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Research Article
The genera Rugonectria and Thelonectria (Hypocreales, Nectriaceae) in China
expand article infoZhao-Qing Zeng, Wen-Ying Zhuang
‡ Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Open Access

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. Rugonectria microconidia 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. Thelonectria guangdongensis 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.

Keywords

Morphology, Multigene analyses, Taxonomy

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 genus Rugonectria P. Chaverri & Samuels, typified by R. rugulosa (Pat. & Gaillard) Samuels, P. Chaverri & C. Salgado, is characterised by perithecia solitary or in groups, seated on or partially immersed in a stroma. The perithecia are orange to red, globose to subglobose and non-papillate, with warted or rugose walls. Ascospores are ellipsoidal to oblong, striate, hyaline and 1-septate; and microconidia are ovoid to cylindrical (Chaverri et al. 2011). Currently, four species are recognised in the genus (Chaverri et al. 2011; Zeng et al. 2012). Thelonectria P. Chaverri & C. Salgado, typified by T. discophora (Mont.) P. Chaverri & C. Salgado, was established by Chaverri et al. (2011) to accommodate the nectriaceous fungi having superficial, globose to subglobose or pyriform to elongated perithecia which do not collapse when dry, with a prominent and darkened papilla; smooth, rarely spinulose or striate ascospores and curved macroconidia with rounded ends (Chaverri et al. 2011; Lombard et al. 2015; Salgado-Salazar et al. 2016). About 44 species are currently accepted in the genus (Chaverri et al. 2011; Salgado-Salazar et al. 2012, 2015, 2016; Zeng and Zhuang 2013; Crous et al. 2018). Species in the genera Rugonectria and Thelonectria are distributed in the tropics, subtropics and temperate regions and occur on early decaying bark, roots, branches, trunks and rarely in soil (Chaverri et al. 2011; Salgado-Salazar et al. 2015). A few species are plant pathogenic, such as R. castaneicola (W. Yamam. & Oyasu) Hirooka & P. Chaverri causing Abies and Acer cankers and T. rubi (Osterw.) C. Salgado & P. Chaverri causing Rubus cankers (Cedeño et al. 2004; Kobayashi et al. 2005; Chaverri et al. 2011; Salgado-Salazar 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.

Materials and methods

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 micromorphological 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.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fresh mycelium following the method of Wang and Zhuang (2004). Four primer pairs, act1-act2 (Samuels et al. 2006), ITS5-ITS4 (White et al. 1990), LR0R-LR5 (Vilgalys and Hester 1990; Rehner and Samuels 1994) and crpb1a-rpb1c (Castlebury et al. 2004) were used to amplify the ACT, ITS, LSU and RPB1 regions, respectively. PCR reactions were performed using an ABI 2720 Thermal Cycler (Applied Biosciences, Foster City, USA) with a 25 μl reaction system consisting of 12.5 μl Taq MasterMix, 1 μl each primer (10 μM), 1 μl template DNA and 9.5 μl ddH2O, based on the procedures detailed in Chaverri et al. (2011). DNA sequencing was carried out in both directions on an ABI 3730XL DNA Sequencer (Applied Biosciences, Foster City, USA).

Sequence alignment and phylogenetic analyses

Newly obtained sequences and those retrieved from GenBank are listed in Table 1. 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 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 substitution models were determined by MrModeltest 2.3 (Nylander 2004). Four Markov chains were run simultaneously for 1000000 generations with the trees sampled 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.

Table 1.

List of species, herbarium/strain numbers and GenBank accession numbers of materials used in this study.

Species Herbarium/strain numbers GenBank Accession numbers
ACT ITS LSU RPB1
Cosmospora coccinea Rabenh. CBS 114050 GQ505967 FJ474072 GQ505990 GQ506020
Nectria cinnabarina (Tode) Fr. AR 4302/AR 4477 HM484627 HM484548 HM484562 HM484577
Rugonectria castaneicola (W. Yamam. & Oyasu) Hirooka & P. Chaverri CBS 128360 MH864901 MH876352
R. microconidia Z.Q. Zeng & W.Y. Zhuang HMAS 254521 MF669044 a MF669050 MF669052 MF669056
R. neobalansae (Samuels) P. Chaverri & Samuels CBS 125120 KM231750 HM364322 KM232146
R. rugulosa (Pat. & Gaillard) Samuels, P. Chaverri & C. Salgado YH 1001 JF832515 JF832661 JF832761 JF832836
R. sinica W.Y. Zhuang, Z.Q. Zeng & W.H. Ho HMAS 183542 MF669046 HM054141 HM042430 MF669058
Thelonectria asiatica C. Salgado & Hirooka MAFF 241576 KC121436 KC153774 KC121500 KC153967
T. beijingensis Z.Q. Zeng, J. Luo & W.Y. Zhuang HMAS 188498 MF669047 JQ836656 MF669054 MF669059
T. blattea C. Salgado & P. Chaverri CBS 95268 KC121387 KC153725 KC121451 KC153918
T. brayfordii C. Salgado & Samuels CBS 118612 KC121381 KC153719 KC121445 KC153912
T. conchyliata C. Salgado & P. Chaverri GJS 8745 KC121401 KC153739 KC121465 KC153932
T. discophora (Mont.) P. Chaverri & C. Salgado AR 4742 KC121376 KC153714 KC121440 KC153907
T. guangdongensis Z.Q. Zeng & W.Y. Zhuang HMAS 254522 MF669045 MF669051 MF669053 MF669057
T. ianthina C. Salgado & Guu GJS 10118 KC121393 KC153731 KC121457 KC153924
T. japonica C. Salgado & Hirooka MAFF 241524 KC121428 KC153766 KC121492 KC153959
HMAS 98327 MK556799 HM054140 HM042434
T. mammoidea (W. Phillips & Plowr.) C. Salgado & R.M. Sanchez IMI 69361 KC121425 KC153763 KC121489 KC153956
T. ostrina C. Salgado & P. Chaverri GJS 9623 KC121418 KC153756 KC121482 KC153949
T. phoenicea C. Salgado & P. Chaverri GJS 85179 KC121398 KC153736 KC121462 KC153929
HMAS 76856 MK556800 JQ836657 DQ119572
T. pinea (Dingley) C. Salgado & P. Chaverri AR 4324 HM352875 HM364294 HM364307 HM364326
T. porphyria C. Salgado & Hirooka MAFF 241515 KC121426 KC153764 KC121490 KC153957
HMAS 98333 MK556798 HM054136 HM042433
T. purpurea C. Salgado & P. Chaverri GJS 10131 KC121394 KC153732 KC121458 KC153925
T. rubi (Osterw.) C. Salgado & P. Chaverri CBS 11312 KC121380 KC153718 KC121444 KC153911
T. sinensis (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang HMAS 183186 MF669048 FJ560441 FJ560436 MF669060
T. tyrus C. Salgado & P. Chaverri GJS 9046 KC121413 KC153751 KC121477 KC153944
T. violaria C. Salgado & R.M. Sanchez AR 4766 KC121377 KC153715 KC121441 KC153908
T. yunnanica Z.Q. Zeng & W.Y. Zhuang HMAS 183564 MF669049 FJ560438 MF669055 MF669061

Results

The sequences of ACT, ITS, LSU and RPB1 from 25 representative taxa of Rugonectria and Thelonectria were analysed. The partition homogeneity test (P = 0.03) indicated that the individual partitions were not highly incongruent (Cunningham 1997), thus these four loci were combined for the phylogenetic analyses. In the MP analysis, the datasets included 2524 nucleotide characters, of which 1836 were constant, 198 were variable and parsimony-uninformative and 490 were parsimony-informative. The MP analysis resulted in three most parsimonious trees (tree length = 1415, CI = 0.6721, HI = 0.3279, RI = 0.6098, RCI = 0.5351). One of them is shown in Figure 1. The ML and BI trees were of similar topology. The final matrix was deposited in TreeBASE with accession no. S23994. The isolate HMAS 254521 grouped with other members of Rugonectria by receiving high bootstrap values (MLBP/MPBP/BIPP = 100%/100%/100%) and the isolate HMAS 254522 clustered with the representatives of Thelonectria (MLBP/MPBP/BIPP = 100%/100%/100%), which support the taxonomic placements of these new species.

Figure 1. 

A Maximum Parsimony tree inferred from the combined ACT, ITS, LSU and RPB1 sequences. Cosmospora coccinea and Nectria cinnabarina were used as outgroup taxa. MLBP (left) and MPBP (middle) above 50%, BIPP (right) above 90% are indicated at nodes.

Taxonomy

Rugonectria microconidia Z.Q. Zeng & W.Y. Zhuang, sp. nov.

Fungal Names: FN570487
Figure 2

Holotype

CHINA. Hunan, Yizhang, Mangshan, (24°57'56.58"N, 112°57'34.63"E), alt. 700 m, on mossy bark, 26 October 2015, Z.Q. Zeng, X.C. Wang, K. Chen, Y.B. Zhang 10266 (HMAS 254521); ex-type culture: HMAS 247232.

Sequences

ACT (MF669044), ITS (MF669050), LSU (MF669052) and RPB1 (MF669056).

Etymology

The specific epithet refers to the microconidia produced in culture.

Description

Mycelium not visible around ascomata or on natural substrata. Ascomata superficial, gregarious, with basal stroma, pyriform to subglobose, non-papillate, yellow to orange, often with a darker red ostiolar area when dry, turning dark red in KOH, becoming slightly yellow in LA, 421–549 × 333–470 μm (n = 8). Perithecial surface warted, 30–93 µm thick, of textura globulosa to textura angularis, cells 10–27 × 8–18 µm, walls 1.5–2.5 µm thick. Perithecial wall of two layers, 45–70 µm thick, outer layer 25–45 µm thick, of textura globulosa to textura angularis; inner layer 7–25 µm thick, of textura prismatica. Asci unitunicate, clavate, 8-spored, 93–130 × (11–)15–25 µm (112.6 ± 12.6 × 18.9 ± 3.2 μm). Ascospores ellipsoid to broadly ellipsoid, 1-septate, striate, uniseriate or biseriate above and uniseriate below, hyaline, 20–28 × 8–12 µm (24.0 ± 2.0 × 10.1 ± 0.9 μm). Colony on PDA 42 mm diameter after 7 d under daylight at 25 °C, surface velvety, with white aerial mycelium, producing pale pinkish pigment in medium. Colony on SNA reaches 40 mm diameter after 7 d under daylight at 25 °C, surface with sparse whitish aerial mycelium. Conidiophores simply branched, 18–50 × 2–3 μm. Microconidia allantoid to rod shaped, slightly curved, 0(1–2)-septate, 3–14(–18) × 1.2–2.5(–3) μm (6.7 ± 3.1 × 1.6 ± 0.4 μm).

Figure 2. 

Rugonectria microconidia a–d ascomata on natural substratum e colony on PDA f colony on SNA g, h median section through perithecium i–k asci with ascospores l–o ascospores p–s conidiophores and conidia t, u conidiogenous cells and conidia v, w microconidia. Scale bars: 0.5 mm (a–d); 50 μm (g, h); 10 μm (i–w).

Habitat

On mossy bark.

Distribution

Asia (China).

Notes

The non-papillate perithecia with warted surface, clavate asci with ellipsoidal to broadly ellipsoidal, uniseptate, striate ascospores, as well as our molecular data, suggest that this species belongs to Rugonectria (Chaverri et al. 2011). Amongst the known species of the genus, R. microconidia is morphologically most similar to the type species, R. rugulosa, in having gregarious, warted, orange perithecia often with a dark red ostiole when dry (Samuels et al. 1990; Samuels and Brayford 1994). The newly described species differs in having asci that are 93–130 × (11–)15–25 µm and larger than those of R. rugulosa that are (53–)64–83(–95) × (7.5–)11.3–15.5(–17) µm. In addition, the ascospores of R. microconidia are also larger, 20–28 × 8–12 µm, while those of R. rugulosa are (10–)13.5–18(–24) × (3.3–)4.7–6.7(–10) µm. Unlike R. microconidia, R. rugulosa does not produce macroconidia in culture (Samuels et al. 1990; Samuels and Brayford 1994). Sequence comparisons reveal that there are 21 bp, 21 bp, 12 bp and 22 bp divergences in the ACT, ITS, LSU and RPB1 regions, respectively, between R. microconidia and R. rugulosa (YH1001). Both morphological and molecular data suggest that these species are distinct.

Rugonectria rugulosa (Pat. & Gaillard) Samuels, P. Chaverri & C. Salgado, in Chaverri, Salgado, Hirooka, Rossman & Samuels, Stud. Mycol. 68: 73, 2011

Nectria rugulosa Pat. & Gaillard, Bull. Soc. Mycol. Fr. 5(4): 115, 1890.

Neonectria rugulosa (Pat. & Gaillard) Mantiri & Samuels, in Mantiri, Samuels, Rahe & Honda, Can. J. Bot. 79(3): 339, 2001.

= Cylindrocarpon rugulosum Brayford & Samuels, in Samuels & Brayford, Sydowia 46(1): 148, 1994.

Specimens examined

CHINA. Henan, Jigongshan, alt. 400 m, on rotten twigs, 14 November 2003, W.Y. Zhuang, Y. Nong 5142 (HMAS 91774). Hainan, Changjiang, Bawangling, alt. 1100 m, on rotten twigs, 7 December 2000, W.Y. Zhuang, X.M. Zhang H25 (HMAS 83349); Ledong, Jianfengling, alt. 1100 m, on rotten twigs, 9 December 2000, W.Y. Zhuang, X.M. Zhang, Z.H. Yu H36, H41 (HMAS 83350, 83370); Qiongzhong, Limushan, alt. 700 m, on rotten twigs, 18 December 2000, W.Y. Zhuang, X.M. Zhang H124 (HMAS 76867); Tongzha, Wuzhishan, alt. 1000 m, on bark, 16 December 2000, W.Y. Zhuang, X.M. Zhang, Z.H. Yu, Y.H. Zhang H105 (HMAS 83371); on rotten twigs, W.P. Wu W7058 (HMAS 183161); Yunnan, Xichou, on rotten twigs, 11 November 1999, W.Y. Zhuang, Z.H. Yu 3407 (HMAS 183160).

Habitat

On rotten twigs, wood of recently dead and dying trees.

Distribution

Africa (Congo), Americas (Venezuela), Asia (China, Indonesia), possibly pantropical.

Notes

The species was formerly placed in Nectria (Fr.) Fr. and Neonectria Wollenw. until Chaverri et al. (2011) introduced Rugonectria with R. rugosa as the type species. The Chinese materials match well the description of the fungus (Samuels and Brayford 1994).

Rugonectria sinica W.Y. Zhuang, Z.Q. Zeng & W.H. Ho, in Zeng, Zhuang & Ho, Mycosystema 31(4): 467, 2013

Specimens examined

CHINA. Hainan, Changjiang, Bawanling, alt. 1100 m, on dead twigs of Quercus sp., 7 December 2000, W.Y. Zhuang, X.M. Zhang H22, H30 (HMAS 76854, 83369); Changjiang, Bawanling, alt. 1100 m, on dead twigs, 7 December 2000, W.Y. Zhuang, X.M. Zhang H28 (HMAS 76865); Lingshui, Diaoluoshan, alt. 1100 m, on bark, 13 December 2000, W.Y. Zhuang, X.M. Zhang, Z.H. Yu H70 (HMAS 76866); Henan, Jigongshan, alt. 400 m, on dead twigs, 14 November 2003, W.Y. Zhuang, Y. Nong 5099 (HMAS 91773); Fujian, Wuyishan, on dead twigs, 21 September 2006, W.Y. Zhuang, J. Luo, W.Y. Li 6846 (HMAS 183542).

Sequences

ACT (MF669046), ITS (HM054141), LSU (HM042430) and RPB1 (MF669058).

Habitat

On bark and dead twigs.

Distribution

Asia (China).

Notes

Morphologically Rugonectria sinica resembles R. castaneicola (W. Yamam. & Oyasu) Hirooka & P. Chaverri in having four-spored asci (Zeng et al. 2012). However, R. castaneicola differs in possessing perithecia that are 250–470 × 350–430 μm and larger than those of R. sinica that are 216–420 × 194–404 μm. In addition, the ascospores of R. castaneicola are larger, 18–28 × 7.5–11 μm, while those of R. sinica are 16–26 × 5.5–11 μm. The sequence analyses of the ITS and β-tubulin regions from type culture confirmed that they are different taxa (Zeng et al. 2012).

Thelonectria guangdongensis Z.Q. Zeng & W.Y. Zhuang, sp. nov.

Fungal Names: FN570488
Figure 3

Holotype

CHINA. Guangdong, Shixing, Chebaling, (24°43'17.38"N, 114°16'39.50"E), alt. 600 m, on branches, 2 November 2015, Z.Q. Zeng, X.C. Wang, K. Chen, Y.B. Zhang 10627 (HMAS 254522); ex-type culture: HMAS 247233.

Sequences

ACT (MF669045), ITS (MF669051), LSU (MF669053) and RPB1 (MF669057).

Etymology

The specific epithet refers to the type locality of the fungus.

Description

Mycelium not visible around ascomata or on natural substrata. Ascomata perithecial, solitary to gregarious, up to 10 in a group, with a well–developed stroma, superficial, subglobose to globose, bright red with a prominently darkened papilla, turning dark red in KOH, becoming slightly yellow in LA, 235–382 × 245–412 μm (n = 8). Perithecial surface slightly roughened. Perithecial wall of two layers, 20–50 µm thick, outer layer 13–37 µm thick, of textura intricata; inner layer 7.5–13 µm thick, of textura prismatica. Asci not observed. Ascospores ellipsoid, 1-septate, smooth, 10–13 × 3–5 µm (11.6 ± 1.3 × 4.2 ± 0.7 μm). Colony on PDA 28 mm diameter after 7 d under daylight at 25 °C, surface velvety, with white aerial mycelium, producing purple pigment in medium. Colony on SNA 35 mm diameter after 7 d under daylight at 25 °C, surface with sparse whitish aerial mycelium. Phialides cylindrical or slightly swollen, 20–58 × 2–4 μm. Macroconidia cylindrical, slightly curved with rounded ends, 2–5-septate, 48–70 × 4.8–5.3 μm (58.9 ± 7.14 × 5.0 ± 0.2 μm). Microconidia and chlamydospores not observed in culture.

Figure 3. 

Thelonectria guangdongensis a–d ascomata on natural substratum e colony on PDA f colony on SNA g median section through perithecium h–m ascospores n, q, r conidiogenous cells and macroconidia o, p, s–u macroconidia. Scale bars: 0.5 mm (a–d); 50 μm (g); 10 μm (h–u).

Habitat

On branches.

Distribution

Asia (China).

Notes

Amongst species of Thelonectria, T. guangdongensis resembles T. phoenicea in having subglobose to globose perithecia with slightly roughened surface, purple colony, lack of microconidia and number of septa in macroconidia (Salgado-Salazar et al. 2015). However, T. phoenicea has much larger perithecia 300–600 × 200–350 μm, wider ascospores that are 4–5.5 μm wide, and wider phialides 3–6.5 μm wide (Salgado-Salazar et al. 2015). Moreover, there are 13 bp, 44 bp, 8 bp and 54 bp divergences in the ACT, ITS, LSU and RPB1 regions, respectively, between the type of T. guangdongensis (HMAS 254522) and that of T. phoenicea (G.J.S. 85–179).

Phylogenetically T. guangdongensis is closely related to T. beijingensis with strong statistical support (MLBP/MPBP/BIPP = 100%/97%/100%) (Figure 1). However, T. beijingensis differs in having larger ascospores that are 13–17 × 4–7 μm, while those of T. guangdongensis are 10–13 × 3–5 µm and form microconidia in culture in addition to macroconidia (Zeng and Zhuang 2013). There are 20 bp, 30 bp, 5 bp and 50 bp divergences in the ACT, ITS, LSU and RPB1 regions between the ex-type culture of T. guangdongensis and that of T. beijingensis (HMAS 188498). Both morphology and molecular data support the establishment of the new species.

Thelonectria beijingensis Z.Q. Zeng, J. Luo & W.Y. Zhuang, Phytotaxa 85(1): 18, 2013

Specimen examined

CHINA. Beijing, on bark of an unidentified tree, 1 September 2010, L. Cai 7604 (HMAS 188498), ex-type culture: HMAS 188566.

Sequences

ACT (MF669047), ITS (JQ836656), LSU (MF669054) and RPB1 (MF669059).

Habitat

On bark.

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).

Thelonectria coronalis C. Salgado & Guu, in Salgado-Salazar, Rossman, Samuels, Capdet & Chaverri, Mycologia 104(6): 1339, 2012

Habitat

On bark of decaying shrubs and trees.

Distribution

Asia (China).

Notes

Salgado-Salazar et al. (2012) described T. coronalis, based on the specimens occurring on bark of decaying shrubs and trees. The fungus is only known from Taipei and Yilan of Taiwan Province.

Thelonectria coronata (Penz. & Sacc.) P. Chaverri & C. Salgado, in Chaverri, Salgado, Hirooka, Rossman & Samuels, Stud. Mycol. 68: 76, 2011

Nectria coronata Penz. & Sacc., Malpighia 11(11–12): 510, 1897.

Specimen examined

CHINA. Hainan, Lingshui, Diaoluoshan, alt. 1050 m, on rotten twigs of Pinus sp., 15 December 2000, W.Y. Zhuang, X.M. Zhang H90 (HMAS 76855).

Habitat

On bark of shrubs and trees, sometimes associated with small cankers.

Distribution

Americas (Costa Rica), Asia (Indonesia, Taiwan), possibly pantropical.

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).

Thelonectria discophora (Mont.) P. Chaverri & C. Salgado, in Chaverri, Salgado, Hirooka, Rossman & Samuels, Stud. Mycol. 68: 76, 2011

Sphaeria discophora Mont., Annls Sci. Nat., Bot., sér. 2 3: 353, 1835.

Neonectria discophora (Mont.) Mantiri & Samuels, in Mantiri, Samuels, Rahe & Honda, Can. J. Bot. 79(3): 339, 2001.

Specimens examined

CHINA. Hainan, Changjiang, Bawangling, alt. 1100 m, 7 December 2000, on rotten twigs, W.Y. Zhuang, X.M. Zhang, Z.H. Yu H24 (HMAS 83351); Lingshui, Diaoluoshan, alt. 1050 m, 15 December 2000, on rotten twigs, W.Y. Zhuang, X.M. Zhang H83, H92-1 (HMAS 83353, 83352). Yunnan, Tengchong, 16 October 2003, W.P. Wu W7097 (HMAS 183180).

Habitat

On decaying bark of shrubs and trees.

Distribution

Americas (Chile), Asia (China), Europe (Scotland).

Notes. Thelonectria discophora is the type species of the genus Thelonectria. Many specimens identified as this species were determined to be species complex until Salgado-Salazar et al. (2015) separated them into at least 16 taxa, based on phylogenetic analyses of six nuclear loci and morphological evidences.

Thelonectria ianthina C. Salgado & Guu, in Salgado-Salazar, Rossman, Samuels, Hirooka, Sanchez & Chaverri, Fungal Diversity 70(1): 12, 2015

Habitat

On decaying bark of trees and shrubs.

Distribution

Americas (Costa Rica), Asia (China).

Notes

This species is known from Heredia Province of Costa Rica and Taiwan Province of China on decaying bark of trees and shrubs (Salgado-Salazar et al. 2015).

Thelonectria japonica C. Salgado & Hirooka, in Salgado-Salazar, Rossman, Samuels, Hirooka, Sanchez & Chaverri, Fungal Diversity 70(1): 14, 2015

Specimens examined

CHINA. Hubei, Wufeng, Houhe, alt. 800 m, 13 September 2004, on rotten twigs, W.Y. Zhuang, Y. Nong 5621 (HMAS 98327); Yunnan, Tengchong, on rotten twigs, W.P. Wu W7104a (HMAS 183155).

Sequences

ACT (MK556799), ITS (HM054140) and LSU (HM042434).

Habitat

On decaying bark of Fagus crenata and possibly on bark of other shrubs and trees.

Distribution

Asia (China, Japan).

Notes

Specimens of this fungus were treated as T. discophora sensu lato until T. japonica was introduced by Salgado-Salazar et al. (2015). The morphological characteristics of the Chinese materials fit the concept of T. japonica. The Hubei and Yunnan collections extend its distribution to China.

Thelonectria lucida (Höhn.) P. Chaverri & C. Salgado, in Chaverri, Salgado, Hirooka, Rossman & Samuels, Stud. Mycol. 68: 76, 2011

Nectria lucida Höhn., Sber. Akad. Wiss. Wien, Math.-naturw. Kl., Abt. 1 118: 298, 1909.

Neonectria lucida (Höhn.) Samuels & Brayford, in Brayford, Honda, Mantiri & Samuels, Mycologia 96(3): 590, 2004.

Habitat

On decaying bark of shrubs and trees.

Distribution

Africa (Cameroon), Americas (Costa Rica), Asia (China, Indonesia), possibly pantropical.

Notes

This is a relatively common species and recorded as Neonectria lucida by Guu et al. (2007) from Taiwan Province.

Thelonectria mamma C. Salgado & P. Chaverri, in Salgado-Salazar, Rossman & Chaverri, Fungal Diversity 80: 444, 2016

Habitat

On decaying bark of shrubs and trees.

Distribution

Americas (French Guiana), Asia (China).

Notes

The specimens of this species were filed under T. lucida (Guu et al. 2007). After re-examinations of the collections from China and French Guiana, Salgado-Salazar et al. (2016) stated that they represent a separate species related to T. discophora sensu stricto.

Thelonectria phoenicea C. Salgado & P. Chaverri, in Salgado-Salazar, Rossman, Samuels, Hirooka, Sanchez & Chaverri, Fungal Diversity 70(1): 16, 2015

Specimen examined

CHINA. Hainan, Lingshui, Diaoluoshan, alt. 1050 m, 15 December 2000, W.Y. Zhuang, X.M. Zhang H86 (HMAS 76856).

Sequences

ACT (MK556800), ITS (JQ836657) and LSU (DQ119572).

Habitat

On decaying Acacia celsa and other plants.

Distribution

Asia (China, Indonesia), Oceania (Australia).

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 (Salgado-Salazar et al. 2015).

Thelonectria porphyria C. Salgado & Hirooka, in Salgado-Salazar, Rossman, Samuels, Hirooka, Sanchez & Chaverri, Fungal Diversity 70(1): 19, 2015

Specimen examined

CHINA. Hubei, Wufeng, Houhe, alt. 800 m, on rotten twigs, 12 September 2004, W.Y. Zhuang, Y. Nong 5542 (HMAS 98333).

Sequences

ACT (MK556798), ITS (HM054136) and LSU (HM042433).

Habitat

On decaying bark of Cryptomeria japonica and other woody substrates.

Distribution

Asia (China, Japan).

Notes

The collection was previously treated as T. discophora sensu lato (Zhuang 2013). The sequence analyses (Figure 1) and morphological characteristics of HMAS 98333 indicate that the correct name for the collection is T. porphyria.

Thelonectria sinensis (J. Luo & W.Y. Zhuang) Z.Q. Zeng & W.Y. Zhuang, Phytotaxa 85(1): 18, 2013

Neonectria sinensis J. Luo & W.Y. Zhuang, Mycologia 102(1): 147, 2010.

Specimen examined

CHINA. Hubei, Shennongjia, alt. 1700 m, on bark of a coniferous (?) tree, 17 September 2003, X.M. Zhang, Y.Z. Wang Z108 (HMAS 183186), ex-type culture: HMAS 173255.

Sequences

ACT (MF669048), ITS (FJ560441), LSU (FJ560436) and RPB1 (MF669060).

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 (Zeng and Zhuang 2013).

Thelonectria veuillotiana (Sacc. & Roum.) P. Chaverri & C. Salgado, Stud. Mycol. 68: 77, 2011

Nectria veuillotiana Sacc. & Roum., Rev. Mycol. 2: 189, 1880.

Neonectria veuillotiana (Sacc. & Roum.) Mantiri & Samuels, Canda. J. Bot. 79: 339, 2001.

Specimens examined

CHINA. Anhui, Jinzhai, Tiantangzhai, alt. 1000 m, on bark, 24 August 2011, W.Y. Zhuang, H.D. Zheng, Z.Q. Zeng, S.L. Chen 7869 (HMAS 266577). Hubei, Shennongjia, alt. 1200 m, on rotten twigs associated with other fungi, 15 September 2004, W.Y. Zhuang, Y. Nong 5686 (HMAS 98332); Shennongjia, alt. 1700 m, on bark associated with other fungi, 15 September 2003, X.M. Zhang, Y. Z. Wang Z196 (HMAS 183188); Xingshan, Longmenhe, alt. 1800 m, on rotten twigs associated with other fungi, 18 September 2004, W.Y. Zhuang, Y. Nong 5832 (HMAS 99207). Jilin, Changbaishan, alt. 800 m, on rotten twigs, 27 July 2012, T. Bau, W.Y. Zhuang, H.D. Zheng, Z.Q. Zeng, Z.X. Zhu, F. Ren 8246 (HMAS 266579); Jiaohe, Qianjin forest farm, alt. 450 m, on rotten twigs, 23 July 2012, T. Bau, W.Y. Zhuang, Z.Q. Zeng, H.D. Zheng, Z.X. Zhu, F. Ren 8087b (HMAS 266578). Yunnan, Tengchong, on rotten twigs associated with other fungi, 16 September 2003, W.P. Wu W7095 (HMAS 183568).

Sequences

ITS (HM054151) and LSU (HM042437).

Habitat

On bark of deciduous trees, Eucalyptus sp., Fagus sp., Gleditschia triacanthos, Salix sp.

Distribution

Asia (China), Europe (France and Germany), Azores Islands.

Notes

The species was first placed in Nectria, then in Neonectria (Mantiri et al. 2001) and recently transferred to Thelonectria by Chaverri et al. (2011). It occurs on bark of recently killed trees, rarely on wood or leaves and is cosmopolitan in distribution (Brayford and Samuels 1993; Zhuang 2013).

Thelonectria yunnanica Z.Q. Zeng & W.Y. Zhuang, Phytotaxa 85(1): 19, 2013

Specimen examined

CHINA. Yunnan, Baoshan, on bark of an unidentified tree, 15 October 2003, W.P. Wu W7122 (HMAS 183564), ex-type culture: HMAS 188567.

Sequences

ACT (MF669049), ITS (FJ560438), LSU (MF669055) and RPB1 (MF669061).

Habitat

On bark.

Distribution

Asia (China).

Notes

Thelonectria yunnanica is only known from the type locality. It is phylogenetically related to T. ostrina (Figure 1). However, T. ostrina has a perithecial wall 25–40 μm while those of T. yunnanica are thicker 49–71 μm and have asci that are (56–)67–86(−98) × 7–12 μm while those of T. yunnanica are larger, 87–120 × 8.2–9.6 μm. Unlike T. yunnanica, T. ostrina does not forming microconidia in culture (Zeng and Zhuang 2013; Salgado-Salazar et al. 2015).

Excluded species

Thelonectria jungneri (Henn.) P. Chaverri & C. Salgado, in Chaverri, Salgado, Hirooka, Rossman & Samuels, Stud. Mycol. 68: 76, 2011

Nectria jungneri Henn., Bot. Jb. 22: 75, 1895.

Neonectria jungneri (Henn.) Samuels & Brayford, Mycologia 96(3): 580, 2004.

Macronectria jungneri (Henn.) C. Salgado & P. Chaverri, in Salgado-Salazar, Rossman & Chaverri, Fungal Diversity 80: 448, 2016.

Specimen examined

CHINA. Guangdong, Dinghushan, on rotten twigs associated with other fungi, 9 October 1998, W.P. Wu W1871-2 (HMAS 183155).

Habitat

On various woody substrates, as well as other plant organic matter.

Distribution

Africa (Cameroon), Americas (Brazil, Costa Rica), Asia (China), possibly pantropical.

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. 1999; Mantiri 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, 2015, 2016; Zeng 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.

Acknowledgements

The authors would like to thank Dr. A.Y. Rossman for her valuable comments and corrections and Drs. X.C. Wang, K. Chen and Y.B. Zhang for collecting specimens jointly for this study. This work was supported by the National Natural Science Foundation of China (nos. 31750001, 31570018, 31870012) and Frontier Key Program of Chinese Academy of Sciences (No. QYZDY-SSW-SMC029).

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