Species Delimitation for Neonectria Coccinea Group Including the Causal Agents of Beech Bark Disease in Asia, Europe, and North America

Species delimitation for Neonectria coccinea group including the causal agents of beech bark disease in Asia, Europe, and North America

Yuuri HIROOKA1* Amy Y. ROSSMAN2 Wen-Ying ZHUANG3 Catalina SALGADO-SALAZAR4 Priscila CHAVERRI4

1Forestry & Forest Products Research Institute, Department of Forest Microbiology, 1 Matsunosato, Tukuba, Ibaraki, 305-8687, JAPAN 2Systematic Mycology and Microbiology Laboratory, United States Department of Agriculture, Agricultural Research Service, 10300 Beltsville Ave., Beltsville, Maryland 20705, USA 3State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China 4University of Maryland, Department of Plant Science and Landscape Architecture, 2112 Plant Sciences Building, College Park, Maryland 20742, USA

Abstract: Neonectria coccinea has historically been known as a serious pathogen of beech trees in North America and Europe. Phylogenetic relationships of this species and its relatives have been determined. Neonectria faginata, previously referred to as Neo. coccinea var. faginata has been delimited from Neo. coccinea sensu stricto and Neo. punicea. Five additional lineages supported by relatively high phylogenetic values are allied with Neo. coccinea and its relatives. Until now, these lineages have not been recognized as species because no significant morphological differences among them were found. Although each phylogenetic species appeared to be unique in host specificity and geography, relatively few specimens of these species existed. We re-evaluated these species using newly obtained specimens and isolates from Asia, Europe, and North America. Based on sequences of multiple loci, specifically act, ITS, LSU, rpb1, tef1 and tub, our results indicate that these isolates represent four phylogenetic species, Neo. coccinea, Neo. faginata, Neo. microconidia, and Neo. punicea. In our phylogeny, each species is well supported by high BI PP, MP BP, and ML BP values. The single-locus analyses of ITS and LSU did not correlate with our species recognition criteria. Based on morphology, phylogeny, host specificity and geographical traits, these four species are conclusively delimited. Each of them is re-described and illustrated. Epitype specimens for Sphaeria coccinea and Neonectria coccinea var. faginata are designated. Because no type specimen of Neo. punicea was found, this name is neotypified. Within Neo. coccinea, Neo. microconidia, and Neo. punicea, some sublineages were supported by high phylogenetic values. In the future these may warrant recognition as distinct species. Key words: invasive pathogens, molecular systematics, Neonectria coccinea, Neonectria faginata, Neonectria microconidia, Neonectria punicea, species concept, typification

*Corresponding author. E-mail: [email protected] Received: 18-12-2012, accepted: 13-03-2013 486 ISSN1672-6472 CN11-5180/Q Mycosystema May 15, 2013 Vol.32 No.3

含亚洲、欧洲、北美洲山毛榉病害病原菌的 Neonectria coccinea 复合群的物种概念 Yuuri HIROOKA1* Amy Y. ROSSMAN2 Wen-Ying ZHUANG3 Catalina SALGADO-SALAZAR4 Priscila CHAVERRI4

1Forestry & Forest Products Research Institute, Department of Forest Microbiology, 1 Matsunosato, Tukuba, Ibaraki, 305-8687, JAPAN [日本] 2Systematic Mycology and Microbiology Laboratory, United States Department of Agriculture, Agricultural Research Service, 10300 Beltsville Ave., Beltsville, Maryland 20705, USA [美国] 3State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China [中国] 4University of Maryland, Department of Plant Science and Landscape Architecture, 2112 Plant Sciences Building, College Park, Maryland 20742, USA [美国]

摘要：对包含亚洲、欧洲、北美洲山毛榉病害病原菌在内的 Neonectria coccinea 复合群中物种之间的系统演化关系以及物种概念进行了探讨。Neonectria faginata（曾处理为 Neo. coccinea var. faginata）与狭义的 Neo. coccinea 和 Neo. punicea 有明显区别。系统树显示 5 个分支与 Neo. coccinea 群的关系较为接近，由于缺乏显著的形态差异，它们始终没有在物种的等级上区分。根据形态学、系统发育、寄主专化性和地理因素，采用多基因 act、ITS、LSU、rpb1、tef1 和 tub 序列分析的方法，对亚洲、欧洲、北美洲的相关材料进行了研究，结果表明，它们代表 4 个独立的物种，即 Neo. coccinea，Neo. faginata，Neo. microconidia 和 Neo. punicea，对每个种进行了详尽的描述和图示。并为 Sphaeria coccinea 和 Neonectria coccinea var. faginata 指定了附加模式。关键词：入侵性病原菌，分子系统学，猩红新丛赤壳，Neonectria faginata，小孢新丛赤壳，Neonectria punicea，物种概念，选模式

INTRODUCTION The beech scale insect was introduced into Nova The beech bark disease (BBD) constitutes one Scotia through seedling beech trees brought from of the greatest global threats to beech forests. This Europe in 1890 (Ehrlich 1934; Hewitt 1914). Recent disease occurs following the interaction between the molecular studies indicate that the causal agents of beech scale insect, Cryptococcus fagisuga Lind. and BBD were introduced from Europe, yet it is unclear one of the three ascomycetous fungi, Neonectria if the beech scale insect and BBD fungal pathogens coccinea (Pers.) Rossman & Samuels, Neo. faginata were introduced into North America at the same (M.L. Lohman, A.M.J. Watson & Ayers) Castl. & time (Mahoney et al. 1999; Gwiazdowski et al. Rossman, and Neo. ditissima (Tul. & C. Tul.) 2006). The disease has now rapidly spread across Samuels & Rossman. The disease was first the northeastern United States and southeastern documented in Germany in 1849 (McIntosh 1849). Canada (Houston 1994; Griffin et al. 2003) with

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80%–90% mortality rates of mature beech (Houston tubercularia-like anamorph (Hirooka et al. 2011, 1994). 2012). Previously considered to be a synonym of The BBD pathogenic fungi are Neonectria Nectria, Rossman et al. (1999) resurrected the genus coccinea on Fagus in Europe, Neo. faginata on Neonectria Wollenw. (Wollenweber 1916), typified Fagus in North America, and Neo. ditissima on by Neo. ramulariae Wollenw., for nectria-like hardwoods in North America and Europe, rarely species having a cylindrocarpon-like anamorph. Asia. These fungi have red, globose, slightly Many nectrioid species having cylindrocarpon-like roughened perithecia, 1-septate ascospores, and anamorphs were included in the genus Neonectria. cylindrocarpon-like anamorphs (Rossman et al. Chaverri et al. (2011) divided Neonectria into five 1999). They are morphologically similar and often genera: Campylocarpon Halleen, Schroers & Crous, misidentified because they are distinguished by only Ilyonectria P. Chaverri & C. Salgado, Neonectria small differences in ascospore size. Castlebury et al. Wollenw., Rugonectria P. Chaverri & Samuels, and (2006) conducted taxonomic studies of these fungi Thelonectria P. Chaverri & C. Salgado. These genera based on morphological observation and combined were defined based on a multiple locus phylogeny phylogenetic analyses of rpb2, tef1, and tub gene and morphological observations. regions. They clarified the differences between Neonectria coccinea with its anamorph Neonectria coccinea, Neo. ditissima, and Neo. Cylindrocarpon candidum (Link: Fr.) Wollenw. has faginata (=Neo. coccinea var. faginata), newly been considered a common species distributed recognized at the species rank. The phylogenetic tree widely in the northern hemisphere, frequently of Castlebury et al. (2006) also showed that reported on twigs and wood of beech trees as well as potentially five distinct species exist that are sister to on other deciduous trees, dung, and soil (Domsch et Neo. coccinea. Although each phylogenetic species al. 2007). The basionym of the anamorphic fungus, seemed to be segregated by host specificity and Fusidium candidum Link, was described by Link geography, no significant morphological differences (1809) and sanctioned by Fries (1832). Wollenweber among them were found due to the limited number (1928) transferred Fusidium candidum to of samples. Cylindrocarpon. Booth (1966) first introduced the Neonectria coccinea was originally described teleomorph-anamorph connection as states of one as Sphaeria coccinea Pers.: Fr. by Persoon (1800). species, and recent studies have confirmed the After Fries (1823) sanctioned Sphaeria coccinea, he connection. The recent study by Castlebury et al. transferred this species to Nectria (Fries 1849). (2006) restricted this species to Fagus in Europe. Because Persoon (1800) and Fries (1823, 1849) did Using combined sequences of several loci, a not typify any specimens, Booth (1959) found number of new species within Neonectria sensu lato several Persoon specimens of S. coccinea and were recently described from China (Luo & Zhuang designated one of them as the lectotype. Since the 2010; Zhao et al. 2011). Neonectria confusa J. Luo 19th century, the broadly conceived genus Nectria & W.Y. Zhuang was placed in the Neo. coccinea has been narrowed. Most recently the genus Nectria group while Neo. microconidia J. Luo, P. Zhao & was determined to include fungi having red W.Y. Zhuang was sister to the Neo. coccinea clade. perithecia without bright yellow scurf and Although Luo & Zhuang (2010) and Zhao et al.

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(2011) recognized Neo. confusa and Neo. low nutrient agar (SNA; Nirenberg 1976). Cultures microconidia as distinct species, the variation on SNA were incubated at 25℃ with alternating between them was relatively low with subtle 12h/12h fluorescent light/darkness for 2–3 wks. morphological differences from other species in the Images were captured with a Nikon DXM1200 Neo. coccinea group. A taxonomic reappraisal of the digital camera. Some composite images were made Neo. coccinea group is needed. with Helicon Focus version 4.21.5 Pro (Helicon To determine the species circumscription within Soft, www.heliconfocus.com). Color terminology the Neo. coccinea group, detailed morphological and used for perithecia, ascospores, conidia, and top and molecular phylogenetic analyses were undertaken. reverse colony, were based on Kornerup & Many isolates from freshly collected and herbarium Wanscher (1978). specimens around the northern hemisphere, Statistical analysis particularly Asia, Europe, and North America, were Measurements of morphological characters used to define the species in the Neo. coccinea such as length and width were made using Scion group. In order to identify the most suitable locus for Image software beta version 4.0.2 (Scion recognition of cryptic species in this group, the Corporation, Frederick, Maryland, USA) and are multi-locus genealogies as well as a single locus based on up to 50 measurements for structures in approach were compared. Each species is described each isolate and specimen. For morphological and illustrated, and a key is provided. structures, descriptive statistics (minimum, mean, 1 MATERIALS AND METHODS median, maximum and standard deviation) were Specimens and isolates computed. All computations were performed using Specimens and isolates of the Neo. coccinea Systat 10 (Systat Software, San José, California, group preserved in herbaria and culture collections USA). Only isolates for which all data existed were were obtained and examined in this study. Fresh included in the analysis. Ranges are reported as specimens of the teleomorphs were collected and mean values ± one standard deviation; the number of sent by authors or collaborators. These specimens items measured is given in parentheses together with are deposited in the U.S. National Fungus maximum and minimum. Collections (BPI), Beltsville, Maryland, United Growth trial States. Single ascospore or conidial cultures were Disks of 5mm diam. were cut from the edge of established as described earlier (Hirooka et al. 2011, young colonies and placed in the center of PDA 2012). Newly isolated cultures are preserved at the plates, then incubated at temperatures from 15℃ to CBS Fungal Biodiversity Centre (CBS, Utrecht, 35℃ at 5℃ intervals in complete darkness. Netherlands), and/or GenBank, National Institute of Diameters of the colonies on three plates for each Agrobiological Sciences (NIAS, Tsukuba, Ibaraki, isolate at each temperature were measured daily for Japan). ten days. This trial was replicated three times for Morphology each isolate. Methods of morphological observation are DNA extraction, PCR, and sequencing described by Hirooka et al. (2011, 2012). To observe In total, 75 cultures of Neo. coccinea group and sporulating structures, the cultures were grown on a three isolates of Neonectria hederae (C. Booth)

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Castl. & Rossman (Table 1) were grown in Difco™ and IMI 058770a, were used as the outgroup for potato dextrose broth in 6cm diam. Petri plates for inferring inter- and intra-specific relationships. about one to two weeks at 20℃. Mycelial mats were JMODELTEST (Posada 2008) was used to obtained in a laminar flow hood and dried with calculate the models of nucleotide substitutions for clean, absorbent paper towels. DNA was extracted each gene/partition for the ML and BI analyses. The with Ultra Clean™ Plant DNA Isolation Kit (MO number of substitution schemes was set to 11, base BIO Laboratories Inc., Solana Beach, California, frequencies +F, rate variation +I and +G, and the USA). base tree for likelihood calculations was set to ‘ML For each isolate, six loci were sequenced, optimized’. Eighty-eight models were compared. namely α-actin (act) (Carbone & Kohn 1999), After calculation of likelihood scores, the models β-tubulin (tub) (O’Donnell & Cigelnik 1997), RNA were selected according to the Akaike information polymerase II subunit one (rpb1) (Castlebury et al. criterion (AIC) (Posada & Buckley 2004). After 2004), the internal transcribed spacer (ITS) (White jMODELTEST, likelihood settings for trees were set et al. 1990), large subunit nuclear ribosomal DNA to each gene (Table 2). (LSU) (Vilgalys n.d.), and translation elongation BI analysis was done using MrBayes 3.1.2 factor 1-α (tef1) (Carbone & Kohn 1999; Rehner (Huelsenbeck et al. 2001, 2002). BI data were 2001). The primers and PCR protocol information partitioned by locus and the parameters of the are listed in Tables 2. PCR products were purified nucleotide substitution models for each partition with ExoSAP-IT® (USB Corporation, Cleveland, were set as described (Table 2). For these analyses, Ohio, USA) following the manufacturer’s two independent analyses of two parallel runs and instructions. Clean PCR products were sequenced at four chains were carried out for 10,000,000 the DNA Sequencing Facility (Center for generations using MrBayes. Analyses were initiated Agricultural Biotechnology, University of Maryland, from a random tree and trees sampled every 100th College Park, Maryland, USA) and at MCLAB generation. The first 20% of the resulting trees were (Molecular Cloning Laboratories, San Francisco, eliminated (=‘burn in’). A consensus tree (‘sumt’ California, USA). Sequences were assembled and option) and posterior probabilities (PP) were edited with Sequencher 4.9 (Gene Codes, Madison, calculated in MrBayes. Wisconsin, USA). Sequences are deposited in For the ML and bootstrap analyses (BP), GenBank (Table 1). GARLI version 0.96 (Zwickl 2006) was computed Phylogenetic analyses through the Grid computing (Cummings & Sequences of the six loci were aligned with Huskamp 2005) and The Lattice Project (Bazinet & MAFFT version 6 (Katoh 2008) and the alignment Cummings 2008), which includes clusters and was visually improved with Mesquite version 2.74 desktops in one integrated network (Myers et al. (Maddison & Maddison 2010). Phylogenetic 2008). In GARLI, the starting tree was made by analyses with aligned sequences were performed stepwise-addition and the number of runs or search using the Bayesian (BI), Maximum likelihood (ML) replicates was set to 50. 2000 ML BP replicates were and Maximum parsimony (MP) analyses. Three done in GARLI, with the starting tree chosen isolates of Neo. hederae, CBS 714.97, A.R. 4523 randomly.

Mycosystema 490 ISSN1672-6472 CN11-5180/Q Mycosystema May 15, 2013 Vol.32 No.3 rpb1 tef1 tef1 rpb1 tub GenBank accession No. ITS LSU act act States KC660742 - - - - States KC660733 - - - - ed it Country Country Un United United States KC660409 KC660519 KC660600 KC660653 KC660457 KC660743 United Slovakia - - - - - KC660720 - - - - Slovakia Canada KC660412 HQ840385* HQ840382* HQ840393* JF268746* JF268730* HQ840393* JF268746* HQ840385* HQ840382* Canada KC660412 United States - United KC660518 KC660560 KC660657 KC660456 DQ789856* United States KC660408 KC660513 KC660562 KC660658 KC660451 KC660735 United Germany KC660422 KC660521 KC660620KC660727 KC660672 JF268734* United States KC660404 KC660509 KC660557 KC660661 KC660447 KC660736 United Slovakia KC660424 KC660506 KC660593 KC660667 KC660444 KC660724 Slovakia KC660415 KC660502 KC660591 KC660663 KC660438 KC660716 United States KC660406 KC660517 KC660555 KC660652 KC660455 KC660740 United Slovakia KC660423 KC660499 KC660609 KC660668 KC660434 KC660721 Slovakia KC660417 KC660504 KC660577 KC660665 KC660440 KC660717 United States - United KC660511 KC660559 KC660651 KC660449 KC660730 Romania KC660416 - Slovakia KC660578 KC660666 KC660441 KC660718 KC660421 - KC660579 KC660671 KC660437 KC660726 Romania KC660425 KC660505 KC660601 KC660664 KC660442 KC660719 France France KC660420 KC660501 KC660619 KC660670 KC660436 KC660723 KC660419 KC660500 KC660607 KC660669 KC660435 KC660725 Japan KC660426 KC660548 KC660552 KC660674 KC660493 KC660729

sp. Scotland KC660418 - KC660581 KC660673 KC660467 KC660722 sp. sp. States KC660407 KC660498 KC660571 KC660655 KC660433 KC660741 United Substrate/Host Substrate/Host Fagus grandifolia Fagus grandifolia Fagus grandifolia Fagus sylvatica sylvatica Fagus Fagus grandifolia Fagus sylvatica Fagus Fagus sylvatica Fagus Fagus sylvatica Fagus Fagus sylvatica Fagus Fagus grandifolia Fagus sylvatica Fagus Fagus sylvatica Fagus Fagus sylvatica Fagus Fagus sylvatica Fagus Fagus grandifolia Fagus Fagus sylvatica Fagus Fagus crenata Fagus Fagus grandifolia Fagus sylvatica Fagus sylvatica Fagus No. No. 878329 871114 871114 871125 871125 871115 871115 871113 871113 871123 871123 870945 870941 870937 802648 870940 870943 870939 871095

BPI 880528 Table 1 Isolates and accession numbers used in the phylogenetic analyses analyses phylogenetic the in used numbers accession and Isolates 1 Table 134254 BPI 134254 119155 - 119155 119154 - 119154 134246 BPI 134246 134253 - 134253 119522 BPI 119522 119199 BPI 119199 119523 BPI 119523 118918 BPI 118918 119150 BPI 119150 119519 BPI 119519 118915 BPI 118915 BPI 119156 118917 BPI 118917 118916 BPI 118916 119161 BPI 119161 3680=CBS No. No. Herbarium 92-33=CBS 346=AR 3682=CBS 3677=CBS 4307=CBS 3712=CBS 3701 BPI 4167=CBS 3691=CBS 4197=CBS 3705=CBS 4149=CBS 3707=CBS 3700=CBS 4152=CBS 3708=CBS 4130=CBS 3687=CBS CBS 217.67=ATCC 16547=IMI CBS 217.67=ATCC 105738 118983 118983 CBS 119158=G.J.S. 98-114 CBS 119158=G.J.S. BPI 748295

A.R. 3696=CBS 119534 BPI 871112 871112 119534 BPI 3696=CBS A.R. A.R. A.R. A.R. A.R. A.R. G.J.S. G.J.S. A.R. MAFF 241561=TPP-h430 BPI 881942 BPI 241561=TPP-h430 MAFF A.R. A.R. A.R. 3694=CBS 118914 BPI 870938 BPI 118914 3694=CBS A.R. A.R. A.R. A.R. = A.R. A.R. A.R. A.R. A.R. NCF NCF A.R. faginata faginata faginata faginata coccinea coccinea coccinea faginata coccinea faginata coccinea coccinea faginata coccinea coccinea coccinea faginata coccinea coccinea coccinea faginata faginata ...... Neo Cylindrocarpon faginatum Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Species Isolate Isolate Species Neonectria coccinea Neo Neo Neo Neo

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KC660430 AJ009254* KC660617 KC660692 DQ789752* DQ789895* Japan KC660388 - - KC660675 KC660471 KC660745 United States KC660405 KC660514 KC660554 KC660656 KC660452 KC660732United States KC660410 KC660512 KC660572 KC660659 KC660450 KC660737United States KC660411 KC660508 KC660614 KC660654 KC660446 KC660739United Japan KC660390 KC660546 KC660580 KC660681 KC660490 KC660752 Netherlands KC660429 - KC660616 KC660693 KC660461 DQ789878* Kingdom Japan KC660394 - KC660584 KC660676 KC660491 KC660753 United States KC660413 KC660510 KC660613 KC660662 KC660448 KC660731 United United States KC660414 KC660516 KC660551 KC660660 KC660454 KC660738 United Japan KC660401 KC660550 KC660576 KC660689 KC660495 KC660755 Japan KC660395 KC660549 KC660608 KC660683 KC660494 KC660751 United France KC660428 KC660520 KC660615 KC660691 KC660459 KC660760

Vitis coignetiaeVitis Weigela coraeensis Fagus grandifolia Fagus grandifolia Fagus sylvatica Fagus grandifolia Twigs China KC660402 KC660530 KC660587 KC660680JF268724* JF268739* Cerasus Jamasakura Fagus crenata Hedera helix Hedera helix wood Fagus sylvatica Hedera helix 871043 BPI871044 and Y. Nong Nong Y. and 5639 (dried culture) (dried culture) 714.97 BPI AFF 241516=TPP-h176 882163 AFF Twigs BPI Japan KC660393 - KC660621 KC660685 KC660475 KC660749 A.R. 4097=CBS 118938 BPI 870942 A.R. 4153=CBS 119163 A.R. 4151=CBS 119162 BPI 871097 BPI 871096 IMI 058770aIMI M A.R. 4523=CBS 125175 BPI 878947 A.R. 4148=CBS 119524 BPI 871122 A.R. 4166=CBS 119198 BPI 871124 241518=TPP-h190 882165 MAFF Twigs BPI 241572=TPP-h542 MAFF Japan BPI 881958 KC660396 KC660534 KC660582 KC660687 KC660476 KC660756 241493=TPP-h64 MAFF BPI 882137 MAFF 241530=TPP-h260 BPI 882098 Bark of dead wood Japan dead 241530=TPP-h260 wood of Japan 882098 MAFF KC660389 - dead Bark BPI 241556=TPP-h385 of 881931 MAFF KC660398 KC660545 KC660585 KC660686 Bark KC660489 KC660758 BPI 241560=TPP-h427 881941 MAFF KC660618 KC660678 KC660478 KC660757 Twigs BPI 241558=TPP-h390 MAFF Japan 241506=TPP-h120 BPI 881933 MAFF KC660391 - KC660547 KC660588 KC660677 KC660492 KC660748 241570=TPP-h521 MAFF Stump BPI 881956 Japan KC660392 - KC660583 KC660684 KC660472 KC660746 HMAS 98294 98294 Zhuang HMAS W.Y. 241559=TPP-h391 MAFF BPI 881934 Bark of dead MAFF 241555=TPP-h378 BPI 881930 Bark of dead wood Japan dead 241555=TPP-h378 of 881930 MAFF KC660399 - Bark BPI KC660590 KC660679 KC660488 KC660750

CBS microconidia microconidia microconidia faginata faginata faginata hederae faginata faginata hederae microconidia microconidia microconidia microconidia microconidia microconidia hederae hederae microconidia microconidia microconidia microconidia ...... Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo

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China KC660367 KC660531KC660558 KC660642KC660714 JF268735* Japan KC660371 KC660539KC660604 KC660636 KC660482 KC660709 Japan KC660368 KC660532KC660594 KC660632 KC660473 KC660705 Japan KC660369 KC660536KC660606 KC660633 KC660479 KC660707 Austria KC660403 KC660515KC660575 KC660647 KC660453 DQ789854* Austria KC660386 KC660496KC660568 KC660649 KC660431 DQ789824* France KC660385 - KC660567 KC660648 KC660458 KC660715 sp. Germany KC660387 KC660522KC660565 KC660650 DQ789730* DQ789873* sp. Germany KC660382 - KC660611 KC660645 KC660460 DQ789867*

Rhamnus fallax Frangula alnus Frangula alnus Twigs of a tree dicotyledon Ulmus Rhamnus Prunus×yedoensis wood Quercus crispula BPI 871038 Bark BPI Germany KC660380 - KC660573 KC660643 DQ789719* DQ789862* Zhuang 5694 871035=Centr aalbureau voor Schimmelcultu res Herbarium H-11427 Y. Nong, W.Y. Nong,W.Y. Y. 125.24=IMI 125.24=IMI 242.29 208.30 BPI

113880=MUCL 9808 113880=MUCL A.R. 4522=CBS 134247 BPI 878875 A.R. 4155=CBS 119527 HMAS 99197 BPI 871062 MAFF 241513=TPP-h166MAFF 882160 BPI Bark dead of 241537=TPP-h290MAFF 882104 BPI MAFF 241546=TPP-h326MAFF 882113 BPI 241522=TPP-h227 MAFF - Twigs Japan KC660427 KC660535KC660596 KC660690 KC660477 KC660759 MAFF 241514=TPP-h171 BPI 882161 Bark of dead wood Japan dead 241514=TPP-h171 of 882161 MAFF KC660400 KC660533KC660597 KC660688 KC660474 KC660754 Bark BPI 241552=TPP-h341 882119 MAFF Stem BPI Japan KC660397 KC660544KC660586 KC660682 KC660487 KC660747

A.R. 3102=CBS 119724 BPI 871063 CBS CBS tree 134255 748311 France Hardwood 98-133=CBS BPI G.J.S. KC660364 KC660529KC660598 KC660624 KC660469 KC660701 241550=TPP-h330 882117 MAFF Twigs BPI Japan 241540=TPP-h296 MAFF KC660374 KC660543 KC660570 - KC660639 KC660486 KC660708 241547=TPP-h327 882114 MAFF Bark BPI 241541=TPP-h297 882107 MAFF Twigs BPI Twigs Japan Japan KC660375 KC660540KC660561 KC660641 KC660483 KC660710 Japan KC660379 KC660538KC660610 KC660635 KC660481 KC660706 KC660370 KC660537KC660595 KC660634 KC660480 KC660712 CBS 241548=TPP-h328 882115 MAFF Twigs BPI Japan KC660372 KC660541KC660569 KC660637 KC660484 KC660713 microconidia microconidia microconidia punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea ...... Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo

http://journals.im.ac.cn/jwxtcn Yuuri HIROOKA et al. / Species delimitation for Neonectria coccinea group including the causal agents of beech bark disease in Asia, … 493 : CBS : Yuuri : Yuuri : Jacques Y.H. Table 1 continued 1 continued Table : Yuuri Hirooka, Toropical Plant Plotection Lab Lab Plotection Plant Toropical Hirooka, : Yuuri : Gary: J. Samuels,USDA-ARS MD USA; J.F. TUA-TPP-h G.J .S. : U.S. National Fungus Collections USDA-ARS MD USA;

BPI

States KC660734 - - - - States KC660744 - - - - United United Scotland KC660378 KC660527 KC660564 KC660631 KC660466 KC660699

Slovakia KC660361 KC660503 KC660563 KC660628 KC660439 KC660728 Switzerland KC660383 KC660526 KC660574 KC660644 KC660465 KC660694 Slovakia KC660362 - KC660566 KC660623 KC660443 KC660697 Slovakia KC660376 KC660507 KC660553 KC660630 KC660445 KC660698 Slovakia KC660360 KC660497 KC660592 KC660622 KC660432 KC660696

sp. France KC660366 KC660523 KC660556 KC660625 KC660462 KC660703 sp. France KC660363 KC660524 KC660603 KC660626 KC660463 KC660700 sp. France KC660365 - KC660589 KC660627 KC660470 KC660702 sp. France KC660377 KC660525 KC660605 KC660629 KC660464 KC660704 sp. France KC660381 - KC660612 KC660646 - KC660695 sp. States KC660384 KC660528 KC660602 KC660640 KC660468 DQ789888* United Fagus grandifolia Acer Fagus grandifolia Acer Fagus sylvatica Castanea Fagus sylvatica Salix Robinia Fagus sylvatica Salix Fagus sylvatica Fagus sylvatica Acer Acer macrophyllum

: Christian Lechat, Ascofrance, Villiers en Bois, France.; France.; Bois, en Villiers Ascofrance, Lechat, Christian : C.L.L. : Yuuri Hirooka, Toropical Plant Plotection University Hirooka, Toropical Japan; Lab Culture Agriculture, Collection, of Tokyo : Yuuri Tokyo 802504 1107108 1107108 871117 871117 802647 870944 871118 871118 871116 : CultureAmerican Collection, Type USA; Manassas, VA, - - - - - TPP-h ATCC : MAFF Genebank, National Institute of Agrobiological Sciences, Ibaraki, Japan; Ibaraki, Sciences, Agrobiological of Institute National Genebank, MAFF : 4828=CBS 4829=CBS 4830=CBS MAFF 119528 BPI BPI 119528 119530 BPI BPI 119530 119529 BPI BPI 119529 119153 - - 119153 119532 - - 119532 BPI 119231 119531 BPI BPI 119531 119525 BPI BPI 119525 119533 BPI BPI 119533 4831=CBS 4832=CBS 10040=A.R. 10042=A.R. 10045=A.R. 93-35=CBS 92-32=CBS 90-29=CBS 90-29=CBS Sequences obtained fromGenBank. 4096=CBS 3675=CBS 3711=CBS 3711=CBS 3704=CBS 3454=CBS 3713=CBS * 11017=A.R. 11023=A.R. 134251 134248 134252 134249 134250

J.F. J.F. C.L.L. A.R. C.L.L. G.J.S. G.J.S. A.R. G.J.S. G.J.S. A.R. C.L.L. J.F. J.F. A.R. A.R. A.R. G.J.S. 241549=TPP-h329 882116 MAFF Twigs BPI Japan KC660373 KC660542 KC660599 KC660638 KC660485 KC660711 : Amy Y. Rossman, USDA-ARS MD USA; : Y. Amy A.R. punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea punicea ...... Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Neo Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; Note: Note: Hirooka, USDA-ARS MD USA. Fournier, Ascofrance, Villiers en Bois, France; France; Bois, en Villiers Ascofrance, Fournier, Japan; University Agriculture, of Tokyo Herbarium, Tokyo

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Table 2 Parameters used and statistical values resulting from the different phylogenetic analyses of individual datasets for members of the Neonectria coccinea group

Locus Act ITS LSU Rpb1 Tef1 Tub Combined

Number of Included sites 663 449 788 650 440 535 3525

characters

MP Phylogenetically 41 (6%) 22 (5%) 11 (1%) 72 (11%) 64 (15%) 64 (12%) 274 (8%)

informative sites (%)

Tree length 68 47 17 97 135 102 514

Consistency Index (CI) 0.912 0.766 0.941 0.825 0.837 0.863 0.768

Retention Index (RI) 0.978 0.893 0.990 0.968 0.960 0.973 0.942

Rescaled consistency 0.892 0.684 0.931 0.798 0.803 0.840 0.724

(RC)

Homoplasy Index (HI) 0.088 0.234 0.059 0.175 0.163 0.137 0.232

ML and BI Nucleotide substitution TrN+I TPM2uf+I+G TIM3 TrNef+I K80+I TIM1+I

models

PCR Primers used (reference) Tact1, Tact2 ITS5, ITS4 LR5, LROR crpb1a, rpb1c tef1-728, Btub-T1,

(Samuels et al. (White et al. (Vilgalys (Castlebury tef1-1567 Btub-T2

2006) 1990) n.d.) et al. 2004) (Carbone & (O’Donnell

Kohn 1999; & Cigelnik

Rehner 2001) 1997)

PCR protocol: annealing 65℃, 30s, 15× 53℃, 1min, 53℃, 1min, 50℃, 2min, 66℃, 55s, 9× 55℃, 30s,

temp. & cycles 48℃, 30s, 30× 35× 35× 40× 56℃, 55s, 35×

35×

MP analyses were performed with PAUP* almost identical topologies in each dataset, the version 4.0 b10 (Swofford 2002), using 1,000 Bayesian trees were taken as representatives (Figs. replicates of heuristic search with random addition 1−7). The number of taxa, substitution models and of sequences and subsequent TBR branch swapping. other statistical values resulting from the different Gaps (insertion/deletions) were treated as missing analyses are presented in Table 2. data. Bootstrap analysis was performed with 1,000 The combined BI, MP, and ML analyses of six replicates, and a 70 % majority rule consensus tree loci using 75 isolates of the Neonectria coccinea was constructed in PAUP*. The three analyses for group resolved four distinct species (Fig. 7). The each locus, act, tub, rpb1, ITS, LSU, and tef1, were first major clade (clade A) occurring only in Asia is also done and produced each tree to identify the here determined as Neo. microconidia. This species most suitable locus in Neo. coccinea group. was supported by moderate values (BI PP 0.73, MP 2 RESULTS BP 77%, ML BP 74%). In the Neo. microconidia Phylogenetic analyses clade, MAFF 241514 and MAFF 241572 formed a Because the BI, MP and ML trees showed clade A-1 supported by high BI PP, MP BP, and ML

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occurs only on Fagus in North America. This species is moderately supported (BI PP 0.77, ML BP 72%, ML BP 94%). The topologies of the individual act, tef1, and rpb1 trees did not contradict each other, and these trees showed the same topologies as the six-locus phylogeny. The topologies of the tub tree showed the

Fig. 1 Bayesian trees with best log likelihoods (Ln−1421.821) derived from analyses of act data. Thickened branches indicate BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML bootstrap ≥70%.

BP values. The second major clade (clade B) is recognized as Neo. punicea although this species is supported by less than 0.70 BI PP, 70% MP BP, and

70% ML BP values. Within the Neo. punicea clade, Fig. 2 Bayesian trees with best log likelihoods (Ln–1007.727) two subclades are highly supported, specifically B-1 derived from analyses of ITS data. Thickened branches indicate with values BI PP 1.00, MP BP 96%, ML BP 94%, BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML and B-2 with values BI PP 1.00, MP BP 100%, ML bootstrap ≥70%. BP 100%. Neonectria punicea subclade B-1 is known only from Japan while Neo. punicea subclade B-2 is known only from Europe. Neonectria punicea subclade B-2 includes the ex-epitype isolate (A.R. 3102=CBS 119724). The third major clade (clade C) contained two distinct species: Neo. coccinea and Neo. faginata. Neonectria coccinea was supported by moderate BI PP, MP BP, and ML BP values (BI PP 0.86, MP BP 76%, ML BP 75%). At the base of the Neo. coccinea clade (clade E), MAFF 241561 is known only from Asia. The Neo. coccinea subclade E-1 including the ex-type isolate was supported by moderate phylogenetic values (BI PP 0.90, MP BP Fig. 3 Bayesian trees with best log likelihoods (Ln−1298.747) 75%, ML BP 72%). A fourth species is recognized as derived from analyses of LSU data. Thickened branches indicate Neo. faginata (clade F), previously recognized by BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML Castlebury et al. (2006). Neonectria faginata bootstrap ≥70%.

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almost similar topologies with the six-locus phylogeny as well as the other protein-coded dataset phylogeny. The topologies of the ITS and LSU trees conflict with the six-locus phylogeny. Among these single locus trees, tef1 provides the best resolution and thus is the most phylogenetically informative.

Fig. 6 Bayesian trees with best log likelihoods (Ln–1458.84) derived from analyses of tub data. Thickened branches indicate BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML bootstrap ≥70%.

Morphological, colony growth and temperature

analyses

Fig. 4 Bayesian trees with best log likelihoods (Ln–1545.111) The four species in the Neo. coccinea group are derived from analyses of rpb1 data. Thickened branches indicate difficult to distinguish based solely on teleomorph BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML morphology. Although perithecial characters, such as bootstrap ≥70%. color, surface, and wall cell structure, are generally reliable for identifying the species or genera of other nectrioid fungi, this is not true for the segregate species of the Neo. coccinea group. The perithecial apex of Neo. coccinea, Neo. faginata, and Neo. punicea tends to be papillate and thus seems to be useful in distinguishing species but this characteristic varies within species. The perithecial wall surface of species in the Neo. coccinea group is slightly roughened. The perithecial walls are about the same thickness and cell walls form similar tissue of textura globulosa to textura angularis; thus, Fig. 5 Bayesian trees with best log likelihoods (Ln−1548.664) perithecial wall structure is not useful in derived from analyses of tef1 data. Thickened branches indicate distinguishing these species. According to BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML Castlebury et al. (2006), the asci of Neo. coccinea bootstrap ≥70%. are narrowly clavate with an apical ring while those

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Fig. 7 Bayesian tree with best log likelihood (Ln−8382.847) derived from analysis of combined data set (act, ITS, LSU, rpb1, tef1 and tub). Values at branches indicate Bayesian posterior probabilities (BI PP)/Maximum Likelihood bootstrap (ML BP)/Maximum parsimony bootstrap (MP BP). BI posterior probabilities ≤0.7, MP bootstrap ≤70%, and ML bootstrap ≤70% indicated by - . Thickened branches indicate BI posterior probabilities ≥0.7, MP bootstrap ≥70%, and ML bootstrap ≥70%. The culture collection number, geography and host are included after each taxon name. “EX” indicates ex-type cultures.

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of Neo. faginata are cylindrical without an apical macroconidia that are more than 80μm long when ring and with uniseriate ascospores. In our study, we 5-septate while the other species produce observed that all species of Neo. coccinea group macroconidia that are less than 80μm long when have uniseriate or occasionally obliquely biseriate 5-septate. ascospores in asci with an apical ring. These Overall, Neo. faginata is easily identified morphological characters were not useful in because it is known only on species of Fagus in distinguishing species. Although size, surface North America and produces macroconidia more structure, and septation of ascospores are considered than 80μm long when 5-septate. Neonectria to be important in distinguishing these species, we coccinea is distinguishable based on host specificity found no significant differences among the four occurring only on Fagus in Europe (except for one species of the Neo. coccinea group. We analyzed anomalous isolate from Japan) and the fast mycelial 95% confidence intervals of length/width ratios of grow at 30℃ for 7d on PDA. Neonectria ascospores on natural substrate (Fig. 8). Although microconidia and Neo. punicea are similar because length of ascospores in Neo. faginata is statistically of the broad host range and the same size of shorter than Neo. coccinea, no differences of the macroconidia; however, the number of septa in the length were found among Neo. faginata, Neo. macroconidia and mycelial growth rate are different. microconidia and Neo. punicea. We were not able to In addition, Neonectria microconidia is known only examine the anamorphic states of species in the Neo. from Asia. coccinea group in nature because we did not find TAXONOMY many specimens with the anamorph fructification. Based on our morphological and molecular As for the anamorph in culture, colony colors analyses, the Neonectria coccinea group is are indistinguishable among species. Colonies are re-determined as four distinct species, each of which white to umber with ochraceous to sienna reverse. is described and illustrated below. A key to these Although the optimal temperature for mycelial four species is provided. growth on PDA for each species is 20℃, the Neonectria coccinea (Pers.: Fr.) Rossman & mycelial growth rates at 30℃ for 7days on PDA Samuels, in Rossman, Samuels, Rogerson & Lowen, were different: Neo. punicea and Neo. faginata are Stud. Mycol. 42: 158. 1999. Fig. 9 less than 15mm diameter while Neo. microconidia ≡Sphaeria coccinea Pers.: Fr., Persoon, Icon. & and Neo. coccinea are greater than 15mm diameter. Descr. Fung. 2: 47. 1800: Fries, Syst. Mycol. 2: 412. The morphology of conidiophores, conidiogenous 1823. cells, chlamydospores, and microconidia are similar ≡Nectria coccinea (Pers.: Fr.) Fr., Summa Veg. for the four species of the N. coccinea group; Scand. 2: 388. 1849. however, these species can be distinguished based =Fusidium candidum Link, Observationes I, on the morphology of the macroconidia. On SNA, Mag. Ges. Naturf. Freunde Berlin 3: 6. 1809. Neo. microconidia and Neo. faginata produce up to ≡Cylindrocarpon candidum (Link) Wollenw., 8-septate macroconidia while Neo. punicea and Neo. Fus. Autogr. Del., ed. 2, no. 655. 1926. coccinea produce up to 7-septate macroconidia. Size =Sphaerostilbe caespitosa Fuckel, Jahrb. of conidia is also reliable; only Neo. faginata forms Nassauischen Vereubs Naturk. 27-28: 33. 1873.

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Fig. 8 Graphs of 95% confidence intervals of length to width ratios of ascospores and conidia.

≡Neonectria caespitosa (Fuckel) Wollenw., caespitose up to 38 on a stroma, subglobose to Angew. Bot. 8: 192. 1926. pyriform, 218–367μm high and 148–399μm diam. =Fusidium fractum Sacc. & Cav., N. Giorn. (n=21), red to sienna, often cupulate upon drying or Bot. ital. 7: 308. 1900. collapsing by lateral pinching, papillate, apical ≡Cylindrocarpon fractum (Sacc. & Cav.) region sometimes slightly darker, KOH+ purple, Wollenw., Fus. Autogr. Del., ed. 1, no. 655. 1924. LA+ yellow, surface with smooth to rough. Teleomorph on natural substrata: Mycelium Perithecial surface cells forming textura globulosa sometimes visible around perithecia and on host. or textura angularis, with walls pigmented, ca. Stromata up to 0.3mm high and 8mm diam., 1.5μm thick. Perithecial wall ca. 17–49μm thick, of erumpent through epidermis, red to bay, KOH+ dark two distinct regions: outer region ca. 22–42μm red, LA+ yellow, pseudoparenchymatous, cells thick, intergrading with stroma, cells forming textura forming textura angularis to textura prismatica with globulosa or textura angularis, walls pigmented, ca. cells oriented more or less vertically; cells 3–10μm 1.5μm thick; inner region ca. 7–21μm thick, of diam., with 1–1.5μm thick walls, intergrading with elongated, thin-walled, hyaline cells, forming textura ascomatal wall. Perithecia superficial on prismatica. Asci unitunicate, (58–)69–97(–124)× well-developed, erumpent stroma, solitary or (7.8–)8.9–12.9(–15.9)μm (n=44), cylindrical to

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narrowly clavate, often with an apical ring at apex, 100.8(–111.0)×(5.1–)5.4–6.2(–6.5)μm (n=17). stipitate, 8-spored, ascospores occasionally obliquely Chlamydospores intercalary in hyphae, globose to biseriate near apex. Ascospores ellipsoidal to fusiform subglobose, sometimes ellipsoidal, smooth, hyaline. with narrowly rounded ends, straight or slightly Perithecia not produced in culture. curved, hyaline, finely spinulose, 1 septate, (9.1–) Distribution: Asia (Japan) and Europe (France, 11.8–14.6(–19.3)×(3.7–)4.9–6.3(–7.6)μm (n=309). Germany, Romania, Scotland, Slovakia). Anamorph in culture: Optimum temperature for Habitat: On Fagus crenata, F. sylvatica and growth on PDA 20℃, colonies 39–56mm (average Fagus sp. 45mm) diam. at 20℃ after 7d. Colony surface on Lectotype of Neonectria coccinea designated PDA, radial, abundant cottony with aerial mycelium, by Booth (1959). Herb. Lugd. Bat. No. 910, 270-56 sometimes wavy, white to saffron or umber, saffron on bark of Fagus, isolectotype BPI 738862. to ochraceous sporodochial conidial masses Epitype of Neonectria coccinea designated producing after one week; reverse ochraceous to here. Germany Saarland, Naturpark Saar-Hundsruck, sienna. Sporulation on SNA from lateral phialidic between Leisel and Schollen, alt. 600m. 49°40'0''N pegs on submerged or commonly on aerial hyphae, 7°10'0''E, on hardwood, Fagus sp., Oct. 13, 1998, 2.0–7.5μm long, 1.5–3.0μm wide at base. coll. G.J. Samuels, H.J. Schroers (BPI 748295; Conidiophores occasionally developing on aerial ex-epitype culture CBS 119158=G.J.S. 98-114). hyphae, unbranched, sometimes verticillate, Additional type specimens: A holotype of 1–3-branched, becoming loosely to moderately Sphaeria caespitosa exists at G, isotype K, and BR, densely branched, 6.5–124μm long, 2.1–3.0μm wide according to Booth (1966) and Seifert (1985). at base. Conidiogenous cells monophialidic, Additional specimens and isolates examined: cylindrical and slightly tapering toward tip or France, on Fagus sylvatica, 7 Sep. 2001, coll. A. narrowly flask-shaped with widest point in middle, Kunca (BPI 871112; culture A.R. 3696=CBS 4–36μm long, 1.5–3.0μm wide at base. Conidia 119534); on Fagus sylvatica, 7 Sep. 2001, coll. A. formed by monophialides on submerged or aerial Kunca (BPI 870938; culture A.R. 3694=CBS hyphae, formed abundantly on slimy heads, 118914). Japan, Kanagawa Prefecture, ellipsoidal, oblong to long cylindrical, hyaline, Ashigarakami-gun, on Fagus crenata, 17 Apr. 2005, smooth, straight or slightly curved with round at coll. H. Masuya, Y. Hirooka (BPI 881942; culture both ends, 0–7-septate; 0-septate: (3.4–)6.0–9.8 MAFF 241561=TPP-h430). Romania, Brasov, (–12.5)×(2.4–)2.7–3.9(–4.6)μm (n=120), 1-septate: Busteni, on Fagus sylvatica, 26 Sep. 2000, coll. A. 10.0–22.6(–43.5)×(3.6–)3.9–5.1(–5.6)μm (n=52), Kunca (BPI 870939; culture A.R. 3708=CBS 2-septate: (22.5–)22.7–29.5(–33.1)×(4.1–)4.4–5.0μm 118916); Brasov, Busteni, on Fagus sylvatica, 26 (n=24), 3-septate: (28.1–)32.1–43.5(–52.2)×4.4–6.0 Sep. 2000, coll. A. Kunca (BPI 870937; culture A.R. (–7.2)μm (n=48), 4-septate: (41.1–)44.5–62.1 3707=CBS 118915). Scotland, Cowal Peninsula, (–62.6)×(5.4–)5.7–6.4(–6.6)μm (n=38), 5-septate: Argyll Forest Park. N. End of Loch Goil, alt. (52.2–)58.9–72.1(–80.4)×(4.2–)5.5–67.1(–8.0)μm 25–250m, on Fagus sp., 12 Apr. 1992, coll. G.J. (n=114), 6-septate: (70.0–)73.6–84.2(–85.7)×(5.3–) Samuels, D. Brayford (BPI 802648; culture G.J.S. 5.3–6.3(–6.4)μm (n=18), 7-septate: (72.9–)88.4– 92-33=CBS 134254). Slovakia, Kacin, Male Karpety,

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Fig. 9 Neonectria coccinea. A, B: Perithecia in the natural environment; C: Median section of perithecium; D: Median section of perithecial apex and wall; E: Ascus apices; F: Asci; G: Ascospores in optical section; H: Ascospores in surface view; I: Colony of A.R. 3700 after 7d at 20℃ on PDA; J: Colony of A.R. 3691 after 7d at 20℃ on PDA; K: Colony of A.R. 3707 after 7d at 20℃ on PDA; L: Colony of G.J.S. 92-33after 7d at 20℃ on PDA; M, N, Q–S: Conidiophores on SNA; O, P: Lateral phialidic pegs; T–V: Micro- and macroconidia on SNA. Bar: A=3mm; B=300µm; C, M=100µm; D=30µm; E–H, N=20µm; I–L=30mm; O–V=10µm.

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on Fagus sylvatica, 14 Apr. 2000, coll. A. Kunca representative isolate of N. coccinea but an epitype (BPI 870940; culture A.R. 3700=CBS 119156); was not designated. Since this species is Muranky planina, Pod Vel’kow stozkou, on Fagus economically important, an epitype of the basionym sylvatica, 26 Oct. 2000, coll. A. Kunca (BPI 870941; Sphaeria coccinea is needed to distinguish Neo. culture A.R. 3705=CBS 119150); Bansky Studenec coccinea from other species in the Neo. coccinea 5, Stiavnicke vrchy, on Fagus sylvatica, 10 Jun. group. The specimen (BPI 748295, ex-epitype 2001, coll. A. Kunca (BPI 871113; culture A.R. culture CBS 119158) is herein designate as epitype 3691=CBS 119523); Bansky Studenec 1, on Fagus of S. coccinea. This specimen was collected on sylvatica, 14 Mar. 2001, coll. A. Kunca (A.R. Fagus sp. from Germany, most likely the type 3687=CBS 134253), Vel’ke Rovne, Javornicky, on locality of Sphaeria coccinea collected by Persoon Fagus sylvatica, 11 Jul. 2001, coll. A. Kunca (BPI (1800) because Persoon moved to Göttingen for 871115; culture A.R. 3712=CBS 119522); Nova botanical research and earned a doctorate from the Bystrica, Kysucke vrchy, on Fagus sylvatica, 20 Academy of Natural Sciences in Erlangen in 1777 Nov. 2000, coll. A. Kunca (BPI 871114; A.R. 3701). (de Zeeuw 1939; Petersen 1977). Notes: Neonectria coccinea is here determined to include specimens collected on Fagus spp. from Neonectria faginata (M.L. Lohman, A.M.J. Watson Europe and one isolate from Japan. To distinguish & Ayers) Castl. & Rossman, in Castlebury, Rossman this species from others in the Neo. coccinea group, & Hyten, Can. J. Bot. 84: 1425. 2006. Fig. 10 cultural characters are required. Neonectria coccinea ≡Neonectria coccinea var. faginata M.L. is similar to Neo. microconidia in having 5-septate Lohman, A.M.J. Watson & Ayers, Lloydia 6: 100. conidia that are 50–70μm long on SNA and a growth 1943. rate in culture of more than 15mm at 30℃ for 7days =Cylindrocarpon faginatum C. Booth, Mycol. on PDA. In addition, septation of macroconidia in Pap. 104: 1. 1966. Neo. coccinea ranges from 1–7-septate while in Neo. Teleomorph on natural substrata: Mycelium microconidia the range is 1–8-septate. Also, Neo. sometimes visible around perithecia and on host. coccinea is known only from Fagus while Neo. Stromata up to 0.3mm high and 8mm diam., microconidia occurs on Fagus and other hosts. erumpent through epidermis, red to bay, KOH+ dark Surprisingly our phylogenetic analyses suggest a red, LA+ yellow, pseudoparenchymatous, cells sister-group relationship between Neo. coccinea and forming textura angularis to textura prismatica with MAFF 241561 collected from Japan (Fig. 7). cells oriented more or less vertically; cells 3–9μm However, based on our observations of the diam., with 1–2μm thick walls, intergrading with morphological characters in the natural environment ascomatal wall. Perithecia superficial on and culture, MAFF 241561 completely matches well-developed, erumpent stroma, solitary or Neo. coccinea. Although there are obvious caespitose up to 50 on a stroma, subglobose to geographical differences, we tentatively regard pyriform, 195–399μm high and 156–302μm diam. MAFF 241561 as Neo. coccinea. (n=45), red to bay, often cupulate upon drying or Castlebury et al. (2006) mentioned that BPI collapsing by lateral pinching, papillate, apical 748295 and the derived CBS 119158 as a region sometimes slightly darker, KOH+ purple,

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LA+ yellow, surface with smooth to rough. smooth, straight or slightly curved with round at Perithecial surface cells forming textura globulosa both ends, 0–8-septate; 0-septate: (2.5–)7.5–11.9 or textura angularis, with walls pigmented, ca. (–13.2)×(2.8–)3.3–4.5(–4.8)μm (n=140), 1-septate: 1.5μm thick. Perithecial wall ca. 31–50μm thick, of (13.2–)13.4–16.6(–19.0)×(3.6–)3.7–4.7(–5.0)μm two distinct regions: outer region ca. 17–42μm thick, (n=40), 2-septate: (13.7–)15.5–25.9(–30.3)×(3.1–) intergrading with stroma, cells forming textura 4.0–5.1(–5.8)μm (n=25), 3-septate: (20.4–)35.8– globulosa or textura angularis, walls pigmented, ca. 50.5(–55.6)×(4.5–)4.8–6.7(–7.0)μm (n=21), 4-septate: 1.5μm thick; inner region ca. 13–20μm thick, of (44.4–)59.1–67.2(–73.4)×(4.4–)4.8–6.2(–6.8)μm elongated, thin-walled, hyaline cells, forming textura (n=22), 5-septate: (75.9–)76.7–92.3(–97.1)×(5.6–) prismatica. Asci unitunicate, (65.8–)69.4–96.4 5.8–6.8(–7.0)μm (n=50), 6-septate: (78.2–)79.5– (–124.5)×(7.0–)8.9–11.9(–13.7)μm (n=47), cylindrical 92.1(–104.5)×(5.1–)5.9–7.2(–8.0)μm (n=14), 7-septate: to narrowly clavate, often with an apical ring at (93.1–)94.1–102.3(–102.7)×5.0–6.6(–7.7)μm (n=35), apex, stipitate, 8-spored, ascospores occasionally 8-septate: (95.5–)97.3–109.0(–114.1)×(5.1–)5.9–7.5 obliquely biseriate near apex. Ascospores (–8.1)μm (n=20). Chlamydospores intercalary in ellipsoidal to fusiform with narrowly rounded ends, hyphae, globose to subglobose, sometimes straight or slightly curved, hyaline, finely spinulose, ellipsoidal, smooth, hyaline. Perithecia not produced 1-septate, (8.5–)10.3–12.3(–14.3)×(4.1–)4.4–6.2 in culture. (–8.0)μm (n=249). Distribution: North America (Canada, United Anamorph in culture: Optimum temperature for States). growth on PDA 20℃, colonies 34–40mm (average Habitat: On Fagus grandifolia, F. sylvatica and 36mm) diam. at 20℃ after 7d. Colony surface on Fagus sp. PDA, radial, abundant cottony with aerial mycelium, Holotype of Neonectria faginata: United sometimes wavy, ochraceous, saffron to ochraceous States, Maine, Meddybemps, on Fagus grandifolia, sporodochial conidial masses producing after one 4 Oct. 1933, coll. T.T. Ayers (BPI 551558). week; reverse ochraceous. Sporulation on SNA from Epitype of Neonectria faginata designated here: lateral phialidic pegs on submerged or commonly on United States, Maine, on Fagus grandifolia, Apr. aerial hyphae, 2.0–6.8μm long, 1.5–2.5μm wide at 2006, coll. M. Kassen (BPI 878329; ex-epitype base. Conidiophores occasionally developing on culture A.R. 4307=CBS 134246). aerial hyphae, unbranched, sometimes verticillate, Ex-holotype culture of Cylindrocarpon 1–3-branched, becoming loosely to moderately faginatum: Canada, New Brunswick York County, densely branched, 6.5–87μm long, 2.1–3.0μm wide Rocky Brook, on Fagus grandifolia (CBS at base. Conidiogenous cells, monophialidic, 217.67=ATCC 16547=IMI 105738). cylindrical and slightly tapering toward tip or Specimens and isolates examined: United narrowly flask-shaped with widest point in middle, States, New Hampshire, Grafton Co., Hubbard 8–29μm long, 1.5–2.5μm wide at base. Conidia Brook Experimental Forest, on Fagus grandifolia, formed by monophialides on submerged or aerial 1998, coll. E.M. Mahoney (culture A.R. 3677=CBS hyphae, formed abundantly on slimy heads, 119154); Maine, Penobscot Co., Penobscot ellipsoidal, oblong to long cylindrical, hyaline, Experimental Forest, on Fagus grandifolia, 1998,

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Fig. 10 Neonectria faginata. A–C: Perithecia in the natural environment; D: Median section of perithecium; E: Median section of perithecial apex and wall; F: Asci; G: Ascospores in optical section; H: Ascospores in surface view; I: Colony of A.R. 4167 after 7d at 20℃ on PDA; J: Colony of A.R. 4307 after 7d at 20℃ on PDA; K–O, Q–U: Conidiophores on SNA; M: Lateral phialidic pegs and conidiophores on SNA; P, V: Micro- and macroconidia on SNA; W: Chlamydospores on SNA. Bar: A=3mm; B, C=300µm; D=100µm; E, F, K=30µm; G, H, L–W=10µm; I, J=30mm.

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coll. E.M. Mahoney (culture A.R. 3682=CBS morphology, geography and phylogeny. Among the 119155); West Virginia, on Fagus sp., coll. E.M. four species in the Neo. coccinea group, the Mahoney (BPI 880528; culture NCF 346=AR morphology of Neo. faginata is relatively 3680=CBS 118983); West Virginia. Burner Mt. BBD distinctive. The five-septate macroconidia of Neo. plot tree 4, on Fagus grandifolia, Oct. 2004, coll. M. faginata are longer than the other species i.e., MacKenzie (BPI 871095; culture A.R. 4130=CBS 80–90μm long in Neo. faginata vs. 50–70μm long in 119161; West Virginia, Burner Mt. BBD plot tree 8, other species of Neo. coccinea group. Neonectria on Fagus grandifolia, Mar. 03 2005, coll. M. faginata is known only on Fagus spp. in North MacKenzie (BPI 870943; culture A.R. 4152=CBS America. According to Castlebury et al. (2006), only 118917; North Carolina, Blue Ridge Parkway Fagus grandifolia was recorded as the host of Neo. National Park, Richland Balsam, on Fagus sylvatica, faginata. Four specimens on Fagus sylvatica are Mar. 25 2004, coll. M. MacKenzie (BPI 871123; reported here. culture A.R. 4149=CBS 119519); Michigan, Luce The holotype specimen of Neo. coccinea var. Co., N of Bass Lake on County Road 421. 46.5°N faginata is preserved in BPI, but it lacks a living 85.78°W, on Fagus grandifolia, Jun. 29 2005, coll. ex-type culture. Therefore, we epitypify this name G. Joseph O’Brien (BPI 870945; culture A.R. with BPI 878329 (ex-epitype culture A.R. 4197=CBS 118918); Pennsylvania, McKean Co., 4307=CBS 134246) collected from the same host Bradford Rd., on Fagus sylvatica, May 12 2005, and state in the United States as the holotype. coll. M. MacKenzie (BPI 871125; culture A.R. Neonectria microconidia J. Luo, P. Zhao & W.Y. 4167=CBS 119199); West Virginia, Burner Mt. BBD Zhuang, in Zhao, Luo, Zhuang, Liu & Wu, Sci. plot, tree gamma, on Fagus grandifolia, Mar. 31 China Life Sci. 54: 671. 2011. Fig. 11 2005, coll. M. MacKenzie (BPI 871097; culture Teleomorph on natural substrata: Mycelium A.R. 4153=CBS 119163; West Virginia, Burner Mt. sometimes visible around perithecia and on host. BBD plot, tree beta, on Fagus grandifolia, Mar. 31 Stromata up to 0.4mm high and 12mm diam., 2004, coll. M. MacKenzie (BPI 871096; culture erumpent through epidermis, red to bay, KOH+ dark A.R. 4151=CBS 119162; North Carolina, Great red, LA+ yellow, pseudoparenchymatous, cells Smoky Mountains National Park. Newfound Gap, forming textura angularis to textura prismatica with on Fagus sylvatica, Dec. 03 2003, coll. B. Jones cells oriented more or less vertically; cells 3–12μm (BPI 871122; culture A.R. 4148=CBS 119524); diam., with 1–2μm thick walls, intergrading with Pennsylvania, Allegheny National Forest, tree 53, on ascomatal wall. Perithecia superficial on Fagus grandifolia, Oct. 07 2004, coll. M. MacKenzie well-developed, erumpent stroma, solitary or (BPI 870942; culture A.R. 4097=CBS 118938; West caespitose up to 300 on a stroma, subglobose to Virginia, Monogahela National Forest. Cheat Mt. pyriform, 203–380μm high and 175–357μm diam. Road, on Fagus sylvatica, May 04 2005, coll. M. (n=30), red to sienna, often cupulate upon drying or MacKenzie (BPI 871124; culture A.R. 4166=CBS collapsing by lateral pinching, sometimes papillate, 119198). apical region sometimes slightly darker, KOH+ Notes: Neonectria faginata was raised to the purple, LA+ yellow, surface with smooth to rough. rank of species by Castlebury et al. (2006) based on Perithecial surface cells forming textura globulosa

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or textura angularis, with walls pigmented, ca. 0–8-septate; 0-septate: (3.1–)5.5–8.9(–12.3)×(1.7–) 1.5μm thick. Perithecial wall ca. 30–44μm thick, of 2.1–3.3(–4.9)μm (n=288), 1-septate: (9.8–)12.2–19.2 two distinct regions: outer region ca. 20–40μm (–31.0)×(2.5–)3.5–4.5(–5.6)μm (n=144), 2-septate: thick, intergrading with stroma, cells forming textura (12.2–)16.7–28.5(–34.8)×(3.4–)4.2–5.4(–5.6)μm globulosa or textura angularis, walls pigmented, ca. (n=37), 3-septate: (24.6–)31.5–43.9(–49.7)×(4.0–) 1.5μm thick; inner region ca. 10–18μm thick, of 4.8–6.2(–7.1)μm (n=84), 4-septate: (39.9–)44.5– elongated, thin-walled, hyaline cells, forming 53.3(–57.7)×(4.4–)4.8–6.2(–6.8)μm (n=42), 5-septate: textura prismatica. Asci unitunicate, (58–)64–94 (47.4–)55.7–69.7(–76.4)×(4.0–)5.5–6.9(–7.6)μm (–102)×(6.3–)8.0–10.6(–11.1)μm (n=35), cylindrical (n=95), 6-septate: (63.9–)69.3–88.7(–101.9)×(5.0–) to narrowly clavate, often with an apical ring at 5.9–7.5(–8.2)μm (n=54), 7-septate: (71.5–)80.3– apex, stipitate, 8-spored, ascospores occasionally 98.3(–118.2)×(5.1–)6.2–7.6(–8.5)μm (n=51), 8-septate: obliquely biseriate near apex. Ascospores ellipsoidal (79.1–)85.2–101.7(–120.0)×(5.2–)6.6–7.2(–8.0)μm to fusiform with narrowly rounded ends, straight or (n=17). Chlamydospores intercalary in hyphae, slightly curved, hyaline, finely spinulose, 1-septate, globose to subglobose, sometimes ellipsoidal, (8.9–)10.6–15.8(–23.1)×(3.8–)4.5–6.5(–9.4)μm smooth, hyaline. Perithecia not produced in culture. (n=192). Distribution: Asia (China, Japan). Anamorph in culture: Optimum temperature for Habitat: On dead woody substrata, known from growth on PDA 20℃, colonies 34–56mm (average Cerasus jamasakura, Fagus crenata, Vitis coignetiae 47mm) diam at 20℃ after 7d. Colony surface on and Weigela coraeensis. PDA, radial, abundant cottony with aerial mycelium, Holotype of Neonectria microconidia: China, sometimes wavy, white to ochraceous, saffron to Hubei, Wufeng, 800m alt., on twigs, 13 Sep. 2004, ochraceous sporodochial conidial masses producing coll. W.Y. Zhuang, Y. Nong (5639) (HMAS 98294). after one week; reverse ochraceous to rust. Specimens and isolates examined: Japan, Sporulation on SNA from lateral phialidic pegs on Fukuoka Prefecture, Oazanatuki, Tagawa city, on submerged or aerial hyphae common, 2.0–7.2μm bark of dead wood, Apr. 02 2004, coll. Y. Hirooka long, 1.5–2.5μm wide at base. Conidiophores (BPI 882098; culture MAFF 241530=TPP-h260); occasionally developing on aerial hyphae, Kanagawa Prefecture, Yamakawa-cho, unbranched, sometimes verticillate, 1–3-branched, Ashigarakami-gun, on bark of dead wood, Oct. 31 becoming loosely to moderately densely branched, 2004, coll. Y. Hirooka (BPI 881931; culture MAFF 6.5–110μm long, 2.1–3.0μm wide at base. 241556=TPP-h385); Kanagawa Prefecture, Conidiogenous cells, monophialidic, cylindrical and Kiyokawa-mura, Aigo-gun, on twigs, Apr. 16 2005, slightly tapering toward tip or narrowly flask-shaped coll. Y. Hirooka (BPI 881941; culture MAFF with widest point in middle, 6–38μm long, 241560=TPP-h427); Kanagawa Prefecture, 1.5–2.5μm wide at base. Conidia formed by Yamakawa-cho, Ashigarakami-gun, on dead wood of monophialides on submerged or aerial hyphae, Cerasus jamasakura, Oct. 31 2004, coll. Y. Hirooka formed abundantly on slimy heads, ellipsoidal, (BPI 881933, culture MAFF 241558=TPP-h390), oblong to long cylindrical, hyaline, smooth, straight Kanagawa Prefecuutre, Atugi city, on stump, May or slightly curved with round at both ends, 03 2003, coll. Y. Hirooka (culture MAFF

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241506=TPP-h120); Kanagawa Prefecture, supported by high BIPP, MPBP and MLBP values. Yamakitagawayose, Ashigarakami-gun, on Fagus Although subclade A-1 includes only Japanese crenata, Jul. 03 2005, coll. Y. Hirooka (BPI 881956; isolates MAFF 241514 and MAFF 241572, no culture MAFF 241570=TPP-h521); Kanagawa morphological differences were found from the Prefecture, Yamakawa-cho, Ashigarakami-gun, other isolates of Neo. microconidia. Thus subclade Genbu river, on bark of dead wood, Oct. 31 2004, A-1 is not considered to be distinct from Neo. coll. Y. Hirooka (BPI 881934, culture MAFF microconidia. 241559=TPP-h391); Kanagawa Prefecture. Neonectria microconidia is known only from Yamakawa-cho, Ashigarakami-gun, on bark of dead Asia although Neo. punicea and one isolate of N. wood, Oct. 31 2004, coll. Y. Hirooka (BPI 881930; coccinea are also known from Asia. Neonectria culture MAFF 241555=TPP-h378); Kochi microconidia can be distinguished from the other Prefecture, Tosakitakaido, Tosa-cho, on twigs, Aug. species of the Neo. coccina group using the 04 2003, coll. Y. Hirooka (BPI 882163; culture morphological characters of the anamorph. MAFF 241516=TPP-h176); Kochi Prefecture, Neonectria microconidia has 5-septate macroconidia Shirono, Kitagawa-mura, on twigs, Aug. 05 2003, that are 50–70μm long, conidia varying from coll. Y. Hirooka (BPI 882165; culture MAFF 1–8-septate, a colony growth rate of more than 241518=TPP-h190); Nagano Prefecture, Sugadaira, 15mm in diameter after 7d at 30℃ and a wide host Ueda city, on twigs of Vitis coignetiae, Sep. 01 2006, range. coll. Y. Hirooka (BPI 881958; culture MAFF 241572=TPP-h542); Saitama Prefecture, Nakaizu, Neonectria punicea (J.C. Schmidt: Fr.) Castl. & Tagata-gun, on twigs of Weigela coraeensis, Oct. 14 Rossman, in Castlebury, Rossman & Hyten, Can. J. 2002, coll. Y. Hirooka (BPI 882137; culture MAFF Bot. 84: 1425. 2006. Fig. 12 241493=TPP-h64); Tokyo, Okutama-gun, on twigs, ≡Sphaeria punicea J.C. Schmidt, in Kunze & Nov. 20 2003, coll. Y. Hirooka (culture MAFF Schmidt, Mykologische Hefte (Leipzig) 1: 61. 1817. 241522=TPP-h227); Yamagata Prefecture, Akakura, =Fusarium album Sacc., Michelia 2: 132. 1880. mogami-cho, on bark of dead wood, Jul. 26 2003, ≡Cylindrocarpon album (Sacc.) Wollenw., coll. T. Tokiwa (BPI 882161; culture MAFF Fusaria autographica delineate 1: no. 473. 1916. 241514=TPP-h171); Yamanashi Prefecture, =Neonectria confusa J. Luo & W.Y. Zhuang, Yamato-son, Higashiyamanashi-gun, Yamato-son, on Mycologia 102: 143. 2010. stem, Sep 26, 2009, coll. T. Tokiwa, Y. Hirooka (BPI Teleomorph on natural substrata: Mycelium 882119; culture MAFF 241552=TPP-h341). sometimes visible around perithecia and on host. Notes: Neonectria microconidia was recently Stromata up to 0.4mm high and 10mm diam., described from China (Zhao et al. 2011). Based on erumpent through epidermis, scarlet to bay, KOH+ our phylogenetic analyses, the type isolate of this dark red, LA+ yellow, pseudoparenchymatous, cells species clustered with many Japanese isolates. This forming textura angularis to textura prismatica with clade is supported by more than 0.70 MI PP, 70% cells oriented more or less vertically; cells 4–14μm MPBP and 70% ML BP values (clade A in Fig. 7). diam., with 1–1.5μm thick walls, intergrading Within the Neo. microconidia clade, subclade A-1 is with ascomatal wall. Perithecia superficial on well-

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Fig. 11 Neonectria microconidia. A–C: Perithecia in the natural environment; D: Median section of perithecium; E: Median section of perithecial wall; F, G: Asci; H: Ascospores in optical section; I: Ascospores in surface view; J: Colony of MAFF 241516 after 7d at 20℃ on PDA; K: Colony of MAFF 241493 after 7d at 20℃ on PDA; L: Colony of MAFF 241530 after 7d at 20℃ on PDA; M: Colony of MAFF 241559 after 7d at 20℃ on PDA; N, P–W: Conidiophores on SNA; O: Lateral phialidic pegs on SNA; X: Micro- and macroconidia on SNA. Bar: A=3mm; B, C=300µm; D=100µm; E, U, V=30µm; F, G=20µm; H, I=5µm; J–M=30mm; N–T, W, X=10µm.

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developed, erumpent stroma, solitary or caespitose cylindrical and slightly tapering toward tip or up to 40 on a stroma, subglobose to pyriform, narrowly flask-shaped with widest point in middle, 211–364μm high and 160–338μm diam. (n=50), red 4–24μm long, 2.5–3.5μm wide at base. Conidia to sienna, often cupulate upon drying, papillate, formed by monophialides on submerged or aerial apical region slightly darker, KOH+ purple, LA+ hyphae, formed abundantly on slimy heads, yellow, surface with smooth to rough. Perithecial ellipsoidal, oblong to long cylindrical, hyaline, surface cells forming textura globulosa or textura smooth, straight or slightly curved with round at angularis, with walls pigmented, ca. 1.5μm thick. both ends, 0–7-septate: 0-septate: (3.2–)6.1–9.7 Perithecial wall ca. 17–46μm thick, of two distinct (–18.3)×(1.5–)2.3–3.7(–6.1)μm (n=505), 1-septate: regions: outer region ca. 16–34μm thick, (8.6–)11.8–20.6(–27.7)×(2.3–)3.6–5.0(–6.6)μm intergrading with stroma, cells forming textura (n=241), 2-septate: (20.3–)22.7–28.7(–31.2)×(4.0–) globulosa or textura angularis, walls pigmented, ca. 4.3–6.1(–7.0)μm (n=50), 3-septate: (25–)31.4–42.2 1.5μm thick; inner region ca. 6–15μm thick, of (–52.5)×(3.5–)4.5–6.1(–8.0)μm (n=233), 4-septate: elongated, thin-walled, hyaline cells, forming textura (37.7–)43.0–54.6(–64.9)×(4.3–)4.9–6.1(–7.2)μm prismatica. Asci unitunicate, (48–)69–93(–109)× (n=94), 5-septate: (40.9–)51.6–67.9(–79.9)×(4.0–) (6.1–)8.1–11.3(–14)μm (n=100), cylindrical to 5.2–6.6(–8.0)μm (n=199), 6-septate: (66.3–)68.6– narrowly clavate, often with an apical ring at apex, 79.2(–84.7)× (4.7–)5.2–7.0(–7.8)μm (n=25), 7-septate: stipitate, 8-spored, ascospores occasionally (80.7–)81.9–102.1(–103.8)×(5.0–)5.2–6.8(–6.9)μm obliquely biseriate near apex. Ascospores ellipsoidal (n=21). Chlamydospores intercalary in hyphae, to fusiform, sometimes long fusiform, with narrowly globose to subglobose, sometimes ellipsoidal, rounded ends, straight or slightly curved, hyaline, smooth, hyaline. Perithecia not produced in culture. finely spinulose, 1-septate, (8.3–)11.0–14.4(–18.5)× Distribution: Asia (China, Japan); Europe (2.9–)4.2–5.8(–7.6)μm (n=675). (Austria, France, Germany, Scotland, Slovakia, Anamorph in culture: Optimum temperature for Switzerland); North America (United States). growth on PDA 20℃, colonies 31–54mm (average Habitat: On dead woody substrata including 41mm) diam at 20℃ after 7d. Colony surface on Acer macrophyllum, Acer sp., Frangula alnus, PDA, radial, abundant cottony with aerial mycelium, Fagus grandifolia, F. sylvatica, Prunus×yedoensis, sometimes wavy, white to saffron or ochraceous, Quercus crispula, Rhamnus fallax, Rhamnus sp., and saffron to ochraceous sporodochial conidial masses Ulmus sp. producing after one week; reverse ochraceous to Neotype of Sphaeria punicea designated here: sienna. Sporulation on SNA from lateral phialidic Austria, Kaernten, St. Margareten i. Ros., "Tumpfi". pegs on submerged or aerial hyphae common, Grid square 9452/4, on Frangula alnus, 26 Sep. 2.0–7.5μm long, 1.5–3.0μm wide at base. 1999, W. Jaklitsch (BPI 871063; ex-neotype culture Conidiophores occasionally developing on aerial A.R. 3102=CBS 119724). hyphae, unbranched, sometimes verticillate, Ex-type of Neonectria confuse: China, Hubei, 1–3-branched, becoming loosely to moderately Wufeng, 1,200m, on twigs of a dicotyledonous tree, densely branched, 6.5–98μm long, 2.1–3.5μm wide 15 Sep. 2004, coll. Y. Nong, W.Y. Zhuang (Y. Nong, at base. Conidiogenous cells, monophialidic, W.Y. Zhuang 5694; HMAS 99197).

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Specimens and isolates examined: Austria, 31 Aug. 2004, coll. Y. Hirooka (BPI 882117; culture Kaernten, Bad Vellach, Vellacher Kotschna. MTB MAFF 241550=TPP-h330); Tokyo, Okutama-gun, 8653/1, on Rhamnus fallax, 17 Aug. 2004, coll. W. on bark of dead wood, 12 Jul. 2003, coll. T. Tokiwa Jaklitsch (BPI 871062; culture A.R. 4155=CBS (BPI 882160; culture MAFF 241513=TPP-h166); 119527). France, Pyrenees Atlantiques, Isle de Miyagi Prefecture, Taihaku-ku, akiu-cho, Sauveterre de Bearn, alt. 100m, on hardwood tree, Akiuootaki, on twigs, 4 Aug. 2004, coll. Y. Hirooka 25 Oct. 1998, coll. G.J. Samuels, F. Candoussau (culture MAFF 241540=TPP-h296); Niigata (BPI 748311; culture G.J.S. 98-133=CBS 134255); Prefecture, Nakauonuma-gun, Tuna-cho, on bark, 31 Foret du St. Sauvant, on dead twigs of Frangula Aug. 2004, coll. Y. Hirooka (BPI 882114; culture alnus, 18 Apr. 2008, coll. A. Rossman (BPI 878875; MAFF 241547=TPP-h327); Miyagi Prefecture, culture A.R. 4522=CBS 134247); Village de Taihaku-ku, akiu-cho, Akiuootaki, on twigs, 4 Aug. Combret, 48 St. Germain du Teil, on Castanea, 19 2004, coll. Y. Hirooka (BPI 882107; culture MAFF Feb. 2011, coll. C. Lechat C.L.L. 10040 (A.R. 241541=TPP-h297); Miyagi Prefecture, Taihaku-ku, 4828=CBS 134248); St. Maixent, on Robinia, 14 akiu-cho, Akiuootaki, on Quercus crispula, 4 Aug. Feb. 2011, coll. C. Lechat C.L.L. 10042 (A.R. 2004, coll. Y. Hirooka (BPI 882104; culture MAFF 4829=CBS 134249); Lauastric, on Salix, 12 Feb. 241537=TPP-h290); Gifu Prefecture, Mizunashi 2011, coll. C. Lechat C.L.L. 10045 (A.R. 4830=CBS city, Kashimo-mura, on twigs, 31 Aug. 2004, coll. Y. 134250); Las Muros, 09 Rimont, on Acer, 14 Mar Hirooka (BPI 882116; culture MAFF 2011, coll. J. Fournier J.F. 11017 (A.R. 4831=CBS 241549=TPP-h329). Scotland, Cowal Peninsula, 134251); La Maille, 09 Rimont, on Salix, 25 May Argyll Forest Park. N. End of Holy Loch, alt. 2011, on Salix, 25 Mar 2011, coll. J. Fournier J.F. 25–250m, on dead bark of Acer macrophyllum, 13 11023 (A.R. 4832=CBS 134252). Germany, on Apr. 1992, coll. G.J. Samuels, D. Brayford (BPI bark, Mar. 1924, coll. H.W. Wollenweber (BPI 802647; culture G.J.S. 92-32=CBS 119530). 871038; culture CBS 125.24=IMI 113880=MUCL Slovakia, Volvec, Volovske vrchy, on Fagus 9808); on Ulmus sp., Jul. 1930, coll. H.W. sylvatica, 29 Oct. 1999, coll. A. Kunca (BPI 871118; Wollenweber (BPI 871035=Centraalbureau voor culture A.R. 3713=CBS 119531); Stagiar, on Fagus Schimmelcultures Herbarium H-11427; culture CBS sylvatica, 1999, coll. I. Mihal, A. Kunca (BPI 208.30); former West-Germany, on bark of Rhamnus 871116; A.R. 3454=CBS 119525); Stagiar, sp., Jul. 1929, coll. H.W. Wollenweber (culture CBS Kremnicke vrchy, on Fagus sylvatica, 16 Jul. 1999, 242.29). Japan, Gifu Prefecture, Mizunashi city, coll. A. Kunca (BPI 871117; A.R. 3711=CBS Kashimo-mura, on twigs, 31 Aug. 2004, coll. Y. 119533); Poruba-N6, on Fagus sylvatica, coll. A. Hirooka (BPI 882115; culture MAFF 241548= Kunca (culture A.R. 3704=CBS 119532). TPP-h328); Niigata Prefecture Nakauonuma-gun, Switzerland, Grisons (Graubunden), Canton, Malan Tuna-cho, Akiyamago, on twigs of Prunus× Vicinity, alt. ca. 600m, on Fagus sylvatica, 5 Sep. yedoensis, 30 Aug. 2004, coll. Y. Hirooka (BPI 1990, coll. K.F. Rodrigues (BPI 1107108; culture 882113; culture MAFF 241546=TPP-h326); Gifu G.J.S. 90-29=CBS 119529). United States, New Prefecture, Mizunashi city, Kashimo-mura, on twigs, York, Ulster Co., Catskill Mts. from Oliverea at end

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Fig. 12 Neonectria punicea. A–C: Perithecia in the natural environment; D, E: Median section of perithecia; F: Asci; G: Ascospores in surface view; H: Colony of MAFF 241513 after 7d at 20℃ on PDA; I: Colony of MAFF 241549 after 7d at 20℃ on PDA; J: Colony of CBS 125.24 after 7d at 20℃ on PDA; K, M–U: Conidiophores on SNA; L: Lateral phialidic pegs on SNA; V, W: Micro- and macroconidia on SNA; X: Chlamydospores on SNA. Bar: A=3mm; B–D=300µm; E, K, R=100µm; F=20µm; G, L–N=5µm; H–J=30mm; O–Q, S–X=10µm.

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of McKinley Hollow Road to Summit of Balsam Mt., neotypified. The one isolate from Germany on alt. 550–1,098m. 42°4'0''N 74°22'0''W, on Acer sp., Rhamnus sp., the original host of Sphaeria punicea, 7 May 1993, coll. W.R. Buck (BPI 802504; culture did not have a teleomorph specimen. A specimen G.J.S. 93-35=CBS 119528); New York, Schuyler with culture from Austria on Frangula alnus Co., Arnot Forest, on Fagus grandifolia,1988, coll. (previously known as Rhamnus frangula; E.M. Mahoney (culture A.R. 3675=CBS 119153); Rhamnaceae) is herein designated the neotype of Pennsylvania, Allegheny National Forest. Tree 6, Sphaeria punicea (BPI 871063; ex-neotype culture on Fagus grandifolia, 7 Oct. 2004, coll. M. A.R. 3102=CBS 119724). MacKenzie (BPI 870944; culture A.R. 4096=CBS Morphologically, Neo. punicea could be difficult 119231). to identify because it has a wide host range and is Notes: The basionym of Neonectria punicea, known in Asia, Europe, and North America. Based on Sphaeria punicea, was described from Germany in our phylogenetic data, at least two cryptic species are 1817 (Kunze & Schmidt 1817), and the type embedded within Neo. punicea. These appear as specimens should be preserved in B. Despite subclades B-1 and B-2 each supported by high MI PP, intensively searching in several herbaria including B MPBP and ML BP values. Although these specimens around Europe, this type specimen could not be and isolates of these subclades were examined located. Because no illustrations exist in the original carefully, we could not find useful characters to paper (Kunze & Schmidt 1817), the name is herein distinguish them.

Key to species in the Neonectria coccinea GROUP 1. Mycelial growth >15mm at 30℃ for 7d on PDA············································································ 2 1. Mycelial growth <15mm at 30℃ for 7d on PDA············································································ 3 2. Macroconidia 1−7-septate; on Fagus; in Europe and Asia (one isolate from Japan) ····Neo. coccinea 2. Macroconidia 1−8-septate; known only from Asia (China and Japan)··················Neo. microconidia 3. Macroconidia 1−7-septate, 5-septate macroconidia 50–70µm long on SNA; known from diverse woody hosts in Asia (China and Japan), Europe, and North America ··························· Neo. punicea 3. Macroconidia 1−8-septate, 5-septate macroconidia 80–90µm long on SNA; known only on Fagus in North America·········································································································Neo. faginata

3 DISCUSSION species (Hirooka et al. 2011). In the N. cinnabarina Species delimitation species complex anamorphic states in nature showed By combining a morphological species concept the most useful characters for defining species. In (John & Maggs 1997; Kirk et al. 2008) with the Neo. coccinea group these characters were rarely genealogical concordance phylogenetic species found. recognition (Taylor et al. 2000), we are able to Our phenotypic and genotypic analyses of the define four species in the Neonectria coccinea Neo. coccinea group delimited four previously group. Similarly this approach has been used to known species, each of which is described and divide the Nectria cinnabarina complex into four illustrated. Based on our analyses as well as the

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phylogenetic tree in Zhao et al. (2011), Neo. segregated from the sensu stricto isolates. Likewise, coccinea, Neo. faginata, Neo. microconidia and Neo. subclade B-1 of Neo. punicea consisting of isolates punicea form a monophyletic group within the genus only from Japan is supported by high MI PP, MP BP Neonectria sensu Chaverri (Chaverri et al. 2011). Of and ML BP values. However, because no the six loci used, two nuclear ribosomal DNA morphological differences could be observed, we do regions, ITS and LSU datasets, did not fully not recognize these two clades as distinct species. distinguish these species, but sequence data from the Similar subclades exist in Neo. coccinea and Neo. four protein-coding genes, act, tef1, tub, and rpb1, microconidia, but again no morphological characters strongly supported them. In the tub dataset one exist on which to base species delimitations. isolate of Neo. punicea, A.R. 4155=CBS 119527, Geography was not nested within the main Neo. punicea clade The Neonectria coccinea group is known from due to transitions of a few base pairs. However, this throughout the world especially in the Northern isolate falls in the Neo. punicea clade in the Hemisphere (Castlebury et al. 2006). Within each phylogeny produced by the other three species, geography is more or less useful. protein-coding genes as well as in the combined Neonectria microconidia is known only in Asia, dataset. This phylogenetic separation resulting from Neo. faginata only in North America, and Nectria a few base pair positions also occurred in the coccinea without MAFF 241561 only from Europe. Grosmannia serpens complex (Duong et al. 2012). Neonectria punicea is widespread in Asia, Europe Among the protein-coding genes used in this study, and North America. These four species have been the tef1 dataset provided the most variable regions reported in tropical regions and the Southern and thus was the most informative. Sequence data of Hemisphere (Doidge 1950); however, none of these the ITS, the official barcoding gene for fungi (Seifert reports could be confirmed. 2009; Schoch et al. 2012), did not reliably resolve Host relationships within the Neo. coccinea group. Known only on Fagus, Neonectria coccinea In our study, Neo. faginata and Neo. coccinea and Neo. faginata are recognized as the causal were segregated based on phylogenetic and agents of beech bark disease in Europe and North morphological data as mentioned in Castlebury et al. America, respectively. On the other hand Neo. (2006). Within Neo. punicea many distinct clades punicea and Neo. microconidia are collected from a are included, although only two of them are wide range of woody shrubs and trees including phylogenetically supported by more than 0.70 MI Fagus. Although we have not examined the PP, 70% MP BP and 70% ML BP values. The pathogenicity of Neo. microconidia and Neo. ex-neotype culture of the Neo. puniciea designated punicea, they could be plant pathogenic. Most herein as A.R. 3102 (CBS 119724) is included in specimens used in this study were collected on Neo. punicea subclade B-2 together with CBS newly killed branches or trunks. These fungi may 242.29 and A.R. 4522 (=CBS 134247) supported by exist as endophytes and then sporulate when the high phylogenetic values. These three isolates could substrate becomes weakened (Chapela & Boddy be regarded as Neo. punicea sensu stricto; the other 1988, Sieber 2007). isolates of Neo. punicea could be taxonomically Knowledge of the geographic distribution and

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host association of species in the Neo. coccinea Canadian forests (Houston 1994; O’Brien et al. group has been limited primarily to North America 2001; MacKenzie & Iskra 2005). and Europe. In our study, many samples of Neo. In our study, MAFF 241561 is identified for the coccinea group from Asia, especially Japan, were first time as Neo. coccinea in Japan based on included; most of these are not species of Neo. phenotypic and genotypic data. This fungus may coccinea and Neo. faginata, cause of BBD. already have spread around Japan and will cause Surprisingly, MAFF 241561 from Fagus crenata in serious damage to Japanese beech trees sooner or Kanagawa, Japan, appears to be basal to the rest of later as has occurred in North America. So far, the the isolates of Neo. coccinea on Fagus in Europe disease caused by this fungus has not been detected (subclade E-1). Subclade E-1 including the ex-type in Japan, possibly because the beech bark scale culture (CBS 119158) was supported by moderate to (Cryptococcus fagisuga) has not colonized Japan high MI PP, MP BP and ML BP values (BI PP 0.90, (Gwiazdowski et al. 2006). The scale insect makes MP BP 75%, ML BP 72%) values. We propose two feeding wounds in the bark that enables infection by hypotheses about the origin of MAFF 241561. First Neonectria spp. In order to determine and prevent MAFF 241561 may have been introduced from the possible threat of BBD, Japanese beech trees Europe to Japan and thus derived from European should be tested for pathogenicity using Neo. coccinea by a mutation. This is suggested by MAFF241561. A careful watch for the beech bark the high number of shared single nucleotide scale insect as well as other isolates of Neo. polymorphisms, the sister relationship between coccinea in Japan should be continued. MAFF 241561 and the E-1 subclade indicates a recent speciation event. The host of MAFF 241561 Acknowledgements: The authors thank Dr. Gary J. is Japanese beech, Fagus crenata, a species that is Samuels (USDA-ARS, Beltsville, Maryland, USA) for sister to the European beech, Fagus sylvatica (Denk using the many specimens and isolates that he collected as 2003). Another hypothesis is that MAFF 241561 well as providing fruitful comments during the course of may have originated in Japan and simply was not this study. We also thank the many mycologists who detected until now. To know the origin of the shared their fresh specimens and/or cultures with us, Japanese isolate, more collections are needed not specifically Matt Kassen, Walter Jaklitsch, Christian only from Japan, but also from other Asian countries. Lechat, Martin MacKenzie, Hayato Masuya, and The potential threat of BBD in Japan Toshiyuki Tokiwa. We express sincere thanks to Sato Non-indigenous fungal pathogens can be highly Toyozo, Takayuki Aoki, and Keisuke Tomioka (NIAS disastrous to hosts that are closely related to the Genebank, National Institute of Agrobiological Sciences, original host species because of host-pathogen Tukuba, Ibaraki, Japan), and Keiko T. Natuaki (Tokyo co-adaptation (Parker & Gilbert 2004). The current University of Agriculture) for quickly depositing and forest inventory data in the United State suggest that sending Japanese cultures. We express sincere thanks to BBD has invaded areas with relatively high densities the curators and staff of the BPI and G. This study was of beech. BBD have degraded American beech trees supported in part by the United States National Science (Fagus grandifolia) in the eastern deciduous forests Foundation (NSF) PEET grant DEB-0731510 of North America and are now spreading in ‘Monographic Studies in the Nectriaceae, Hypocreales:

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