Three new species of Dicephalospora from China as revealed by morphological and molecular evidences

Abstract Three new species of Dicephalospora are introduced based on morphological characters and DNA sequence analyses (maximum parsimony and neighbor-joining methods), viz. D.albolutea, D.shennongjiana, and D.yunnanica. All of them lack mucilaginous caps at ascospore poles. Dicephalosporaalbolutea is distinguished by cream to yellowish white apothecia and slightly curved ascospores. Dicephalosporashennongjiana is characterized by yellow apothecia, elliptical-fusoid ascospores 19−22 × 7−8.8 μm, and J+ asci 130−150 × 14−16.5 μm. Dicephalosporayunnanica is distinguished by orange apothecia and fusoid ascospores 16.5−25.3 × 3.3−3.5 μm. Descriptions and illustrations of the new species as well as a key to the known species in the genus are provided.


Introduction
Dicephalospora Spooner is a small genus established by Spooner (1987) with D. calochroa (Syd. & P. Syd.) Spooner as the type species. The poles of ascospores with a mucilaginous cap and J+ asci were treated as two important features to delimitate the genus, but a later study proved they are not reliable features at the generic level (Zhuang et al. 2016). The emended diagnostic characters of the genus are that apothecia erumpent or superficial, stipitate, yellow, orange, red to blackish, ectal excipulum of textura prismatica with refractive walls, medullary excipulum of textura intricata, asci J+ or J-in Melzer's reagent, ascospores hyaline, subellipsoid to fusoid, guttulate, poles either with a mucilaginous cap or not, paraphyses filiform, straight or slightly curved at apex, and occurring on rotten wood, twigs, and leaf petioles (Zhuang et al. 2016). The genus was once treated as a member of Rutstroemiaceae (Kirk et al. 2008), Helotiaceae (Wijayawardene et al. , 2018, or Sclerotiniaceae (Index Fungorum 2019). Including Dicephalospora in Helotiaceae is more reasonable in view of the phylogenetic studies of related groups in recent years (Han et al. 2014;Zhao et al. 2016). Zhuang et al. (2016) carried out a comprehensive study on taxonomy of Dicephalospora in China and provided a key to the known species of the genus. Approximately, 10 species are currently accepted in the genus and nine of them have been found in China (Zhuang 1995a(Zhuang , 1995b(Zhuang , 1999Verkley 2004;Zhuang et al. 2016). Dicephalosterol was discovered from the culture of D. rufocornea (Hosoya et al. 1999). This compound is a new testosterone 5α-reductase inhibitor and has a potential to be developed as a drug to prevent and cure prostatic hypertrophy (Hosoya et al. 1999). Additional information about utilization of the Dicephalospora spp. was rarely published maybe due to the minimal biomass in nature, difficulty of getting pure culture, and slow-growth if cultured.
During the examinations of helotialean fungi from China, three species fit well with the emended generic concept of Dicephalospora (Zhuang et al. 2016). However, new collections are found to differ from hitherto known species of Dicephalospora. To confirm their affinities and investigate their relationships with other species, phylogenetic analyses were conducted based on the internal transcribed spacers and 5.8S of nuclear ribosomal DNA (ITS).The results support their placement within the genus and their distinctions from any known species.

Materials and methods
Specimens were collected, recorded, and photographed by a Canon PowerShot G16 digital camera in the field. Descriptions of gross morphology and substrate were according to field notes and photos. Dried apothecia were rehydrated with distilled water and sectioned at a thickness of 15−20 μm with a Yidi YD-1508A freezing microtome (Jinhua, China). Measurements were taken from longitudinal sections and squash mounts in lacto-phenol cotton blue solution using an Olympus BH-2 microscope (Tokyo, Japan). Iodine reactions of ascal apparatus were tested with or without 3% KOH solution pretreatment in Melzer's reagent and Lugol's solution (Baral 2009). Microscopic images were taken using a Canon G5 digital camera (Tokyo, Japan) attached to a Zeiss Axioskop 2 Plus microscope (Göttingen, Germany). Voucher specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). Names of the new species were formally registered in the database Fungal Names (http://www. fungalinfo.net/fungalname/fungalname.html).
Pure cultures were obtained from some specimens following the method provided by Webster and Weber (2001) and preserved in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences.
Genomic DNA was extracted from dried apothecia or pure culture, using Plant Genomic DNA Kit (TIANGEN Biotech. Co., Beijing, China). ITS region was amplified and sequenced using the primer pair ITS1/ITS4 (White et al. 1990). PCR reactions had a final volume of 30 μl, containing 15 μl 2×Taq MasterMix (Beijing CWBiotech, China), 1.5 μl of each primer (10 mM), 2 μl DNA, and 10 μl deionized water. PCR reactions were carried out in an Applied Biosystems 2720 thermocycler (Foster City, CA, USA) under the following conditions: 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 53 °C for 30 s and 30 s at 72 °C, and a final extension of 72 °C for 10 min. The PCR products were purified and sequenced at Beijing Tianyi Huiyuan Bioscience and Technology, China. Newly generated sequences were assembled and edited using BioEdit 7.0.5.3 (Hall 1999) or SeqMan (DNASTAR, Lasergene 7.1.0). The new sequences were deposited in GenBank and additional sequences were downloaded from GenBank (Table 1).
Lachnum pygmaeum (Fr.) Bres. and L. spartinae S.A. Cantrel were chosen as outgroup taxa. The ITS sequence matrix was aligned and manually edited using BioEdit 7.0.5.3 (Hall 1999). Phylogenetic analyses were performed using maximum parsimony (MP) and neighbor-joining (NJ) methods with PAUP* 4.0b10 and parameters were set according to Zheng and Zhuang (2015). The topological confidence of the NJ and MP trees was assessed with bootstrap analysis using 1,000 replications, each with 10 replicates of random stepwise addition of taxa. The resulting trees were viewed via TreeView 1.6.6 (Page 1996).

Phylogenetic analyses
The ITS dataset included 37 sequences from eight Dicephalospora species, 11 related fungi and two outgroup taxa. The final alignment resulted in 634 characters including gaps, of which 252 were parsimony-informative, 38 were variable and parsimonyuninformative, and 344 were constant. In the MP analysis, eight most parsimonious trees were generated (tree length = 790, consistency index = 0.5899, homoplasy index = 0.4101, retention index = 0.8126, rescaled consistency index = 0.4793) and one of them was shown in Figure 1. MP and NJ bootstrap proportions (BP) greater than 50% were labeled at the nodes.
From topology of the phylogenetic tree ( Fig. 1), Dicephalospora species clustered together with a medium supporting value (56% MPBP). The three putative new species were clearly distinct from the known and sequenced species of the genus. Dicephalospora albolutea appeared as an independent lineage distinct from any other members of the genus. Dicephalospora shennongjiana was resolved as a sibling species of D. huangshanica (97% MPBP and 99% NJBP). ITS sequences of the three collections of D. yunnanica were identical and formed a well-supported group with D. aurantiaca (100% MPBP and 100% NJBP).  Description. Apothecia scattered, discoid, stipitate, with even margin, 1−2.5 mm in diameter; hymenium surface cream to yellowish white; receptacle surface concolorous. 9.5−10.5 μm. Ascospores sausage-shaped to subfusoid, with anterior end rounded and posterior end narrower, slightly curved, aseptate, hyaline, smooth, lacking a gel cap at each end, multiguttulate, with a dark-stained area when mounted in cotton blue solution, biseriate, 26−31 × 3.8−5.0 μm. Paraphyses filiform, straight, slightly enlarged at apex, hyaline, septate, 3−3.5 μm broad at upper portion and 1.5−2 μm below, equal to or very slightly exceeding the asci.
Notes. The diagnostic features of D. albolutea are cream to yellowish white apothecia and sausage-shaped ascospores. The apothecial color of earlier known Dicephalospora species varied from yellow, orange, red to dark, but never as pale as that in D. albolutea. Dicephalospora calochroa (Syd. & P. Syd.) Spooner is somewhat similar in length of asci and ascospores, but differs by vivid orange apothecia, wider asci (125−150 × 12−15 μm) and ascospores (20−25 × 6−8 μm), which are pointed at both ends (Spooner 1987). Dicephalospora albolutea differs from any investigated species by at least 45 bp in sequences of ITS region, and appeared as an independent lineage in the phylogenetic tree (Fig. 1), which further confirmed its distinction from others in the group.
A taxonomic key to the known species of Dicephalospora

Discussion
Identification of Dicephalospora species is mainly based on morphological features, such as color of apothecia, anatomic structure, and characteristics of asci and ascospores. DNA sequence data are sometimes considered, which play an important role in the delineation of fungal species (Hibbett et al. 2016;Jeewon and Hyde 2016). In the present study, three new species were introduced based on morphology and ITS phylogeny. So far, the genus comprises 13 species, of which 12 have been reported from China. Dicephalospora chrysotricha (Berk.) Verkley originally described from, and endemic to, New Zealand, is the only exception and known only from the type locality (Verkley 2004).
In the phylogenetic analyses, only some species possessing fusoid to sausage-shaped and elliptic-subfusoid ascospores were involved due to limitation of the available sequences. The ITS barcodes seem to be useful for distinguishing Dicephalospora species, as they grouped as well-separated clades (Fig. 1). Seven of the eight species were together receiving moderate statistic supports (86% MPBP and 80% BIPP) and formed the core group. However, D. chrysotricha joined them as a distantly separated lineage with very low support (Fig. 1, 56% MPBP). Dicephalospora chrysotricha is distinct from any other taxa of the genus in having hair-like projections on receptacle surface. Dicephalospora chrysotricha was previously treated as a member of Trichopeziza Fuckel (Saccardo 1889) and then Chlorosplenium Fr. (Dennis 1961). The transfer of this species to Dicephalospora might have been because of presence of polar mucilaginous caps of ascospores and the more or less similar ectal excipulum structure except for hairs (Verkley 2004). However, it does not fit well the generic concept of Dicephalospora. Further study is required to clarify the taxonomic position of this fungus.
As to the phylogenetic position of Dicephalospora, Figure 1 shows its close relationship with Hymenoscyphus Gray, which agrees with the treatment of . Similar results were also achieved in other recent studies (Han et al. 2014;Zhao et al. 2016). In the phylogenetic study of Hyaloscyphaceae and related helotialean cup-fungi, D. huangshanica and D. rufocornea grouped together with some genera of Helotiaceae, such as Hymenoscyphus, Crocicreas Fr. and Cudoniella Sacc., as a highly supported clade in the maximum-likelihood tree inferred from combined sequence data of ITS, the large subunit nrDNA gene (LSU), the second largest subunit of RNA polymerase II gene (RPB2), and mitochondrial small subunit (mtSSU) (Han et al. 2014). Zhao et al. (2016) carried out phylogenetic analyses of Lambertella Höhn. and allied genera including Dicephalospora and Hymenoscyphus, as inferred from ITS, LSU and RPB2 sequence data. In their phylogenetic trees, D. rufocornea was also associated with the clade consisting of Hymenoscyphus species. In view of the above results, close relationship of Dicephalospora with genera of Helotiaceae is obvious. Comprehensive work containing more genera and more genes are required to obtain an accurate conclusion on phylogenetic placement of Dicephalospora.