A taxonomic study of Nemania from China, with six new species

Abstract During an investigation of Xylariaceae from 2019 to 2020, isolates representing eight Nemania (Xylariacese) species were collected from Yunnan, Guizhou and Hainan Provinces in China. Morphological and multi-gene phylogenetic analyses, based on combined ITS, α-actin, rpb2 and β-tubulin sequences, confirmed that six of them are new to science, viz. Nemaniacamelliae, N.changningensis, N.cyclobalanopsina, N.feicuiensis, N.lishuicola and N.rubi; one is a new record (N.caries) for China and one is a known species (N.diffusa). Morphological descriptions and illustrations of all species are detailed. In addition, the characteristics of Nemania are summarised and prevailing contradictions in generic concepts are discussed.


Introduction
Nemania Gray was established by Gray (1821) for a heterogeneous assemblage of taxa and was affiliated with Xylariaceae Tul. & C. Tul. Since the early taxonomic description of this genus was ambiguous, taxonomists have often regarded some species of Nemania as synonyms of Hypoxylon Bull. For example, Nemania angusta (Petch) Y.M. Ju & J. D. Rogers was regarded as a synonym of Hypoxylon angustum Petch. (Miller 1961;Whalley et al. 1983;Ju and Rogers 2002). Subsequently, the generic concept of Nemania was modified by Pouzar (1985a, b) and Petrini and Rogers (1986). Granmo et al. (1999) and Ju and Rogers (2002) provided a comprehensive background to Nemania and accepted 37 species. Sánchez-Ballesteros et al. (2000) used the internal transcribed spacers (ITS) sequence to perform a phylogenetic study of Nemania, which supported the segregation of Nemania from Hypoxylon. However, their conclusion was based only on ITS sequences and Xylaria Hill & Schrank was not included in this study. Hence, the generic placement of Nemania in the Xylariaceae was unclear. Hsieh et al. (2005) used β-tubulin and α-actin to evaluate the phylogenetic relationship of several xylariaceous genera. It was found to be particularly useful in xylariaceous fungi as limited success in using ribosomal DNA genes to delineating genera and resolving generic relationships (Tang et al. 2007). Tang et al. (2007) re-established the phylogenetic relationships of Nemania with related genera, based on the combined dataset of ITS and rpb2 which supported the separation of Nemania from Hypoxylon. However, Tang et al. (2007) stated that Nemania is closely related to Xylaria and phylogenetically distinct from Annulohypoxylon Y.M. Ju et al., Daldinia Ces. & De Not. and Hypoxylon. Ultimately, the boundaries of the genus became relatively clear and Nemania has been accepted as a distinct genus in Xylariaceae (Ju and Rogers 2002). The major morphological characteristics of Nemania include dark brown to black stromata, carbonaceous or at least brittle and not yielding pigments in 10% potassium hydroxide (KOH) (Ju and Rogers 2002), white soft tissue existing between or below the perithecia, ascospores usually pale brown and most of them have no obvious germ-slit and spore dehiscence in 10% KOH (Tang et al. 2007).
Nemania accepted 37 species by 2002, which occurs mainly distributed on the rotting wood of angiosperms (Ju and Rogers 2002;Tang et al. 2007). There are a few species introduced from China in recent years. Two new species (N. flavitextura Y.M. Ju, H.M. Hsieh & J.D. Rogers and N. primolutea Y.M. Ju, H.M. Hsieh & J.D. Rogers), collected from Taiwan, were reported by Ju et al. (2005). One new species and two new record species were discovered and described by Du et al. (2016) and Ariyawansa et al. (2015) in China. Recently, two new species (N. yunnanensis Tibpromma & Lu and N. aquilariae Tibpromma & Lu), collected from Yunnan Province, China, were discovered by Tibpromma et al. (2021). Ninety-three epithets of Nemania are listed on Index Fungorum (2021) (accession date: 06. 2021). Only 17 species of Nemania with gene sequences were retrieved from the NCBI database (https://www.ncbi.nlm.nih. gov) and morphological methods are the main distinguishing method for Nemania. Morphologically, it is mainly distinguished according to the germ slit, the size of the ascospores and the characteristics of the stromata.
In this study, eight species of Nemania, collected from Guizhou, Hainan and Yunnan Provinces in China, are introduced. Six new species are identified, based on morpho-molecular analyses, while N. caries is reported as a new record for China; N. diffusa has been previously reported from China (Du 2015). Detailed morphological descriptions, illustrations and phylogenetic information of all species are provided in this paper.

Collection, isolation and morphology
Samples of rotting wood with fungi were collected from October 2019 to December 2020 in various nature reserves of Guizhou, Hainan and Yunnan Provinces, China. These samples were placed in sealed bags and the coordinates of sampling sites (such as latitude, longitude and altitude) were recorded. Specimens were taken to the laboratory for examination. Microscopic observations were made with fungi mounted in distilled water. A drop of Melzer's Reagent was added to determine whether or not the ascus apical ring blued (the amyloid iodine reaction) and the reaction and morphology of the ring could be observed. Fragments of stroma and perithecial wall were placed in 10% KOH on a microscope slide and the extractable pigment observed. Pure cultures were obtained with the single spore isolation method (Long et al. 2019) and the cultures were grown on oatmeal agar (OA) and potato dextrose agar (PDA).
Morphological examination of fungi on the rotting wood followed the methods of Xie et al. (2020). The characteristics of the stromata were observed with an Olympus SZ61 stereomicroscope and photographed using a fitted Canon 700D digital camera. The photomicrographs of asci and ascospores were taken with a Nikon digital camera (700D) fitted to a light microscope (Nikon Ni). Adobe Photoshop CS6 was used to arrange all the microphotographs. Measurements were performed using the Tarosoft image framework (v. 0.9.0.7). At least 30 ascospores, asci and ascus apical apparatus were measured for each specimen.
To prepare herbarium materials, the colonies grown on PDA were transferred to three 1.5 ml microcentrifuge tubes filled with sterile water and stored at 4 °C or with 10% glycerol at -20 °C. Herbarium materials were deposited in the Herbarium of Guizhou Medical University (GMB) and Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (KUN). Living cultures were deposited at Guizhou Medical University Culture Collection (GMBC).

DNA extraction, PCR amplification and sequencing
The BIOMIGA Fungal Genomic DNA Extraction Kit (GD2416, Biomiga, USA) was used to extract genomic DNA from fresh fungal mycelium, according to the manufacturer's instructions. The extracted DNA was stored at -20 °C.
Target regions of internal transcribed spacers (ITS) and RNA polymerase II second largest subunit (rpb2) regions were amplified symmetrically using primers of ITS4/ ITS5 (White et al. 1990;Gardes and Bruns 1993) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. ACT512F and ACT783R ) and T11 and T22 (Tanaka et al. 2009;Hsieh et al. 2010) primers were used for the amplification of the α-actin gene (ACT) and β-tubulin (TUB2), respectively. The components of the polymerase chain reaction (PCR) mixture and thermal cycling programme were performed as described by Pi et al. (2020). The amplified PCR fragments were sent to Sangon Biotech (Shanghai) Co., China, for sequencing. All newly-generated sequences of ITS, α-actin, rpb2 and β-tubulin regions were uploaded to the GenBank database and the accession numbers are shown in Table 1.

Sequence alignment and phylogenetic analyses
Except for newly-generated sequences, all sequences used for phylogenetic analysis were downloaded from GenBank, based on published literature and the highest hit rate of ITS in the GenBank database. Sequence data for the construction of the phylogenetic tree are listed in Table 1. Sequence alignments were generated using the MAFFT v.7.110 online programme (http://mafft.cbrc.jp/alignment/server/, Katoh and Standley 2013) under default settings. Multiple sequence alignments of ITS, α-actin, rpb2 and β-tubulin were analysed individually and in combination, manually adjusted to achieve the maximum alignment and to minimise gaps using the BioEdit v.5 (Hall 1999). The file formats were converted in ALTER (Alignment Transformation Envi-Ronment) (http://www.sing-group.org/ALTER/). The Maximum Likelihood analysis was carried out with GTR+G+I model of site substitution by using RAxML 7.4.2 black box (https://www.phylo.org/, Stamatakis et al. 2008) and Bayesian Inference
Culture characteristics. The colony grows on PDA medium with a diameter of 6 cm after one week at 25 °C; white, cottony, circular, flocculent or velvety, with light yellow to slightly yellow at the centre. Not sporulating on OA nor on PDA.

Nemania changningensis
Culture characteristics. Colonies on PDA medium in size with a diameter of 6 cm after two weeks at 25 °C; the surface is white, intermediate thick, cottony, dense, with undulate or ring edge, flat, low, whitish-yellow, reverse of the colony yellow at the centre. Not sporulating on OA nor on PDA.
Culture characteristics. Colonies grow slowly on PDA medium with a diameter of 5 cm after 10 days at 25 °C. Colonies surface were white to pale orange, circular, cottony, low, dense, cottony mycelium, reverse with light orange mycelium. Not sporulating on OA nor on PDA.
Notes. In our phylogenetic analysis, Nemania rubi formed a distinct branch, which is sister to N. changningensis and N. caries (Fig. 1). In morphology, N. rubi is similar to N. caries, but is distinct in having a long-cylindrical apical apparatus and the inequilateral ascospores lacking a germ slit (Miller 1961;Ju and Rogers 2002). In addition, the perithecia of N. caries are obovoid (0.3-0.6 × 0.5-0.7 mm) and its height is greater than the width (Tang et al. 2007). The ascomata surface of N. rubi ascomata is uneven with inconspicuous perithecial mounds, which is similar to those of N. plumbea, but the latter has larger ascospores (13-16 × 5.4-6.6 μm) with germ slits on the concave side (Tang et al. 2007).

Discussion
In this study, newly-collected Nemania species from Hainan, Yunnan and Guizhou Provinces were subjected to morpho-molecular analyses. Six new species were introduced while reporting one new record from China. Nemania showed a closer affinity to Roselinia than to Kretzschmaria Fr. and Xylaria (U'Ren et al. 2016), which is also supported in the phylogenetic analysis, based on ITS, rpb2, β-tubulin and α-actin sequences. Although no asexual morphs were observed in this study, Nemania has geniculisporium-like asexual morphs which are a common character in members of Xylariaceae (Fournier et al. 2018).
Nemania forms a single branch in the phylogenetic analysis, which supports that it is a monophyletic genus. However, Nemania genus is separated into six clades (N1-N6, Fig. 1), each of which have relatively-uniform morphological characteristics. N1 clade is represented by N. bipapillata and taxa in this clade have carbonaceous interior to the stromata, ostioles encircled with a disc and dark brown ascospores with a long germ slit. The species within clade N2 are distinguished from other Nemania species with fusoidinequilateral and pale brown ascospores and by having white soft tissues between the perithecia. The species in clades N3, N4 and N5 have little difference in morphology and may be confused. Most taxa in clades N4 and N5 have usually brown, dark brown or blackish-brown ascospores with a germ slit longer than 2/3 spore length (Granmo et al. 1999;Ju and Rogers 2002;Fournier et al. 2018). The taxa in N6 clade have light brown or medium brown ascospores with a germ slit shorter than 2/3 spore length or seemingly lacking (Ju and Rogers 2002). Interestingly, the ascospores of most taxa in N6 clade are olivaceous brown when fresh, turning medium brown after desiccation.
Separation of members of Nemania, based on morphology, is relatively difficult and confusing (Fournier et al. 2018). In some early literature, the new species lacked the description of some key morphological characteristics (Du et al. 2016). Moreover, sequences are available for only a few species in GenBank, thus species identification, based on DNA sequences, is also problematic. Hence, it is essential to re-collect old species that lack ex-type cultures and DNA sequences and to epitypify them.
The similarity of morphological features between species is high, which makes it difficult for existing morphological taxonomic features to identify species. For example, species in clade N3, which includes N. diffusa and N. cyclobalanopsina, are difficult to identify, based solely on morphological characteristics, although their ITS sequence differences can reach more than 3% (Jeewon and Hyde 2016;Vu et al. 2019). In this clade, we tentatively use multiple-genes sequence as the main classification basis for species. Molecular data should be the main identification basis for Nemania species, especially for clade N3. It is worth noting that we should compare sequences with that from type or authoritative strains.