The genera Rugonectria and Thelonectria (Hypocreales, Nectriaceae) in China

Abstract Recent collections and herbarium specimens of Rugonectria and Thelonectria from different regions of China were examined. Using combined analyses of morphological and molecular data, 17 species are recognised including three species of Rugonectria and 14 species in Thelonectria. Amongst them, R.microconidia and T.guangdongensis are new to science. Rugonectriamicroconidia on mossy bark is characterised by superficial, yellow to orange, pyriform to subglobose perithecia with a warted surface; ellipsoidal to broadly ellipsoidal, striate, uniseptate ascospores; and allantoid to rod-shaped, aseptate microconidia. Thelonectriaguangdongensis possesses bright red perithecia with a slightly roughened surface and a prominently dark papilla; ellipsoidal, smooth, uniseptate ascospores; and subcylindrical, slightly curved, multiseptate macroconidia. Morphological distinctions and sequence divergences between the new species and their close relatives are discussed. Name changes for the previously recorded species in China are noted.


Introduction
The family Nectriaceae was introduced in 1865 and circumscribed to accommodate the hypocrealean species having ascomata that are generally yellow, orange-red to purple and usually changing colour in potassium hydroxide (KOH) and lactic acid (LA) (Rossman et al. 1999). About 55 genera containing 900 species are included in the family (Lombard et al. 2015). A phylogenetic backbone for Nectriaceae was constructed based on DNA sequences of 10 loci by Lombard et al. (2015).
The first record of Rugonectria from China dates back to 2000 when R. rugulosa (as Nectria rugulosa Pat. & Gaillard) was reported by Lu et al. (2000) based on a specimen collected on dead petioles of king palm. Research on Thelonectria in China was started by Teng (1936) when T. discophora (as N. discophora Mont.) was first reported on bark of fallen branches from Yunnan Province. In connection with our current work on the Chinese fungus flora, fresh materials and herbarium specimens of the two genera were examined. Based on morphology and phylogenetic analyses of the partial sequences of α-actin (ACT), internal transcribed spacer (ITS), nuclear ribosomal large subunit (LSU) rDNA and the largest subunit of RNA polymerase II (RPB1), 17 species were identified, including two undescribed species. Morphological and molecular diagnostic features between the new taxa and their closely related fungi are discussed.

Sampling and morphological studies
Specimens were collected from Beijing, Fujian, Guangdong, Hainan, Henan, Hubei, Hunan and Yunnan provinces and are deposited in Herbarium Mycologicum Academiae Sinicae (HMAS) and cultures are kept in the State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences. The methods used by Luo and Zhuang (2010) and Chaverri et al. (2011) were followed for morphological observations. The ascomatal wall reactions to 3% KOH and 100% LA were tested. To observe micro-morphological characteristics of perithecial walls, sections were made with a freezing microtome (YD-1508-III, Jinhua, China) at a thickness of 6-8 μm. Lactophenol cotton blue solution was used as mounting medium for examination of anatomic structures and measurements of perithecia, asci and ascospores. Photographs were taken with a Leica DFC450 digital camera (Wetzlar, Germany) attached to a Leica M125 stereomicroscope (Milton Keynes, UK) for gross morphology and a Zeiss AxioCam MRc 5 digital camera (Jena, Germany) attached to a Zeiss Axio Imager A2 microscope (Göttingen, Germany) for microscopic features. Descriptive statistics of ascospores and conidia (minimum, maximum, mean and standard deviation) were calculated following the methods of Hirooka et al. (2012). Measurements of individual structures were based on 30 units, except as otherwise noted. Morphology of colonies were characterised using potato dextrose agar (PDA, 20% w/v potato + 2% w/v dextrose + 2% w/v agar) and synthetic nutrient-poor agar (SNA; Nirenberg 1976) at 25 °C in an incubator with alternating periods of light and darkness (12 h/12 h). Colony growth rates were measured after 7 d.

Sequence alignment and phylogenetic analyses
Newly obtained sequences and those retrieved from GenBank are listed in Table I. The sequences were assembled, aligned and the primer sequences were trimmed via BioEdit 7.0.5 (Hall 1999) and converted to NEXUS files by ClustalX 1.8 (Thompson et al. 1997). A partition homogeneity test was performed with 1,000 replicates in PAUP*4.0b10 (Swofford 2002) to evaluate statistical congruence amongst the four loci. The aligned ACT, ITS, LSU and RPB1 sequences were combined in BioEdit and analysed with Bayesian Inference (BI), Maximum Parsimony (MP) and Maximum Likelihood (ML) methods to determine the phylogenetic positions of the new species. The MP analysis was performed with PAUP 4.0b10 (Swofford 2002) using 1000 replicates of heuristic search with random addition of sequences and subsequent TBR (tree bisection and reconnection) branch swapping. Topological confidence of the resulting trees was tested by Maximum Parsimony bootstrap proportion (MPBP) with 1000 a The GenBank numbers in bold type were newly generated in this study.
replications, each with 10 replicates of random addition of taxa. The BI analysis was conducted by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) using a Markov chain Monte Carlo algorithm. Nucleotide substi tution models were determined by MrModeltest 2.3 (Nylander 2004). Four Markov chains were run simultaneously for 1000000 generations with the trees sam pled every 100 generations. A 50% majority rule consensus tree was computed after excluding the first 2500 trees as 'burn-in'. Bayesian Inference posterior probability (BIPP) was determined from the remaining trees. ML analysis was conducted with IQ-Tree 1.6.10 (Nguyen et al. 2015) using the best model for each locus chose by ModelFinder (Chernomor et al. 2016). Branch support measures were calculated with 1000 bootstrap replicates. Trees were examined by TreeView 1.6.6 (Page 1996). Cosmospora coccinea Rabenh. and Nectria cinnabarina (Tode) Fr. were used as outgroup taxa. Maximum Likelihood bootstrap proportion (MLBP) and MPBP greater than 50% and BIPP greater than 90% were shown at the nodes.

Distribution. Asia (China).
Notes. This species was introduced by Zeng and Zhuang (2013) and only known from the type locality. The phylogenetic analyses indicate that the species is associated with T. guangdongensis (Figure 1). Habitat. On bark of shrubs and trees, sometimes associated with small cankers. Distribution. Americas (Costa Rica), Asia (Indonesia, Taiwan), possibly pantropical.

Thelonectria coronalis C. Salgado & Guu, in
Notes. The morphology and molecular data indicated that T. coronata is a species complex. Salgado-Salazar et al. (2012) divided it into five taxa on the basis of multigene phylogeny. The Chinese collection matches well the concept of T. coronata sensu stricto by Salgado-Salazar et al. (2012). Notes. Re-examination of HMAS 76856 indicated that T. phoenicea is the correct name for the specimen which was previously identified as T. discophora. It is distributed also in Taiwan Province . Habitat. On bark of a coniferous (?) tree. Distribution. Asia (China). Notes. The species was originally placed in Neonectria by Luo and Zhuang (2010). The anatomic structures and DNA data support its placement in Thelonectria  Notes. This fungus was originally described as Nectria jungneri and was transferred to Neonectria (Brayford et al. 2004) and Thelonectria (Chaverri et al. 2011). The recent work by Salgado-Salazar et al. (2016) indicated that it belongs to a separate genus Macronectria C. Salgado & P. Chaverri.

Discussion
The genus Rugonectria is characterised by the non-papillate, orange to red, conspicuously warted to rugose perithecial surface (Chaverri et al. 2011). The ascomatal anatomy, perithecial wall reactions to KOH and LA, features of asci and ascospores and asexual states indicate the placement of R. microconidia in this genus. The multi-locus sequence analyses confirm our morphological observations ( Figure 1) and it is here described as a new species.
Historically, the nectriaceous fungi with cylindrocarpon -like asexual states were assigned to Neonectria. The accumulated morphological and phylogenetic data suggest that the genus was heterogeneous (Mantiri et al. 2001). Efforts were made towards establishment of a monophyletic Neonectria as well as its allies (Booth 1966, Rossman et al. 1999Mantiri et al. 2001;Brayford et al. 2004). The previously recognised infrageneric groups within Neonectria are now recognised as separate genera, i.e. Ilyonectria for the N. radicicola-group, Neonectria sensu stricto for the N. coccinea-group, Rugonectria for the N. rugulosa-group and Thelonectria for the N. mammoidea/N. veuillotiana-groups (Chaverri et al. 2011). Since the establishment of Thelonectria, 45 species have been placed in the genus (www.indexfungorum.org). Salgado-Salazar et al. (2012, 2015 suggested that the criteria formerly used for generic differentiation were of insufficient sensitivity to accurately reflect the degree of species diversity within the group. Subsequently, Salgado-Salazar et al. (2016) emended the generic concept of Thelonectria by excluding T. jungneri, based on the molecular data and morphological characteristics.
The type species of Thelonectria, T. discophora, previously considered to be cosmopolitan, was first described based on material collected from Chile and was determined to be heterogeneous (Brayford et al. 2004). Salgado-Salazar et al. (2015) provided a revisionary treatment of the T. discophora species complex and recognised 16 cryptic species on the basis of the combined analyses of phylogeny and morphology. In this study, the new species T. guangdongensis is determined to be congeneric with T. discophora, while both the molecular data and morphological characteristics indicate that T. guangdongenis is distinct from other species of Thelonectria. To date, 11 species of Thelonectria have been recorded from China (Teng 1936;Salgado-Salazar et al. 2012, 2015Zeng and Zhuang 2012;Zhuang 2013). China is extremely diverse in its climate, vegetation, geographic structures and multiple niches. Our understanding of species diversity of the nectriaceous fungi will be significantly broadened in the near future.