Morphology and phylogeny reveal two novel Coryneum species from China

Abstract Coryneum is currently the sole genus of Coryneaceae in Diaporthales, distinguished from other diaporthalean genera by transversely distoseptate brown conidia. However, Coryneum species are presently difficult to identify because of variability and overlap of morphological characters and the lack of sequence data for most described species. During fungal collection trips in China, 13 Coryneum isolates were obtained from cankered branches of Ilex and Quercus. Morphological and phylogenetic analyses (ITS, LSU, TEF1-α and RPB2) revealed that these strains belong to two new species (viz. Coryneumilicissp. nov. and C.songshanensesp. nov.), and three known species, C.gigasporum, C.sinense, and C.suttonii. Coryneumilicis has larger conidia and more distosepta than most Coryneum species. Coryneumsongshanense was similar to C.sinense from the same host genus, Quercus, in conidial length, but distinct in conidial width and by molecular data.


Introduction
The genus Coryneum Nees is currently the only accepted genus in Coryneaceae and it forms a distinct phylogenetic lineage in Diaporthales , Voglmayr et al. 2017, Fan et al. 2018a, Jiang et al. 2018, Senwanna et al. 2018. The genus Coryneum was introduced based on the asexual morph, with C. umbonatum Nees as the type species (Nees von Esenbeck 1816), and the sexual morph Pseudovalsa Ces. & De Not. was introduced later, based on P. lanciformis (Fr.) Ces. & De Not. (Cesati & De Notaris 1863). Coryneum was recommended to be adopted due to priority and the need of fewer new combinations (Rossman et al. 2015).
Most Coryneum species were considered as phytopathogens, which were discovered from cankers and dieback of shoots and twigs (Wijayawardene et al. 2016, Jiang et al. 2018. However, diseases are commonly mild and only rarely cause serious symptoms in the hosts. Additionally, pathogenicity tests have not yet been conducted. Coryneum species are generally considered highly host-specific, and 28 species and a variety were accepted in this genus before this study (Sutton 1975, 1980, Wijayawardene et al. 2016, Jiang et al. 2018, Senwanna et al. 2018. Coryneum terrophilum was the only species isolated from soil, and the others were reported from dead branches (Table 1). Fagales species are the major hosts of Coryneum species, and host trees from other orders are also hardwoods with rough barks (Table 1).
Molecular phylogenies based on multi-gene loci including the internal transcribed spacer (ITS) and the large subunit (LSU) regions of the nuclear rDNA, translation elongation factor-1α (TEF1-α) and the second largest subunit of the RNA polymerase II (RPB2) have been widely used to infer species delimitation within many genera in Diaporthales (Voglmayr et al 2012, Voglmayr and Jaklitsch 2014, Fan et al. 2018b, Jiang et al. 2019, and are particularly important in speciose genera like Coryneum. Hence, DNA extraction from known species and fresh collections from the potential hosts will greatly improve the elucidation of species concept and circumscription in Coryneum. Thus, the main objectives of the present study were to identify Coryneum taxa based on morphology and phylogenetic evidence, and to analyse the relationships between Coryneum species and host genera.

Sample collection and isolation
Sample collection trips were conducted in Beijing, Hebei and Shaanxi Provinces of China during June to October in 2017 and 2018, aiming to collect fresh specimens with Coryneum-like taxa. Fagales plants were the main hosts and other hardwoods with rough barks were also investigated. Healthy branches and twigs were covered by green leaves, hence the dying and dead materials were conspicuous during our investigations. Asexual fruiting bodies were easily discovered as black spots on the host barks. Tree tissues with fruiting bodies were cut into small pieces, packed in paper bags and taken to the laboratory for further studies. Isolations were obtained by removing the ascospores or conidial masses from the fruiting bodies on to clean potato dextrose agar (PDA) plates, which were incubated at 25 °C until spores germinated. Single germinating spores were transferred on to new PDA plates, which were kept at 25 °C in the dark. Specimens were deposited at the Museum of the Beijing Forestry University (BJFC) and axenic cultures are maintained at the China Forestry Culture Collection Centre (CFCC).

Morphological analysis
Species identification was based on the morphological characters of the sexual and asexual morphs produced on natural substrates. Cross-sections were prepared manually using a double-edged blade under a Leica stereomicroscope (M205 FA). Photomicrographs 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 3.00. Measurements of ascospores and conidia are reported as the maximum and minimum in parentheses and the range representing the mean ± standard deviation of the number of measurements is given in parentheses (Voglmayr et al. 2017). Cultural characteristics of isolates incubated on MEA in the dark at 25 °C were recorded. Recognition and identification of Coryneum species were based on fruiting bodies formed on tree bark, supplied by conidiomata produced on PDA plates. Ascomata and conidiomata from tree bark were sectioned by hand using a double-edged blade, and conidiomata from PDA plates were picked using a needle, which were observed under a dissecting microscope. At least 10 conidiomata/ascomata, 10 asci, and 50 conidia/ascospores were measured to calculate the mean sizes and standard deviation. Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA plates using a modified CTAB method (Doyle and Doyle 1990). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer sets ITS1/ITS4 (White et al. 1990) were used to amplify the ITS region. The primer pair LR0R/LR5 (Vilgalys and Hester 1990) was used to amplify the LSU region. The primer pairs EF1-688F/EF1-986R or EF1-728F/TEF1-LLErev (Carbone and Kohn 1999, Jaklitsch et al. 2006, Alves et al. 2008) were used to amplify TEF1-α gene. The primer pair dRPB2-5f/dRPB2-7r  was used to amplify the RPB2 gene. The polymerase chain reaction (PCR) assay was conducted as described by Fan et al. (2018a). PCR amplification products were assayed via electrophoresis in 2 % agarose gels. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China). Novel sequences generated in the current study were deposited in GenBank (Table 2).
A partition homogeneity test with heuristic search and 1000 replicates was performed using PAUP v. 4.0b10 to assess incongruence among the ITS, LSU, TEF1-α, and RPB2 sequence datasets in reconstructing phylogenetic trees. MP analysis was run using a heuristic search option of 1000 search replicates with random-addition of sequences with a tree bisection and reconnection (TBR) algorithm; branches of zero length were collapsed (collapse = minbrlen), and all equally most parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). ML analysis was performed using 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 using a bootstrapping method of 1000 bootstrap replicates (Hillis and Bull 1993). The MP bootstrap analyses were done with the same settings as for the heuristic search, but with 10 rounds of heuristic search during each bootstrap replicate. Phylograms were shown using FigTree v. 1.4.3 (Rambaut 2016).

Phylogenetic analyses
The alignment based on the combined sequence dataset (ITS, LSU, TEF1-α, and RPB2) included 30 ingroup taxa and two outgroup taxa (Stilbospora macrosperma and Stegonsporium pyriforme), comprising 3544 characters in the aligned matrix. Of these, 2570 characters were constant, 267 variable characters were parsimony-uninformative and 706 characters were parsimony informative. The partition homogeneity test resulted in an insignificant value (level 95%), indicating that ITS, LSU, TEF1-α and RPB2 sequence dataset could be combined. The MP analysis resulted in 2 equally most parsimonious trees; the first tree (TL = 1624, CI = 0.784, RI = 0.822, RC = 0.645) is shown in Fig. 1. The two MP trees were identical, except for an interchanged position of C. ilicis and C. songshanense (not shown). Tree topology of the best tree revealed by the ML analyses was identical to that of the MP tree shown. The phylogram based on the four gene sequences showed that the accessions here studied represented 2 new and 3 known species in Coryneum (Fig. 1).
Culture characters. On PDA at 25 °C, colonies growing slowly and unevenly, reaching 70 mm diam. within 25 d, gradually becoming brownish dark grey in colour with scant cottony aerial mycelium, asexual morphs developed after 35 d.
Notes. Coryneum ilicis is the sole species known from the host genus Ilex; it can be easily recognised by host association and phylogeny (Fig. 1). Morphologically, conidia of Coryneum ilicis are larger and have more distosepta than in most of the other species (Table 1). Diagnosis. Coryneum songshanense can be distinguished from the morphologically similar C. sinense by its narrower conidia.
Etymology. Named after the mountain on which it was collected, Songshan Mountain.
Culture characters. On PDA at 25 °C, colonies growing slowly and unevenly, reaching 70 mm diam. within 30 d, gradually becoming brownish dark grey in colour with scant cottony aerial mycelium, asexual morphs developed after 40 d.

Discussion
In this study, fresh Coryneum specimens were collected in China and identified based on combined morphological amd molecular data. Additional accessions of three recently described Coryneum species, C. gigasporum, C. sinense, and C. suttonii (Jiang et al. 2018), were identified, with matching conidial characteristics and sequences (Fig.  1). The new species C. ilicis was discovered on Ilex pernyi (Aquifoliaceae, Sapindales), which represents a new host family and genus for Coryneum. Coryneum cesatii was reported from the same host order, Sapindales, on branches of Aesculus (Hippocastanaceae) (Sutton 1975). The second new species, Coryneum songshanense, was discovered on dead twigs of Quercus dentata (Fagaceae, Fagales). Host species belonging to Fagales show higher diversity of Coryneum species (Table 1), and it is likely that additional taxa will be discovered by molecular data, considering that in many regions suitable hosts have not yet been adequately studied.
However, most of the Coryneum species are lacking DNA sequences, thus species identification based on DNA sequence analyses is presently difficult. Hence, polyphasic approach, i.e. incorporating morphological characters (such as conidial sizes and numbers of distosepta), as well as host associations are important for species identification (Sutton 1975, 1980, Jiang et al. 2018. However, host identifications may be incorrect and many geographical areas remain insufficiently studied. In addition, the morphological characters often significantly overlap between species, which makes identifications solely by morphology challenging. Hence, studies based on the types of already described species and new collections from potential hosts are important to achieve a reliable species classification and circumscription within Coryneum.