (five from this study and 18 retrieved from  GenBank) was produced, which comprised 2 736 characters including gaps (2 427 constant, 216 variable and parsimony-uninformative, 93 parsimony-informative). A heuristic MP search generated nine equally most parsimonious trees (TL = 332, CI = 0.976, RI = 0.985, RC = 0.961), one of which is shown in Fig. 2. Isolates of Juglanconis clustered in four clades, corresponding to the four known species in this genus. The five Chinese strains sequenced in this study were revealed to belong to Juglanconis juglandina (3) and J. oblonga (2).
Culture characteristics. On PDA, cultures are initially white, becoming straw after 3-5 d and grey olivaceous after 7-10 d. The colonies are felty with an irregular edge; sterile.
Notes. Juglanconis oblonga is based on Melanconium oblongum (= Melanconis juglandis) (Voglmayr et al. 2017). This species can be distinguished from J. juglandina by on average longer length of conidia (22 × 12.5 vs. 20 × 13 μm). However, there is a substantial size overlap between both species and sequence data are sometimes necessary for reliable species identification. It was also recorded to cause canker and dieback disease of Juglans regia in China (China Microbiology and Virology Databases, http:// www.micro.csdb.cn/).  (1863) Notes. Melanconidaceae was introduced by Winter (1886) and subsequently involved many genera with perithecia immersed in a well-developed stroma with ostioles (beaks) that emerge through an ectostromatic disc (Barr 1978). Castlebury et al. (2002) and Rossman et al. (2007) reduced this family to the type genus Melanconis based on LSU rDNA sequences. In this paper, we provide an updated tree with additional isolates of Melanconis (Melanconidaceae) from China (Fig. 5). All species have been described and illustrated by Fan et al. (2016).  (1863) Notes. The type genus Melanconis was established by Tulasne and Tulasne (1863) based on Sphaeria stilbostoma Fr. This genus is characterised by circularly arranged perithecia immersed in well developed to reduced entostromata with a concolourous central column and ostioles erumpent through a light-coloured ectostromatic disc with hyaline, one-septate ascospores; acervuli with light-coloured central column producing brown to olive-brown, fusiform to pyriform alpha conidia and hyaline, cylindrical or allantoid beta conidia (Barr 1978;Castlebury et al. 2002;Voglmayr et al. 2012;Fan et al. 2016). Melanconis has approximately 105 species epithets recorded in Index Fungorum (2018), whereas Rossman et al. (2007) suggested that many of the species previously residing in Melanconis may belong somewhere else. Fan et al. (2016) provided an account on this genus including five species (Melanconis alni, Ms. betulae, Ms. marginalis, Ms. itoana and the type species Ms. stilbostoma), which were restricted to hosts in Betulaceae.  Table 1.

Melanconis
Notes. Melanconis stilbostoma is the type species of Melanconis and is thus far only known to occur on Betula spp. with a worldwide distribution (Fan et al. 2016). Betula pendula, B. rotundifolia and B. tianschanica are recorded as hosts in China (Zhuang 2005). The current investigation suggested that this species is restricted to and widespread on Betula platyphylla in China.  (Wehmeyer 1937(Wehmeyer , 1941Barr 1987). Melanconiella has 37 species epithets recorded in Index Fungorum (2018). Voglmayr et al. (2012) revised the generic circumscriptions of Melanconiella with 13 accepted species, excluded numerous species and confirmed that it is genetically distinct from the genus Melanconis based on morphology and multi-gene phylogeny (ITS,LSU,. Melanconiella is characterised by forming circularly arranged perithecia immersed in the substrate with oblique or lateral ostioles convergent and erumpent through an ectostromatic disc with dark coloured or hyaline ascospores; acervuli with light-coloured central column, producing dark brown melanconium-like or hyaline discosporina-like conidia (not in the same species) (Barr 1978; Figure 6. Phylogram of Melanconiellaceae obtained from an MP analysis from a combined matrix of ITS, LSU, RPB2 and TEF1-α. MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 80 changes. Type species are in bold. Strains obtained in the current study are in blue. Voglmayr et al. 2012). Melanconiella species were observed to be highly host-specific, as they were found to be confined to a single genus or sometimes even species within the host family Betulaceae from Europe and North America (Voglmayr et al. 2012). Fig. 7 Etymology. betulicola (Lat.): referring to the host genus on which it was collected, Betula.
Culture characteristics. On PDA, cultures are initially white, becoming greyishsepia after 3 d and distensible radially after 10 d. The colonies are felty with an irregular edge; texture uniform; sterile.
Additional material examined. CHINA. Shaanxi Province: Ningshan County,Huoditang Forest Farm,Huodi Valley,33°26'37.53"N,108°26'44.14"E, 3 August 2015, on twigs and branches of Betula albosinensis, Q. Yang (CF 20150847; living culture, CFCC 52483); Notes. Melanconiella betulicola is associated with canker disease of Betula albosinensis in China. It is similar to M. ellisii but differs by larger ascospores (18-22 × 4-6 vs. 12.5-16 × 4.0-5.5 μm) with hyaline, broad cap-like appendages at both ends (Voglmayr et al. 2012), distribution (China vs. eastern North America) and a different host, Betula albosinensis vs. Carpinus caroliniana. Melanconiella decorahensis also occurs on Betula (in Europe and North America) and it can be distinguished from M. betulicola based on dark brown ascospores without appendages and dark brown conidia (Vogl-mayr et al. 2012). The clear phylogenetic position confirmed a distinction from all other available strains included in this study and we therefore result in our decision to describe this species as new, based on DNA sequence data and morphology.
Culture characteristics. On PDA, cultures are initially white, becoming fuscous black in the centre and edge after 5 d. The colonies are felty with an irregular edge; texture uniform; sterile.
Culture characteristics. Colony growth on PDA originally white, becoming pale yellowish after 7-10 days. Colony flat, felty-like, with a uniform texture and yellowish to dark brown conidiomata irregularly scattered on the colony surface.
Additional specimens examined (paratypes). CHINA. Shaanxi Province: Ankang City, Ningshan County, Huoditang Forest Farm, 36°26'13.30"N, 108°26'48.32"E, 3 August 2015, on twigs and branches of Juglans regia, Q. Yang (BJFC-S1345 paratype; living ex-paratype culture CFCC 51991). Notes. Sheathospora cornuta is proposed here as a new combination for Melanconiella cornuta. It is the type and currently only species of Sheathospora and so far known from Cornus controversa and Juglans regia in China. The sexual morph of this species is unknown and further collections are required to elucidate its life cycle.

Discussion
During the investigation of melanconis-like fungi in China, we identified eight species residing in three families (Juglanconidaceae, Melanconidaceae and Melanconiellaceae) of Diaporthales. It includes Juglanconis juglandina, J. oblonga, Melanconis betulae, Ms. itoana, Ms. stilbostoma, the two new species Melanconiella betulicola and M. corylina and the new combination Sheathospora cornuta in the new genus Sheathospora.
All specimens in the current study were collected from symptomatic branches and twigs associated with canker or dieback diseases, of which Juglanconis (Juglanconidaceae) species were isolated from Juglans regia (Juglandaceae), Melanconiella (Melanconiellaceae) species from Betula albosinensis and Corylus mandshurica (Betulaceae) and Melanconis (Melanconidaceae) species from Betula albosinensis and Betula platyphylla (Betulaceae). It may indicate that many melanconis-like species have obvious host specificity. The type species of the new genus Sheathospora (Melanconiellaceae) was isolated from Cornaceae (Cornus controversa) and Juglans regia (Juglandaceae), suggesting a low host specificity and that additional undiscovered hosts species of this taxon may exist in China.
As the morphological features in previous melanconis-like fungi are highly overlapping, phylogenetic studies using DNA sequences have been useful to elucidate the diversity and systematics in this group. The current results indicated that Juglanconis and Melanconis are still unique, the only genera in Juglanconidaceae and Melanconidaceae, respectively, due to the lacking of extensive fresh collections. The family Melanconiellaceae was recently proposed by Senanayake et al. (2017) to accommodate Dicarpella, Greeneria, Melanconiella, Microascospora and Tubakia based on morphological features and phylogenetic analyses. In this study, the phylogenetic affinity of Dicarpella, Greeneria and Tubakia was evaluated in Diaporthales (Fig. 1), which conformed to the recently described family Tubakiaceae (Diaporthales) (Braun et al. 2018). We here establish a new genus within Melanconiellaceae, Sheathospora, which is characterised by typical diaporthalean-like pycnidia and aseptate, cylindrical to ellipsoidal conidia with distinct hyaline sheath. Thus Melanconiellaceae is here restricted to the three genera Melanconiella, Microascospora and Sheathospora (Fig. 6).
As shown in this paper, future studies addressing the fungal diversity associated with canker or dieback diseases should routinely include sequence data for proteincoding genes to achieve stable, supported topologies in phylogenetic trees. It is hoped that the classification proposed here will also provide an updated phylogenetic framework that will facilitate further revision of the families with melanconis-like asexual morphs. Although the current study provides additional new data on melanconis-like genera, typification, species concept and taxonomic affiliation of many described Melanconium species are yet unclear, including the type species M. atrum, which currently represents a doubtful taxon (Rossman et al. 2015). In addition, sequence data are missing for most described Melanconium species. Thus, a thorough revision of the genus Melanconium based on robust sampling, reliable identification, cultures and DNA data is urgently needed. The fact that new records and species from three related families of Diaporthales were recorded in China further suggests that Asia may harbour many more species awaiting collections and descriptions. 2017YFD0600105). All authors want to thank the Experimental Teaching Centre (College of Forestry, Beijing Forestry University) for providing installed scientific equipments during the whole process.