A new species, Chlorovibrissea korfii, is described and illustrated, which is distinct from other members of the genus by the overall pale greenish apothecia 0.8–2.0 mm in diam. and 0.6–1.5 mm high, J+ asci 70–83 × 4.5–5.5 μm, and non-septate ascospores 44–52 × 1.2–1.5 μm. This is the first species of Chlorovibrissea reported from northern hemisphere. Vibrissea flavovirens is reported from China for the first time. Sequence analyses of the internal transcribed spacer of nuclear ribosomal DNA are used to confirm the affinity of the two taxa.

morphology, sequence analysis, taxonomy, Vibrisseaceae

Vibrisseaceae was erected by Korf in 1990 to accommodate the genera Vibrissea Fr., Chlorovibrissea L.M. Kohn and Leucovibrissea (A. Sánchez) Korf. Members of the family are characterized by aquatic or semi-aquatic habitat, apothecia sessile to long stipitate, disc color range from whitish, yellowish, brownish, olivaceous to blackish green, ectal excipulum composed of textura globosa, textura angularis to textura prismatica, and filiform ascospores (Korf 1990). The three genera are distinguishable by the color of apothecia, structure of ectal excipulum and ascal apex apparatus (Korf 1990; Sandoval-Leiva et al. 2014). Besides the above three genera, Acephala Grünig & T.N. Sieber and Phialocephala W.B. Kendr. known only by asexual states, are also included in the family (Kirk et al. 2008; Johnston et al. 2014). Acephala was erected for the non-sporulating species of Phialocephala that contain a small group of dark septate root endophytes (Grünig and Sieber 2005; Grünig et al. 2008; Grünig et al. 2009; Münzenberger et al. 2009; Wang et al. 2009; Day et al. 2012). About 80 species are currently accepted in the family, among which two species are in Acephala, six in Chlorovibrissea, one in Leucovibrissea, 33 in Phialocephala, and 36 in Vibrissea (Grünig et al. 2008; Kirk et al. 2008; Grünig et al. 2009; Münzenberger et al. 2009; Wang et al. 2009; Day et al. 2012; Johnston et al. 2014; Sandoval-Leiva et al. 2014; Index Fungorum 2017). The previous phylogenetic studies on the vibrisseaceous fungi showed that Chlorovibrissea and Vibrissea are not closely related, and the family was presumably polyphyletic (Wang et al. 2006; Sandoval-Leiva et al. 2014).

In China, only two Vibrisseaceous fungi were recorded, i.e. V. cf. sporogyra (Ingold) A. Sánchez from Hainan Province (Zhuang et al. 2002) and V. truncorum (Alb. & Schwein.) Fr. from Guizhou Province (He 1988). During our studies of the helotialean fungi from China, two newly collected specimens fit the circumscription of Vibrisseaceae and represent two species. Based on morphology and molecular data, one is proposed as a new species of Chlorovibrissea, and the other is identified as V. flavovirens (Pers.) Korf & J.R. Dixon which is new to China.

Specimens were collected from Beijing and Yunnan Province in 2016, and apothecial gross morphology when fresh was photographed by a Canon PowerShot G16 digital camera. Dried apothecia were rehydrated with distilled water and sectioned at a thickness of 15 μm with a Yidi YD-1508A freezing microtome (Jinhua, China). Measurements were taken from longitudinal sections and from squash mounts in lactophenol cotton blue solution using an Olympus BH-2 microscope (Tokyo, Japan). Iodine reactions of ascus apparatus were tested in Melzer’s reagent and Lugol’s solution with or without 3% KOH solution pretreatment according to Baral (2009). Images were captured using a Canon G5 digital camera (Tokyo, Japan) attached to a Zeiss Axioskop 2 Plus microscope (Göttingen, Germany) for anatomical structure. Voucher specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). Name of the new species was formally registered in the database Fungal Names, one of the three official nomenclatural repositories for fungal names (Redhead and Norvell 2012).

Genomic DNA was extracted from dried apothecia using Plant Genomic DNA Kit (TIANGEN Biotech. Co., Ltd., Beijing, China). Materials were crushed in liquid nitrogen before extraction. The internal transcribed spacer of nuclear ribosomal DNA (ITS) were amplified and sequenced using primer pair ITS1 and ITS4 (White et al. 1990) according to Zheng and Zhuang (2016). Newly generated sequences were deposited in GenBank, and other sequences used in the phylogenetic analyses were retrieved from GenBank (Table 1). Holwaya mucida (Schulzer) Korf & Abawi was selected as outgroup taxon. Alignment was generated and manually edited with BioEdit 7.0.5.3 (Hall 1999). Maximum parsimony (MP) and Neighbor-joining (NJ) analyses were carried out using PAUP*4.0b10 with parameters used by Zheng and Zhuang (2014). 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 in TreeView 1.6.6 (Page 1996).

The three sexual genera in Vibrisseaceae are mainly differentiated by color of apothecia and structure of ectal excipulum. The excipular cells of Vibrissea are more or less angular to globose and lying at a high angle to the receptacle surface, while those of Chlorovibrissea and Leucovibrissea are rectangular and axes of cells are nearly parallel to the receptacle surface. Chlorovibrissea is different from Leucovibrissea in having greenish apothecia instead of the whitish ones. Differences between Chlorovibrissea and Vibrissea were found in ascal apex, which is round to somewhat truncate and with the apical ring placed subapically in the former genus, while that of the latter is acute with apical ring placed at the tip (Sandoval-Leiva et al. 2014). The ascal apex of C. korfii is somewhat conical and with apical ring placed apically, broader at tip and narrower downward (Fig. 2g), unlike that of other known Chlorovibrissea species. Further studies are needed to evaluate whether ascal apical apparatus is phylogenetically informative in Vibrisseaceae.

Chlorovibrissea was assumed to have a southern hemisphere distribution because species of the genus was never reported from north hemisphere (Kohn 1989; Sandoval-Leiva et al. 2014). The discovery of C. korfii from China expands the distribution of the genus to north hemispheres, which might break the assumption “the origin of the southern hemisphere vibrisseaceous fungi could be independent from the northern hemisphere representative” (Wang et al. 2006).

In the family Vibrisseaceae, sequence data of Leucovibrissea are not available. The two asexual genera Vibrisseaceae are Phialocephala and Acephala. Due to the heterogeneity of Phialocephala, only the type species, P. dimorphospora, and two closely related taxa were included in our phylogenetic analyses. Vibrissea, Acephala and Phialocephala clustered as a highly supported group (MPBP/NJBP = 100%/100%, Fig. 1), while Chlorovibrissea were distantly related (Fig. 1). The results coincided with those of the previous studies (Wang et al. 2006; Sandoval-Leiva et al. 2014), in which Chlorovibrissea and Vibrissea appeared as two separate clades. As to relationships among species of Chlorovibrissea, C. albofusca and C. chilensis were very closely related (MPBP/NJBP = 100%/100%), and C. korfii is associated with the rest species of the genus, which did not receive a reasonable support (MPBP = 81%). The inter-specific relationships of Vibrissea were hardly demonstrated (Fig. 1) since very few species were sampled.

The two asexual genera are recognizable and associated each other with low supports (Fig. 1). The two existing species of the genus Acephala were sisters (Fig. 1, MPBP/NJBP = 99%/99%). Acephala was thought to be congeneric with Phialocephala by some authors, and differentiated only by the lack of observed sporulation in culture (Grünig et al. 2009; Münzenberger et al. 2009; Tanney et al. 2016). Phialocephala species are commonly isolated as dark-septate endophytes from coniferous tree roots or from decomposing wood (Menkis et al. 2004), and attributed to Vibrisseaceae mainly based on sequence data (Wang et al. 2006; Kirk et al. 2008). Connections of some Phialocephala species with Mollisia or mollisioid sexual states were reported recently (Grünig et al. 2009; Tanney et al. 2016). It seems that a lot of work needs to be done to establish the generic concepts.

In conclusion, Vibrisseaceae established based on morphology is quite possibly polyphyletic. Sequence data of Leucovibrissea are desirable to get a more comprehensive outline of the family. Phialocephala s.l. is heterogeneous. Its generic concept needs to be clarified. As more sequences of vibrisseaceous fungi are available, the circumscription of the family will become monophyletic.

This work was supported by the National Natural Science Foundation of China (nos. 31570018, 31300021). The authors would like to thank Dr. X.H Wang and Dr. S.H. Li for their invaluable help during the field work.