A New Species and a New Record of Thyronectria (Nectriaceae, Hypocreales) in China

Phytotaxa 376 (1): 017–026 ISSN 1179-3155 (print edition) http://www.mapress.com/j/pt/ PHYTOTAXA Copyright © 2018 Magnolia Press Article ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.376.1.2

A new species and a new record of Thyronectria (Nectriaceae, Hypocreales) in China

SHENG-NAN LI1, YING ZHAO1, CHENG-MING TIAN2, THEMIS J. MICHAILIDES3 & RONG MA1* 1College of Forestry and Horticulture, Xinjiang Agricultural University, Urumqi, 830052, China 2The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China 3Department of Plant Pathology, University of California-Davis, Kearney Agricultural Research and Extension Center, Parlier, CA93648, USA *Corresponding author: [email protected]

Abstract

A new species, Thyronectria berberidis, is described and illustrated based on collections on Berberis heteropoda from the Ili area of Xinjiang Uygur Autonomous Region in China. Thyronectria berberidis is characterized by superficial, gregarious ascomata that become cupulate upon drying, with (1–6)7-septate, ellipsoidal to fusiform ascospores. In addition, T. lamyi is reported for the first time in China. The morphology characteristics of T. berberidis and T. lamyi were compared to those of close relatives. Their phylogenetic positions were confirmed by analyses of combined sequences of alpha-actin, the internal transcribed spacer, the large nuclear ribosomal RNA subunit, translation elongation factor 1-alpha and beta-tubulin. This is the first record that Thyronectria is established in the B. heteropoda in China.

Key words: Morphology, multigene analyses, phylogeny, taxonomy, Thyronectria, Xinjiang

Introduction

The family Nectriaceae (Hypocreales, Sordariomycetes, Ascomycota) contains approximately 56 genera (Lechat & Fournier 2015; Lombard et al. 2015; Voglmayr et al. 2016; Aiello et al. 2017). Two major genera, Nectria (Fr.) Fr. and Pleonectria Sacc., have been described in detail by Hirooka et al. (2012). In their comprehensive study, they suggested that the genus Pleonectria was the oldest available name for this group of fungi; however, Jaklitsch and Voglmayr (2014) demonstrated that Pleonectria and Thyronectria Sacc. are congeneric and argued that Thyronectria is older and, therefore, has priority over Pleonectria. The generic name Thyronectria was thus retained (Jaklitsch & Voglmayr 2014). Voglmayr et al. (2016) argued for the inclusion of Allantonectria Earle in Thyronectria, and to formally combine the three accepted species of Allantonectria in Thyronectria. To date, 41 species of Thyronectria were accepted. (Hirooka et al. 2012; Jaklitsch & Voglmayr 2014; Checa et al. 2015; Voglmayr et al. 2016; Zeng & Zhuang 2017; Lechat et al. 2018). The genus Thyronectria is mainly characterized by perithecia generally covered with yellow scurf; the upper part of the perithecia usually becoming cupulate when dry. Ascospores are diverse in shape and size; sometimes ascospores bud and produce ascoconidia inside the asci. Thyronectria is a cosmopolitan genus, and mainly distributed in temperate and subtropical areas, growing on dead branches, mostly saprophytically, rarely pathogenically (Hirooka et al. 2012; Jaklitsch & Voglmayr 2014; Checa et al. 2015; Lombard et al. 2015; Voglmayr et al. 2016). The study of Thyronectria in China began in 1979: ten species were discovered using modern taxonomic methods. Thyronectria rosellinii (Carestia) Jaklitsch & Voglmayr [as Ophionectria cylindrospora (Sollm.) Berl. & Voglino] was reported by Tai (1979) on Larix in Heilongjiang Province; T. balsamea (Cooke & Peck) Seeler (as Nectria balsamea Cooke & Peck) was found on decaying twigs in Liaoning Province (Zhang & Zhuang 2003; Zhuang 2013); T. pinicola (Kirschst.) Jaklitsch & Voglmayr was detected on Pinus taiwanensis in Taiwan Province (Hirooka et al. 2012) and on Pinus bungeana in Shanxi Province (He et al. 2016). Thyronectria zangii (Z.Q. Zeng & W.Y. Zhuang) Voglmayr & Jaklitsch (as Nectria zangii Z.Q. Zeng & W.Y. Zhuang) was collected on Populus branches in Donglingshan in western Beijing (Zeng & Zhuang 2012; Voglmayr et al. 2016); T. orientalis (Z.Q. Zeng & W.Y. Zhuang), T. sinensis (Z.Q.

Accepted by Wen-Ying Zhuang: 10 Oct. 2018; published: 15 Nov. 2018 17 Zeng & W.Y. Zhuang) and T. atrobrunnea (Z.Q. Zeng & W.Y. Zhuang) were collected on Pinus sp. in Henan, on Pinus sp. in Sichuan, and on Eleutherococcus senticosus in Heilongjiang, respectively, and identified using a combination of morphological and molecular data analyses (Zeng & Zhuang 2017). Furthermore, T. cucurbitula (Tode) Jaklitsch & Voglmayr was collected in Heilongjiang and T. strobi (Hirooka, Rossman & Chaverri) Jaklitsch & Voglmayr in Hubei (Zeng & Zhuang 2017). In addition, T. coryli (Fuckel) Jaklitsch & Voglmayr was collected on rotten twigs in Tibet (Zeng et al. 2018). However, there were no records of the genus Thyronectria in Xinjiang Uygur Autonomous Region. Four specimens were collected from Berberis heteropoda in Xinjiang Uygur Autonomous Region. Based on morphology and combined sequence analyses of the partial alpha-actin (act), internal transcribed spacer regions (ITS4-5.8S-ITS5), large nuclear ribosomal RNA subunit region (LSU), translation elongation factor 1-alpha (tef1) and beta-tubulin (tub) regions using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP), they were identified as a species of Thyronectria and new to science. The objective of this study was to enrich our knowledge regarding the species of Thyronectria that occur in Xinjiang Uygur Autonomous Region.

Materials and methods

Isolates and specimens Single ascospores or conidia were isolated from fresh samples of infected B. heteropoda. Isolate numbers, substrate, geographical origin and NCBI GenBank accession numbers for sequences used in the phylogenetic analyses are listed in Table 1. Representative isolates have been preserved at the China General Microbiological Culture Collection Center (CGMCC) and Centraalbureau voor Schimmelcultures (CBS). Specimens were deposited in the Forest Protection Laboratory of the Forestry and Horticultural College, Xinjiang Agricultural University (XJ-FPL).

Culture and phenotype analysis Cultures of T. berberidis were prepared based on the methods described by Jaklitsch (2009): germinating ascospores were placed on potato dextrose agar (PDA). Cultures used for the study of the asexual morph micro-morphology were grown on malt extract agar (MEA) and incubated at 25 °C under alternating 12 h daylight and 12 h darkness. Chemical tests were performed on the ascomatal wall using 3% potassium hydroxide (KOH) and lactic acid (LA). The morphological analysis was based on the microscopic characteristics of the fungal isolates. Data collection and measurement were performed using a Nikon digital camera and NIS-Elements v. 3.0 software. Colony features were examined using stereomicroscopy (Nikon SMZ25) and Nomarski differential interference contrast (DIC) microscopy using a compound microscope (Nikon Eclipse E600). The stacking software Zerene Stacker v.1.04 (Zerene Systems LLC, Richland, WA, USA) was used to construct some of the ascomata images. Measurements are reported as maxima and minima in parentheses and the range represents the mean plus and minus the standard deviation of the total number of measurements.

DNA extraction, PCR amplification and sequencing Genomic DNA was extracted from fresh mycelium using a modified CTAB method (Doyle & Doyle 1990; Fan et al. 2016). The act, ITS, LSU, tef1 and tub regions were amplified using Tact1 and Tact2 (Samuels et al. 2006), ITS4 and ITS5 (White et al. 1990), LROR and LR5 (Vilgalys & Hester 1990; Rehner & Samuels 1994), tef1-728F and tef1- 1567R (Carbone & Kohn 1999; Rehner & Buckley 2005), and βtub-T1 and βtub-T2 (O’Donnell & Cigelnik 1997), respectively. The amplification reaction was performed in a 30μL reaction volume containing 15 μL of Taq PCR

Master Mix, 10.5 μL of double-distilled water (ddH2O), 1.5 μL of DNA template, and 1.5 μL of each of the forward and reverse primers. The quality of the PCR products was checked by performing 1% agarose gel electrophoresis stained with ethidium bromide. Purification and sequencing of PCR products were carried out at Sangon Biological Engineering (Shanghai) Company Limited (Shanghai, China).

Analyses of sequence data Sequences were aligned using MAFFT v.6 (Katoh & Toh 2010). All sequences were subjected to BI analysis, ML and MP analyses: each locus was analysed separately and then with the combined/concatenated data sets. Gaps were treated as missing data (Hirooka et al. 2011). Using the methods described by Voglmayr et al. (2016), Septofusidium berolinense (Ola’h & H.-W. Ackerm.) Samson and Septofusidium herbarum (A.H.S. Br. & G. Sm.) Samson were

18 • Phytotaxa 376 (1) © 2018 Magnolia Press LI et al. tub HM484604 HM484606 HM484603 JF832888 KM232112 KM232113 JF832842 KJ570639 KT423105 HM484600 HM484597 JF832846 MH015331 MH015332 MH015333 HM484594 JF832871 KJ570642 KX514399 KX514400 HM484609 HM484596 ...continued on next page tef1 ― HM484527 HM484534 ― KM231978 KM231979 JF832548 KJ570760 KX372534 HM484521 HM484520 JF832556 MH015326 MH015328 HM015329 HM484517 JF832552 KJ570762 KX514396 KX514397 HM484524 HM484536 LSU HM484563 HM484562 HM484561 JF832687 KM231722 KM231723 JF832718 KJ570690 KT423108 HM484573 GQ505988 JF832719 MH015322 MH015324 MH015325 HM484568 JF832755 KJ570692 KX514385 KX514386 HM484572 HM484566 ITS HM484701 HM484548 HM484557 JF832630 KM231841 KM231842 JF832597 KJ570690 KT423111 HM484551 HM484555 JF832598 MH015314 MH015316 MH015317 HM484543 JF832617 KJ570692 KX514385 KX514386 HM484547 HM484539 * GenBank Accession No. GenBank act HM484505 HM484503 HM484502 JF832488 KM231250 KM231251 JF832444 KJ570664 ― HM484511 GQ505960 JF832453 MH015318 MH015320 MH015321 HM484510 JF832475 KJ570667 KX514382 KX514383 HM484514 HM484509 Country Japan France USA USA ― UK UK Spain China France USA USA China China China Austria Slovakia Greece Ukraine Canada Italy USA sp. Aesculus heteropoda heteropoda heteropoda

Substrate/Host Bark of dead wood Dead twigs of Bark Elaeagnus angustifolia ― Urtica dioica Ilex aquifolium ilex Quercus senticosus Eleutherococcus Twigs Gleditsia triacanthos Abies fraseri Berberis Berberis Berberis Ribes rubrum Picea abies Caraganae arborescens Berberis cretica Ulmus americana Agave americana Twigs a Herbarium No. BPI 879972 BPI 879981 BPI 749337 ― ― ― BPI 550125 WU32124 HMAS 271280 BPI 841465 BPI 746395 BPI 746322 2177 XJ-FPL 2433 XJ-FPL 2441 XJ-FPL BPI 746346 BPI 881052 WU 35938 WU 32130 WU 35942 BPI 878442 BPI 880697 Isolate No. MAFF 241439 CBS 125165 CBS 126570 CBS 128988 CBS 731.70 CBS 265.58 CBS 307.34 CBS136000 HMAS 271280 CBS 109874 CBS 126114 CBS 125132 CBS 144512 CGMCC3.18998 XJAU 2433-3 XJAU 2441-6 CBS 126112 CBS 128977 TCA CBS136003 CBS 141087 CBS 121121 CBS 129156 Isolates and GenBank accession numbers used in the phylogenetic analyses. 1. Species Nectria asiatica Nectria cinnabarina Nectria dematiosa Nectria nigrescens Septofusidium berolinense Septofusidium herbarum aquifolii Thyronectria asturiensis Thyronectria atrobrunnea Thyronectria aurigera Thyronectria Thyronectria austroamericana balsamea Thyronectria berberidis Thyronectria berberidis Thyronectria berberidis Thyronectria berolinensis Thyronectria boothii Thyronectria caraganae Thyronectria caudata Thyronectria Thyronectria chrysogramma concentrica Thyronectria coryli Thyronectria a ble No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 T

tub KR057951 JF832845 HM484593 MH015330 ― JF832879 JF832862 KT423104 KR057952 JF832880 JF832870 KJ570655 HM484598 KT423103 HM484595 JF832858 KM225701 KM232107 JN997421 JF832884 TUA-TPP-h tef1 KR057949 JF832551 HM484518 MH015327 KJ570766 JF832586 JF832572 KX372535 KR057950 JF832581 JF832578 KJ570775 HM484519 KX372533 HM484531 JF832567 KM225696 ― JX843454 JF832590 Forest protection laboratory of the Forestry the of laboratory protection Forest LSU KR057943 JF832757 HM484569 MH015323 KJ570699 JF832752 JF832748 KT423107 KR057944 JF832743 JF832739 KJ570708 HM484570 KT423106 GQ506001 JF832734 KM225684 KM231717 JX843458 JF832753 XJ-FPL ITS KR057943 JF832673 HM484544 MH015315 KJ570699 JF832675 JF83267 KT423110 KR057944 JF832624 JF832614 KJ570708 HM484545 KT423109 HM484542 JF832604 KM225684 KM231836 JN997424 JF832627 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands;

CBS GenBank Accession No. GenBank act KR057941 JF832447 HM484507 MH015319 KJ570673 JF832452 JF832469 ― KR057942 JF832450 JF832474 KJ570682 HM484512 ― GQ505973 JF832465 KM225678 ― JX843456 JF832510 Country Spain France Austria China Austria Japan Japan China Spain Spain USA Spain Austria China Netherlands USA France USA China USA Herbarium of the University of Vienna, Austria; Vienna, of University the of Herbarium WU

ssp. ssp. rotundifolia sp. MAFF Genebank, National Institute of Agrobiological Sciences, Ibaraki, Japan; Ibaraki, Sciences, Agrobiological of Institute National Genebank, MAFF

heteropoda

sp. sp. sp. MAFF

Substrate/Host iles Quercus Ilex aquifolium Berberis vulgaris Berberis tetrandra Tamarix Castanopsis Pinus koraiensis Pinus Pistacia lentiscus ilex Quercus Abies balsamea Retama sphaerocarpa Acer campestre Pinus Hedera helix Pinus strobus Ostrya carpinifolia elata Yucca Populus Unid. Dead bark a Herbarium No. AH 47011 BPI 879857 BPI 746349 2365 XJ-FPL WU 32142 TUA-TPP-h93 BPI 881061 HMAS 252896 AH 45402 BPI 871328 BPI 881066 WU32153 BPI 746398 HMAS 271282 CBS H-19479 BPI 1107115 WU33426 ― HMAS 251258 BPI 881069 U. S. National Fungus Collections USDA-ARS, Beltsville, MD, USA; BPI Isolate No. CBS 139474 CBS 128978 CBS 115034 XJAU 2365-4 CBS 136923 CBS 129745 MAFF 24145 HMAS 252896 CBS 139475 CBS 128976 CBS 129162 MA CBS 125131 HMAS 271282 CBS 462.83 CBS 102036 NP10 CBS 125499 HMAS 251258 CBS 129157 (Continued) Species giennensis Thyronectria ilicicola Thyronectria lamyi Thyronectria lamyi Thyronectria obscura Thyronectria okinawensis Thyronectria pinicola Thyronectria orientalis Thyronectria pistaciae Thyronectria quercicola Thyronectria rosellinii Thyronectria roseovirens Thyronectria rhodochlora Thyronectria sinensis Thyronectria sinopica Thyronectria strobi Thyronectria virens Thyronectria yuccae Thyronectria zangii Thyronectria Zanthoxyli Thyronectria Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; Beijing, Sciences, of Academy Chinese Microbiology, of Institute Table 1. Table No 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Universidad de Alcalá, Madrid, Spain; AH Numbers in bold indicating the newly submitted sequences. * a HMAS Japan; Tokyo, Agriculture, of University Tokyo Herbarium, Lab Protection Plant Tropical Xinjiang, China. Agricultural University, and Horticultural College, Xinjiang

20 • Phytotaxa 376 (1) © 2018 Magnolia Press LI et al. selected as outgroups for inferring the taxonomic positions of the new species. JMODELTEST (Posada 2008) was used to calculate nucleotide substitution models for each gene/partition for the ML and BI analyses. After the likelihood scores were calculated, the models were selected according to the Bayesian Information Criterion (BIC) (Posada & Buckley 2004). After JMODELTEST was run, likelihood settings for trees were set for each gene. BI analysis was performed using MrBayes 3.2.6. Two independent analyses of two parallel runs and four chains were carried out for 5,000,000 generations and sampled every 5000 generations, resulting in 1000 trees in total. The first 25% of the resulting trees were eliminated as the burn-in phase of each analysis. Branches with significant Bayesian inference posterior probabilities (BIPP) were estimated for the remaining 750 trees. ML analyses were performed with RAxML (Stamatakis 2006), using the ML and rapid bootstrap setting with 1000 bootstrap replicates. Substitution model parameters were calculated separately for the different gene regions included in the combined analyses. MP analysis was performed with PAUP 4.0b10 (Swofford 2003), using the heuristic search option of 1000 random-addition sequences with tree bisection and reconnection (TBR) branch-swapping. Descriptive tree statistics for parsimony tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated for the maximum parsimonious tree (MPT). MP bootstrap analysis was conducted using 1000 replicates, each with ten replicates of a random stepwise addition of taxa (Felsenstein 1985).

Results

The sequence datasets for act, ITS, LSU, tef1 and tub from 41 taxa belonging to genera of Thyronectria and Nectria were analyzed. The combined data sets comprised 2467 characters, of which 1372 characters were constant, 310 variable characters were parsimony-uninformative, and 785 were parsimony informative. In the MP analysis, the reconstructed trees were 3176 steps long with CI = 0.475, RI = 0.593, RC = 0.282, HI = 0.525. MP and ML bootstrap support values above 50% and BIPP above 90% are given in Fig. 1 above or below the branches.

FIGURE 1. Phylogram of combined act, ITS, LSU, tef1 and tub genes generated from maximum parsimony (MP) analyses. Values above or below the branches indicate Bayesian inference posterior probabilities (BI PP≥90%)/maximum likelihood bootstrap (ML BP≥50%)/ maximum parsimony bootstrap (MP BP≥50%). Strains that were isolated in this study are marked in bold.

Thyronectria berberidis R.Ma & S.N.Li, sp. nov. Fig. 2. MycoBank # MB 825056

FIGURE 2. Thyronectria berberidis. A–B: Perithecia on natural substrata. C: Median section of perithecium in water. D: Median section of perithecium in LA. E: Asci. F–K: Ascospores. L: Colonies on PDA after 4 days (left) and 9 days (right). Scale bars: A = 2 mm; B = 100 μm; C–D = 50 μm; E–K = 10 μm.

Holotype:—CHINA, Xinjiang Uygur Autonomous Region, Ili, Huocheng County, 61 groups of natural protected forest, 44°27’37.40’’N, 80°21’46.68’’E, alt. 1184 m, on twigs of Berberis heteropoda, R. Ma, 22 July 2017, (XJ-FPL 2177, living ex-type culture, CGMCC3.18998 and CBS 144512). Etymology:—berberidis, named after the host genus, Berberis. Host/Distribution:—from Berberis heteropoda in northwestern China. Original description:—Asexual state: Undetermined. Sexual morph: mycelium not visible around the ascomata or on the host. Stromata erumpent through the epidermis, (0.37–)0.5–0.78(–0.95) mm high (n = 40), 0.3–1.7(–4.6) mm in diam., brown to black, becoming purple red in KOH and yellow in LA, pseudoparenchymatous, cells forming textura angularis, intergrading with the ascomatal wall. Ascomata were superficial, aggregated in groups of 3–28, brown dark, becoming purple in KOH and yellow in LA, globose to obovoid, slightly rough, 235–286 μm high, 253–352 μm diam., cupulate upon drying. The ascomatal wall was 38–59 μm thick and composed of two regions: outer region 34–45-μm thick, intergrading with the stroma, cells forming textura globulosa or textura angularis, with pigmented walls about 1.5-μm thick; inner region 17–27μm thick and composed of elongate, thin-walled, hyaline cells, forming textura prismatica. Asci were clavate, increasing in size as the ascospores matured, eight-spored, biseriate above, uniseriate below, (55.5–)55.7–69.3(–73.7) × (9.4–)9.6–11.9(–12.8) μm (n = 15). Ascospores were ellipsoidal to fusiform, slightly curved, (1–6)7-septate, (11.9–)18.6–26.4(–32.4) × (4.3–)6.2–8.4(–9.8) μm (n = 80), hyaline and smooth. Culture characteristics:—In culture, the colony grew on average 7.3 mm/d on PDA at 25 °C. On day 4, the mycelium appeared white. After 9 days, concentric circles of a pale-orange pigment were visible in the medium, and by day 15, the mycelia filled the plate. Other specimens examined:—Xinjiang Uygur Autonomous Region, Ili, Huocheng county, Fushou mountain, 44°25’37.94’’N, 80°47’17.31’’E, alt. 1182 m, on twigs of Berberis heteropoda, R. Ma, 15 August 2017 (XJ-FPL 2433, paratype; living culture, XJAU 2433-3); Xinjiang Uygur Autonomous Region, Ili, Huocheng county, Fushou mountain, 44°25’35.27’’N, 80°47’21.45’’E, alt. 1197 m, on twigs of Berberis heteropoda, R. Ma, 15 August 2017 (XJ- FPL 2441, paratype; living culture, XJAU 2441-6).

22 • Phytotaxa 376 (1) © 2018 Magnolia Press LI et al. Notes:—The major taxonomic characters of the fungus, such as the well-developed stromata on natural substrates and multiseptate ascospores in asci are strong evidence that this species belongs to the genus Thyronectria. Among the known species of the genus, T. berberidis is closely related to T. aurigera which has clavate asci and transversely septate ascospores not forming ascoconidia. However, T. aurigera differs from T. berberidis in having wider asci [10–20 μm vs. (9.4–)9.6-11.9(–12.8) μm] and smaller ascospores [(14.9–)17–20.8(–24.7) × (4.4–)5.0–6.4(–7.3) μm vs. (11.9–)18.6–26.4(–32.4) × (4.3–)6.2–8.4(–9.8) μm] (Hirooka et al. 2012). Moreover, the five-locus phylogeny indicates that T. berberidis is a phylogenetically separate taxon.

Thyronectria lamyi (Desm.) Seeler, J. Arnold Arbor. 21: 449. 1940. Fig. 3. Basionym: Sphaeria lamyi Desm., Ann. Sci. Nat., Bot., sér. 2, 6: 246, 1836. = Nectria lamyi (Desm.) De Not., Sfer. Ital., 1: 13. 1863. = Pleonectria lamyi (Desm.) Sacc., Mycotheca Ven. No. 688. 1876.

FIGURE 3. Thyronectria lamyi. A–C: Perithecia on natural substrata. D: Ascus. E: Budding ascospores. F: Ascoconidia. G: Colony on PDA after 5 days at 25 °C. H: Median section of perithecium in water. I: Median section of perithecium in LA. J: Conidiophores and lateral phialidic pegs on MEA. K: Conidia on MEA. Scale bars: A = 200 μm; B–C = 100 μm; D–F, J–K= 10 μm; H–I = 50 μm.

Host/Distribution:—Pathogen on twigs and branches of Berberis heteropoda. Known from Berberis aquifolium, B. candidula, B. gagnepainii, B. vulgaris, B. thunbergii in Austria and B. hispanica in Spain. Descriptions:—Mycelium not visible around the ascomata or on the host. Stromata erumpent through the epidermis, 0.5 mm high, 0.8 mm diam., black brown, becoming dark red in KOH and yellow in LA pseudoparenchymatous, cells forming textura angularis, intergrading with the ascomatal wall. Ascomata superficial on well-developed stromata, aggregated in groups of 4–9, subglobose to globose, 189–228 μm high, 202–244 μm diam., not cupulate when dry,

A new species and a new record of Thyronectria Phytotaxa 376 (1) © 2018 Magnolia Press • 23 blackish brown in colour, becoming purple in KOH and yellow in LA, sometimes the surface with green or slightly yellowish scurf or scaly. Asci widely clavate and increasing in size as ascospores mature, 72.1–100.6(–112.8) × (10.3–)14.2–26.7(–30.6) μm (n = 15), eight-spored, ascospores mainly biseriate. Ascospores ellipsoidal to fusiform, hyaline, constricted at septa, with 5 transverse septa, 1(–2) longitudinal septum, (15.4–)23.7–36.7(–45.1) × (5.1–)6.4– 9.0(–10.5) μm (n = 70), smooth, budding to produce hyaline, thin-walled, bacillar ascoconidia, (3.2–)3.9–4.7(–5.7) × (1.0–)1.3–1.8(–2.8) μm, l/w = (1.65–)2.4–2.8(–3) (n = 100), that fill asci. Asexual morph:—Not observed. Culture characteristics:—In culture, the colony grew on average 8.8 mm/d on PDA at 25 °C; the colour of the colony was slightly orange with concentric round grains after 5 days. Colony growth on MEA 9 mm/d at 25 °C, pale- orange pigment, with white aerial mycelium. Conidiophores developed on aerial mycelium, 10–23.5 μm long, 1.8–2.7 μm wide at base. Conidia ellipsoidal, oblong to cylindrical, hyaline, straight or slightly curved, rounded at both ends, non-septate, smooth, 4.3–6.7 × 1.3–1.9 μm (n=150). Specimen examined:—CHINA, Xinjiang Uygur Autonomous Region, Ili, Huocheng County, Apeiying ditch, 44°25’54.94’’N, 80°46’37.37’’E, alt. 1304 m, on twigs of Berberis heteropoda, R. Ma, 14 August 2017 (XJ-FPL 2365, XJAU 2365-4). Notes:—Thyronectria lamyi used to be called Pleonectria lamyi, and has only been recorded on Berberis spp. The fungus was previously known only from Asia (Pakistan), Europe and North America (Hirooka et al. 2012; Jaklitsch & Voglmayr 2014). This is the first collection of Thyronectria lamyi in China and is the first report from Berberis heteropoda. Compared with the description of Hirooka et al. (2012), the Chinese collection has slightly smaller asci [72.1–100.6(–112.8) × (10.3–)14.2–26.7(–30.6) μm vs. 70–145 × 10–40 μm], larger ascospores [(15.4–)23.7–36.7(– 45.1) × (5.1–)6.4–9.0(–10.5) μm vs. (14.5–)18.9–26.1(–32.2) × (5–)5.2–8(–10.8) μm], and the colour of ascomata is slightly different (blackish brown vs. bay to scarlet), which are treated as infraspecific variation.

Discussion

Thyronectria berberidis and T. lamyi were identified by morphology and phylogenetic analyses that combined five loci (act, ITS, LSU, tef1 and tub) and are associated with on Berberis heteropoda in China. T. berberidis appeared as an independent lineage and is distinct from all other currently described and sequenced species in the phylogenetic tree (Fig. 1). Thronectria berberidis can be distinguished from T. aurigera in having wider asci and smaller ascospores (Hirooka et al. 2012) and distinguished from T. caudata and T. lamyi in ascospores not budding in asci to produce ascoconidia, and by the analysis of sequence data (Hirooka et al. 2012; Jaklitsch & Voglmayr 2014). Thyronectria lamyi is grouped with T. caudata in the same subclade; however, the ascoconidium l/w ratio of the latter is larger [(2.5–)3.0–4.0(–5.0) vs. (1.65–)2.4–2.8(–3)], and the ascospores are longer [(21–)25–33(–39) μm vs. (15.4–)19.1–36.5(–36.8) μm long] (Jaklitsch & Voglmayr 2014). The ascoma colour of T. lamyi appears to be variable: Hirooka et al. (2012) recorded the ascomata bay to scarlet and with the apical region nearly black, Jaklitsch & Voglmayr (2014) reported that as orange over red to brown, and we observed blackish brown ascomata. This suggests that ascomatal colour is not a reliable character for morphological identification of T. lamyi, but the character of micro- morphology: the colour reaction of perithecia in KOH and LA, characteristic of ascospores budding in asci to produce ascoconidia, and the l/w radio of ascoconidia can be better to identified it. Reaction of perithecial wall to KOH is considered to be an important character in the taxonomy of hypocrealean fungi (Rossman et al. 1999). The perithecia of most species of Thyronectria turn dark in KOH, becoming blood-red or purple, and turn yellow in LA; however, some species display a negative reaction to KOH (Zeng & Zhuang 2017). The middle layer of the perithecial wall of T. abieticola (Lechat, Gardiennet & J. Fourn) becomes greenish grey in KOH and red in LA, whereas, the inner layer turns yellow in both KOH and LA (Lechat et al. 2018). The perithecia of T. berberidis appears to be purple in KOH and yellow in LA. Our phylogenetic tree indicates that T. caudata, T. lamyi and T. berberidis form a well-separated group (Fig. 1). Zeng and Zhuang (2017) indicated that the species of Thyronectria mostly show host-specificity. Thyronectria austroamericana (Speg.) Seeler, J. Arnold Arbor is known only species from Fabaceae; T. aquifolii (Fr.) Jaklitsch & Voglmayr and T. ilicicola (Hirooka, Rossman & P. Chaverri) Jaklitsch & Voglmayr are restricted to Ilex aquifolium (Aquifoliaceae); T. balsamea (Cooke & Peck) Seeler and T. rosellinii (Carestia) Jaklitsch & Voglmayr is found on Abies; T. berolinensis (Sacc.) Seaver is restricted to Ribes; T. cucurbitula, T. strobi and T. pinicola are restricted to Pinus (Hirooka et al. 2012; Jaklitsch & Voglmayr 2014; Zeng & Zhuang 2017). Although ten species of Thyronectria have been reported in China, only T. lamyi and T. berberidis have been collected from Berberis heteropoda (Tai 1979;

24 • Phytotaxa 376 (1) © 2018 Magnolia Press LI et al. Zhang & Zhuang 2003; Hirooka et al. 2012; Zeng & Zhuang 2012; Zhuang 2013; Zeng & Zhuang 2017; Zeng et al. 2018). More collections are needed to update our knowledge of host specificity of the genus.

Acknowledgements

This study was supported by the National Key Research and Development Plan of China (grant number: 2016YFC05011501) and the National Natural Science Foundation of China (grant number: 31460198). We are grateful to the China General Microbiological Culture Collection Center (CGMCC) and Centraalbureau voor Schimmelcultures (CBS) for supporting strain preservation during this study. The Dean of College of Life Science in Xinjiang University is thanked for letting us use her photographic equipment.

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