Mycological Progress https://doi.org/10.1007/s11557-018-1395-4 ORIGINAL ARTICLE Diaporthe from walnut tree (Juglans regia) in China, with insight of the Diaporthe eres complex XinLei Fan 1 & Qin Yang 1 & Jadson D. P. Bezerra 2 & Lourdes V. Alvarez 3 & ChengMing Tian 1 Received: 27 June 2017 / Revised: 10 March 2018 / Accepted: 14 March 2018 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract Species of Diaporthe are important plant pathogenic fungi that commonly occur on a wide range of hosts. They are relatively difficult to identify due to their extreme similarity in morphology and confusing multigene phylogeny, especially in the Diaporthe eres complex. In the present study, isolates were collected from diseased branches of Juglans regia in China. Most strains were clustered into the D. eres species complex based on the combined internal transcribed spacer (ITS) region, partial calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (TUB) genes. To focus on this complex, CAL, TEF1-α and TUB were selected in further phylogenetic analyses that showed a better topology compared with combined five-gene phylogeny. Results revealed that all strains which clustered in the Diaporthe eres complex from Juglans regia in China were Diaporthe eres. Results suggested a revised species criterion in the Diaporthe eres complex. The current study uncovered a new species here described as Diaporthe. tibetensis. Keywords Diaporthales . Molecular phylogeny . New species . Species complex . Taxonomy Introduction Members of Diaporthe Nitschke (syn. Phomopsis (Sacc.) Bubák) are plant pathogens, endophytes or saprobes on a wide range of hosts and are responsible for diseases, some of these causing damage in plants of economic importance (Gomes et al. 2013; Chepkirui and Stadler 2017; Guarnaccia et al. 2018). The sexual morph of Diaporthe is characterised by ascomata immersed in the substrate, often erumpent through a stroma with more or less elongated perithecial necks; asci are unitunicate, clavate to cylindrical, eight-spored; Section Editor: Roland Kirschner * ChengMing Tian chengmt@bjfu.edu.cn 1 The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China 2 Departamento de Micologia Prof. Chaves Batista, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n, Centro de Biociências, Cidade Universitária, CEP, Recife, PE 50670-901, Brazil 3 Department of Biology, College of Science, Polytechnic University of the Philippines, Sta. Mesa, Manila, Philippines ascospores are hyaline, biseriate to uniseriate in the ascus, fusoid, ellipsoid to cylindrical, straight, inequilateral or curved, septate and sometimes with appendages (Wehmeyer 1933; Udayanga et al. 2011; Gomes et al. 2013). The asexual morph is characterised by ostiolate conidiomata pycnidial, with cylindrical phialides producing up to two types of conidia; alpha conidia are hyaline, aseptate, fusiform and usually biguttulate, but sometimes aguttulate or having more than two guttules; beta conidia are hyaline, aseptate, filiform, straight or more frequently curved (Wehmeyer 1933; Udayanga et al. 2011; Gomes et al. 2013). More than 1033 epithets for Diaporthe and 976 for Phomopsis were listed in Index Fungorum (January 2018) (http://www.indexfungorum.org/). However, a majority of them may be synonyms due to the unclear linkage of sexual and asexual morphs and the historical species recognition criteria based on morphology, culture characteristics and host affiliation (Wehmeyer 1933; Uecker 1988; Rehner and Uecker 1994; Santos and Phillips 2009). It is widely known that a single Diaporthe species often occurs on taxonomically unrelated hosts, and more than one species can co-occur on the same host plant (Santos and Phillips 2009; Gomes et al. 2013). Taylor et al. (2000) proposed Genealogical Concordance Phylogenetic Species Recognition (GCPSR), Mycol Progress which was used as an alternative to morphological species recognition (MSR) and biological species recognition (BSR). The recent taxonomic process to define Diaporthe species has been established using molecular techniques (Castlebury et al. 2002; Santos and Phillips 2009; Santos et al. 2010; Udayanga et al. 2011, 2012a, b; Tan et al. 2013). Gomes et al. (2013) reviewed the genus Diaporthe using extype material and provided updated backbone trees based on five-gene phylogeny. The results showed a high genetic diversity, and species related to Diaporthe eres, the type species of Diaporthe, are regarded as a species complex (Gomes et al. 2013). Udayanga et al. (2014b) designated the strain AR5193 as the epitype of Diaporthe eres from its original habitat (Ulmus, Germany) and proposed seven genes to determine the species boundaries in the Diaporthe eres complex. Walnut (Juglans regia L.) is the most widely distributed nut tree in the world (Taha and Al-wadaan 2011). It is commercially cultivated throughout southern Europe, northern Africa, eastern Asia, the USA and western South America. China is the leading world producer of walnuts, followed by France, India, Iran, Romania, Turkey, Ukraine and the USA (Martínez et al. 2010). The high protein and oil contents of the kernels of Juglans regia made the fruit indispensable for human nutrition. The walnut therefore is classified as a strategic species for human nutrition and is included in the FAO list of priority plants (Gandev 2007). However, Juglans regia is usually infected by a range of canker disease pathogens, which often cause serious losses (Fan et al. 2015b). During an investigation of pathogens causing tree cankers or dieback diseases in China, several diaporthalean fungi associated with disease symptoms were collected. Hence, the objectives of the present study were to (i) identify Diaporthe taxa associated with disease symptoms of Juglans regia in China and (ii) provide a multi-gene phylogeny delineating the species concerned. Materials and methods Isolation Fresh specimens of Diaporthe spp. were collected from the infected branches of Juglans regia in Beijing, Gansu, Henan, Ningxia, Sichuan and Tibet, China. Single conidial isolations were established from the fruiting body by removing a mucoid conidial mass from pycnidial ostioles, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) and incubating at 25 °C for up to 24 h. Single germinating conidia were removed and plated onto fresh potato dextrose agar (PDA) plates. Fifteen strains were used in the phylogenetic analysis (Table 1). Specimens and isolates were deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures were maintained in the China Forestry Culture Collection Centre (CFCC). Morphology Morphological features of the fruiting bodies produced on infected plant tissues were examined as well as cultural characteristics. Cross-sections were prepared by hand using a double-edge blade under a stereomicroscope. Morphological characteristics of the fruiting bodies included the size of conidiomata and locules and the size and shape of conidiophores and conidia. These were determined using a Leica compound microscope (LM, DM 2500). More than 20 fruiting bodies were sectioned, and 50 spores were selected randomly for measurement. Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were observed and recorded at 3, 7 and 30 days, including colony colour, texture and the arrangement of the conidiomata. Colony diameters were measured, and the colony colours described after 3 weeks according to the colour charts of Rayner (1970). DNA extraction, PCR amplification and sequencing Fungal mycelium from pure cultures of representative isolates was harvested from PDA plates with cellophane using a modified CTAB method (Doyle and Doyle 1990). The DNA was estimated by electrophoresis in 1% agarose gels, and the quality was measured by NanoDrop™ 2000 (Thermo, USA) according to the user’s manual (Desjardins et al. 2009). The PCR amplifications were performed in DNA Engine (PTC-200) Peltier Thermal Cycler (Bio-Rad Laboratories, CA, USA). The CAL gene was amplified with the primers CAL228F/CAL737R (Carbone and Kohn 1999), the HIS gene with the primers CYLH4F (Crous et al. 2004a) and H3-1b (Glass and Donaldson 1995), the ITS region with the primers ITS1/ITS4 (White et al. 1990), the TEF1-α gene with the primers EF1-728F/EF1-986R (Carbone and Kohn 1999) and the TUB gene with the primers Bt2a/Bt2b (Glass and Donaldson 1995). The PCR amplification products were estimated visually by electrophoresis in 2% agarose gels. The DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyzer with BigDye® Terminater Kit v.3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China). Molecular data analyses The DNA sequences generated by forward and reverse primers were used to obtain consensus sequences using SeqMan v.7.1.0 in the DNASTAR Lasergene Core Suite software (DNASTAR Inc., Madison, WI, USA). Reference sequences were selected based on ex-type or ex-epitype sequences available in GenBank and from relevant published Details of the strains included for molecular study Species name D. acaciigenaT D. alleghaniensisT D. alneaT D. ampelinaT D. amygdaliT D. apiculatumT D. arctiiT D. aseanaT D. australafricanaT D. batatasT D. betulaeT D. betulae D. betulicolaT D. betulicola D. biconisporaT D. bicinctaT D. biguttulataT D. biguttusisT D. brasiliensisT D. canthiiT D. carpini D. castaneae-mollisimaeT D. caulivoraT D. celastrinaT D. citriT D. citrichinensisT D. convolvuliT D. cotoneastriT D. crotalariaeT D. cuppateaT D. cynaroidisT D. cytosporellaT D. daoigenaT D. detrusa D. discoidisporaT D. ellipicolaT D. endophyticaT D. eresT D. eres D. eres D. eres D. eres Strain number1 CBS 129521 CBS 495.72 CBS 146.46 CBS 114016 CBS 126679 LC3418 DP0482 MFLUCC 120299 CBS 111886 CBS 122.21 CFCC 50469 CFCC 50470 CFCC 51128 CFCC 51129 ICMP20654 CBS 121004 ICMP20657 CGMCC 3.17081 CBS 133183 CBS 132533 CBS 114437 DNP 128 CBS 127268 CBS 139.27 CBS 135422 ZJUD34 FAU 649 CBS 439.82 CBS 162.33 CBS 117499 CBS 122676 FAU 461 MFLUCC 160097 CBS 109770 ICMP20662 CGMCC 3.17084 CBS 133811 AR5193 CFCC 52003 CFCC 52005 CFCC 52006 CFCC 52007 Substrate Acacia retinodes Betula alleghaniensis Alnus sp. Vitis vinifera Prunus dulcis Camellia sinensis Arctium lappa dead leaf Vitis vinifera Ipomoea batatas Betula platyphylla Betula platyphylla Betula albosinensis Betula albosinensis Citrus maxima Juglans sp. Citrus limon Lithocarpus glabra Aspidosperma tomentosum Canthium inerme Carpinus betulus Castanea mollissima Glycine max Celastrus scandens Citrus sp. Citrus unshiu Convolvulus arvensis Cotoneaster sp. Crotalaria spectabilis Aspalathus linearis Protea cynaroides Citrus limon Prunus persica Berberis vulgaris Citrus unshiu Lithocarpus glabra Schinus terebinthifolius Ulmus sp. Juglandis regia Juglandis regia Juglandis regia Juglandis regia Origin Australia Canada Netherlands France Portugal China Austria Thailand Australia USA China China China China China USA China China Brazil South Africa Sweden China Croatia USA USA China Canada United Kingdom USA South Africa South Africa Spain China Austria China China Brazil Germany China China China China GenBank accession number2 CAL HIS ITS TEF1-α TUB KC343005 KC343007 KC343008 AF230751 KC343022 KP267896 KJ590736 KT459414 KC343038 KC343040 KT732950 KT732951 KX024653 KX024654 KJ490597 KC343134 KJ490582 KF576282 KC343042 JX069864 KC343044 JF957786 KC343045 KC343047 KC843311 JQ954648 KJ590721 KC343090 KC343056 KC343057 KC343058 KC843307 KU557547 KC343061 KJ490624 KF576270 KC343065 KJ210529 – MF279899 MF279900 MF279901 KC343247 KC343249 KC343250 AY745026 KC343264 – KJ612133 KT459464 KC343280 KC343282 KT732997 KT732998 KX024659 KX024660 – KC343376 – – KC343284 KC843174 KC343286 JX197430 KC343287 KC343289 KC843157 KC357494 KJ612130 KC343332 KC343298 KC343299 KC343300 KC843141 KU557595 KC343303 – – KC343307 KJ434999 MF279830 MF279840 MF279841 MF279842 KC343489 KC343491 KC343492 – KC343506 – KJ659218 – KC343522 KC343524 KT732999 KT733000 KX024661 KX024662 KJ490539 KC343618 KJ490524 – KC343526 – KC343528 – KC343529 KC343531 – – KJ659210 KC343574 KC343540 KC343541 KC343542 – – KC343545 KJ490566 – KC343549 KJ420850 MF279845 MF279855 MF279856 MF279857 KC343731 KC343733 KC343734 AY745056 KC343748 KP267970 KJ590776 KT459448 KC343764 KC343766 KT733016 KT733017 KX024655 KX024656 KJ490476 KC343860 KJ490461 KF576257 KC343768 KC843120 KC343770 JX275401 KC343771 KC343773 KC843071 JQ954666 KJ590765 KC343816 KC343782 KC343783 KC343784 KC843116 KU557615 KC343787 KJ490503 KF576245 KC343791 KJ210550 MF279860 MF279870 MF279871 MF279872 KC343973 KC343975 KC343976 JX275452 KC343990 KP293476 KJ610891 KT459432 KC344006 KC344008 KT733020 KT733021 KX024657 KX024658 KJ490418 KC344102 KJ490403 KF576306 KC344010 KC843230 KC344012 JX275438 KC344013 KC344015 KC843187 – – KC344058 KC344024 KC344025 KC344026 KC843221 KU557571 KC344029 KJ490445 KF576291 KC344033 KJ420799 MF279875 MF279885 MF279886 MF279887 Mycol Progress Table 1 Table 1 (continued) Species name CFCC 52008 CFCC 52009 CFCC 52010 CFCC 52011 CFCC 52012 CFCC 52013 CFCC 52014 CFCC 52015 CFCC 52016 CBS 109751 CBS 123208 BRIP 54781 CBS 288.56 MFLUCC 120542 CBS 592.81 AR5211 CBS 115448 CBS 114434 BRIP 54031 BRIP 54900 CGMCC 3.17089 ATCC 60325 CBS 123212 CGMCC 3.15181 DAOMC 250563 MFLUCC 160113 ICMP20656 CBS 144. 27 CBS 109490 CBS 113470 CBS 157.29 BRIP 54801 CBS 127270 LC3166 ICMP20659 CGMCC 3.17092 CBS 133186 CBS 114649 CBS 133184 BRIP 54847 LC3353 MFLUCC 160105 Substrate Juglandis regia Juglandis regia Juglandis regia Juglandis regia Juglandis regia Juglandis regia Juglandis regia Juglandis regia Juglandis regia Rhamnus cathartica Foeniculum vulgare Fraxinus angustifolia subsp. oxycarpa Gardenia jasminoides dead leaf Helianthus annuus Hedera helix Dichroa febrifuga Sorbus aucuparia Helianthus annuus Litchi chinensis Lithocarpus glabra Glycine max Foeniculum vulgare Lithocarpus glabra Picea rubens Prunus persica Citrus maxima Spiraea sp. Ambrosia trifida Castanea sativa Morus sp. Nothofagus cunninghamii Glycine max Camellia sinensis Citrus limon Lithocarpus glabra Maytenus ilicifolia Alnus glutinosa Maytenus ilicifolia Persea americana Camellia sinensis Prunus persica Origin China China China China China China China China China Austria Portugal Australia Italy Thailand Serbia Germany China Sweden Australia Australia China USA Portugal China Canada China China USA USA Korea Japan Australia Croatia China China China Brazil Sweden Brazil Australia China China GenBank accession number2 CAL HIS ITS TEF1-α TUB MF279898 MF279896 MF279897 MF279894 MF279895 MF279890 MF279891 MF279892 MF279893 KC343099 KC343104 JX862528 KC343113 KT459423 KC343115 KJ210538 KC343119 KC343121 JF431301 JX862533 KF576267 KJ590728 KC343136 KC153096 KU552025 KU557563 KJ490633 KC343144 KC343145 KC343146 KC343154 JX862530 KC343156 KP267863 KJ490628 KF576264 KC343164 KC343170 KC343171 JX862532 KP714505 KU557555 MF279839 MF279837 MF279838 MF279835 MF279836 MF279831 MF279832 MF279833 MF279834 KC343341 KC343346 – KC343355 KT459470 KC343357 KJ435043 KC343361 KC343363 – – – KJ612124 KC343378 – – KU557611 – KC343386 KC343387 KC343388 KC343396 – KC343398 – – KF576222 KC343406 KC343412 KC343413 – – KU557603 MF279854 MF279852 MF279853 MF279850 MF279851 MF279846 MF279847 MF279848 MF279849 KC343583 KC343588 – KC343597 – KC343599 KJ420875 KC343603 KC343605 – – – KJ659188 KC343620 – – – KJ490575 KC343628 KC343629 KC343630 KC343638 – KC343640 KP293517 KJ490570 – KC343648 KC343654 KC343655 – KP714493 – MF279869 MF279867 MF279868 MF279865 MF279866 MF279861 MF279862 MF279863 MF279864 KC343825 KC343830 JX862534 KC343839 KT459457 KC343841 KJ210559 KC343845 KC343847 JN645797 JX862539 KF576242 KJ590767 KC343862 KC153087 KU552023 KU557631 KJ490512 KC343870 KC343871 KC343872 KC343880 JX862536 KC343882 KP267937 KJ490507 KF576239 KC343890 KC343896 KC343897 JX862538 KP714517 KU557623 MF279884 MF279882 MF279883 MF279880 MF279881 MF279876 MF279877 MF279878 MF279879 KC344067 KC344072 KF170920 KC344081 KT459441 KC344083 KJ420828 KC344087 KC344089 KJ197272 KF170925 KF576291 KJ610883 KC344104 KF576312 KU574615 KU557587 KJ490454 KC344112 KC344113 KC344114 KC344122 KF170922 KC344124 KP293443 KJ490449 KF576288 KC344132 KC344138 KC344139 KF170924 KP714529 KU557579 Mycol Progress D. eres D. eres D. eres D. eres D. eres D. eres D. eres D. eres D. eres D. fibrosa D. foeniculaceaT D. fraxini-angustifoliaeT D. gardeniae D. garethjonesiiT D. helianthiT D. helicisT D. hongkongensisT D. impulsa D. kongiiT D. litchicolaT D. longicicolaT D. longicollaT D. lusitanicaeT D. mahothocarpusT D. maritimaT D. momicolaT D. multigutullataT D. neilliaeT D. neoarctiiT D. nobilis D. nomurai D. nothofagiT D. novemT D. oracciniiT D. ovalisporaT D. ovoicicolaT D. oxeT D. padi var. padi D. paranensisT D. pascoeiT D. penetriteumT D. pescicolaT Strain number1 Species name D. perjunctaT D. phaseolorumT D. pseudophoenicicolaT D. pterocarpiT D. pterocarpicolaT D. pullaT D. rostrataT D. rostrate D. rudisT D. schiniT D. scobina D. sojaeT D. stewartiiT D. subclavataT D. taoicolaT D. terebinthifoliiT D. thunbergiiT D. thunbergiicolaT D. tibetensisT D. tibetensis D. toxicaT D. ueckeraeT D. unshiuensisT D. vacciniiT D. virgiliaeT D. woolworthii Diaporthella corylina Strain number1 CBS 109745 AR4203 CBS 462.69 MFLUCC 100571 MFLUCC 100580 CBS 338.89 CFCC 50062 CFCC 50063 AR3422 CBS 133181 CBS 251.38 FAU 635 CBS 193.36 ICMP20663 MFLUCC 160117 CBS 133180 MFLUCC 100576 MFLUCC 120033 CFCC 51999 CFCC 52000 CBS 534.93 FAU 656 CGMCC3.17569 CBS 160.32 CMW 40755 CBS 148.27 CBS 121124 Substrate Ulmus glabra Phaseolus vulgaris Phoenix dactylifera Pterocarous indicus Pterocarpus indicus Hedera helix Juglans mandshurica Juglans mandshurica Laburnum anagyroides Schinus terebinthifolius Fraxinus excelsior Glycine max Cosmos bipinnatus Citrus unshiu Prunus persica Schinus terebinthifolius Thunbergia laurifolia Thunbergia laurifolia Juglandis regia Juglandis regia Lupinus angustifolius Cucumis melo Citrus unshiu Vaccinium macrocarpon Virgilia oroboides Ulmus americana Corylus sp. Origin Austria USA Spain Thailand Thailand Yugoslavia China China Austria Brazil UK USA – China China Brazil Thailand Thailand China China Australia USA China USA South Africa. – China GenBank accession number2 CAL HIS ITS TEF1-α TUB KC343172 KJ590738 KC343184 JQ619899 JQ619887 KC343152 KP208847 KP208848 KC843331 KC343191 KC343195 KJ590719 FJ889448 KJ490587 KU557567 KC343216 JQ619893 KP715097 MF279888 MF279889 KC343220 KJ590726 KJ490587 KC343228 KP247573 KC343245 KC343004 KC343414 KJ612135 KC343426 JX197451 JX197433 KC343394 KP208849 KP208850 KC843146 KC343433 KC343437 KJ612116 JX197415 – – KC343458 JX197440 – MF279828 MF279829 KC343462 KJ612122 – KC343470 – KC343487 KC343246 KC343656 KJ659220 KC343668 – – KC343636 KP208851 KP208852 – KC343675 KC343679 KJ659208 – KJ490529 – KC343700 – – MF279843 MF279844 KC343704 KJ659215 KJ490529 KC343712 – KC343729 KC343488 KC343898 KJ590739 KC343910 JX275416 JX275403 KC343878 KP208853 KP208854 KC843090 KC343917 KC343921 KJ590762 GQ250324 KJ490466 KU557635 KC343942 JX275409 KP715098 MF279858 MF279859 KC343946 KJ590747 KJ490466 KC343954 – KC343971 KC343730 KC344140 KJ610893 KC344152 JX275460 JX275441 KC344120 KP208855 KP208856 KC843177 KC344159 KC344163 KJ610875 JX275421 KJ490408 KU557591 KC344184 JX275449 – MF279873 MF279874 KC344188 KJ610881 KJ490408 KC344196 KP247582 KC344213 KC343972 Strains isolated from the current study are in bold. Ex-type/ex-epitype isolates are marked by T 1 ATCC American Type Culture Collection, Virginia, USA; AR, DP, FAU isolates in culture collection of Systematic Mycology and Microbiology Laboratory, USDA-ARS, Beltsville, Maryland, USA; BRIP Australian plant pathogen culture collection, Queensland, Australia; CBS Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CFCC China Forestry Culture Collection Center, China; CGMCC China General Microbiological Culture Collection; CMW culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute; ICMP International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand; LC working collection of Lei Cai, housed at Institute of Microbiology, CAS, China; MFLUCC Mae Fah Luang University Culture Collection; ZJUD Zhe Jiang University, China 2 CAL partial calmodulin region, HIS partial histone H3 region, ITS internal transcribed spacer, TEFl-α partial translation elongation factor 1-alpha region, TUB beta-tubulin region Mycol Progress Table 1 (continued) Mycol Progress literature (Gomes et al. 2013; Udayanga et al. 2014b; Gao et al. 2014, 2015, 2016, 2017; Huang et al. 2015; Tanney et al. 2016) (Table 1). All sequences were aligned using MAFFT v.6 (Katoh and Toh 2010) and edited manually using MEGA6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v.4.0b10 for maximum parsimony (MP) analysis (Swofford 2003), MrBayes v.3.1.2 for Bayesian inference (BI) analysis (Ronquist and Huelsenbeck 2003) and PhyML v.7.2.8 for maximum likelihood (ML) analysis (Guindon et al. 2010). The first analyses were performed on the combined multi-gene dataset (CAL, HIS, ITS, TEF1-α, TUB) to compare Diaporthe species from another ex-type reference of this genus in recent studies (Table 1). Diaporthella corylina (CBS 121124) was selected as outgroup in this analysis (Gomes et al. 2013). The second analyses using a three-gene matrix (CAL, TEF1-α, TUB) were performed to focus on the Diaporthe eres complex according to recent publications (Udayanga et al. 2014b; Gao et al. 2014, 2015, 2016; Tanney et al. 2016). Diaporthe citri (AR3405) and Diaporthe citrichinensis (ZJUD034A and ZJUD034B) were selected as outgroup (Udayanga et al. 2014b). Trees were shown using FigTree v.1.3.1 (Rambaut and Drummond 2010). The MP analysis was run using a heuristic search option of 1000 random addition sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). The branch support was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993). The BI analysis with a Markov chain Monte Carlo (MCMC) algorithm was performed (Rannala and Yang 1996). The models of evolution were estimated by MrModeltest v.2.3 (Posada and Crandall 1998). Two MCMC chains were run from random trees for 1,000,000 generations and stopped when the average standard deviation of split frequencies fell below 0.01. Trees were saved for each 1000 generations. The first 25% of trees were discarded as the burn-in phase of each analysis, and the posterior probabilities (BPP) were calculated to assess the remaining trees (Rannala and Yang 1996). The ML analysis was performed with a GTR site substitution model, including the gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993). Sequence data generated from this study were deposited in GenBank (Table 1). The multilocus sequence alignment files were deposited in TreeBASE (www.treebase.org; accession number: S21214). The taxonomic novelty was deposited in MycoBank (Crous et al. 2004b). Results Molecular data analyses The first analysis based on the combined datasets (CAL, HIS, ITS, TEF1-α and TUB) was performed from the current study and other ex-type strains in previous studies (Gomes et al. 2013; Udayanga et al. 2012a, b, 2014b; Gao et al. 2014, 2015, 2016; Huang et al. 2015; Fan et al. 2015a; Du et al. 2016; Tanney et al. 2016). The alignment included 129 ingroup taxa, comprising 2482 characters after alignment. Of these, 1083 characters were constant, 344 variable characters were parsimony uninformative and 1055 characters were parsimony informative. The MP analysis resulted in 80 equally most parsimonious trees (TL = 7753, CI = 0.340, RI = 0.699, RC = 0.238), and the first tree is shown in Fig. 1. The phylogenetic tree obtained from ML and Bayesian analyses with the MCMC algorithm was consistent with the previous MP tree. MP and ML bootstrap support values above 50% are shown at the first and second positions. The branches with significant Bayesian posterior probability (≥ 0.90) in Bayesian analyses were thickened in the phylogenetic tree. The results based on the five genes indicated that 14 strains in the current study were residing in the Diaporthe eres complex, whereas the strains CFCC 51999 and 52,000 indicated one novel species, which formed an individual clade (MP/ML/BI = 100/100/1) compared other Diaporthe species (Fig. 1). The second analysis based on the combined datasets (CAL, TEF1-α and TUB) was performed to compare the Diaporthe eres complex species. The alignment included 82 ingroup taxa, comprising 1189 characters after alignment. Of these, 932 characters were constant, 91 variable characters were parsimony uninformative and 186 characters were parsimony informative. The MP analysis resulted in 103 equally most parsimonious trees (TL = 429, CI = 0.660, RI = 0.865, RC = 0.571), and the first one is shown in Fig. 2. The phylogenetic tree obtained from ML and Bayesian analyses with the MCMC algorithm was consistent with the previous MP tree. Based on the multi-locus phylogeny and morphology, 14 strains were identified as Diaporthe eres. The MP and ML bootstrap support values above 50% were shown at the first and second positions. The branches with significant Bayesian posterior probability (≥ 0.90) were thickened in the phylogenetic tree. The current results based on the three genes (CAL, TEF1-α and TUB) suggest that the Diaporthe eres clade could be separated from other species in this complex (Fig. 2). However, Diaporthe biguttusis (CGMCC 3.17081), Diaporthe ellipicola (CGMCC 3.17084), Diaporthe longicolla (CGMCC 3.17089) and Diaporthe mahothocarpus (CGMCC 3.15181) were clustered in Diaporthe eres clade and thus treated as the synonyms of D. eres in the current study. Mycol Progress Fig. 1 Phylogram of Diaporthe based on a combined analysis of ITS, CAL, HIS, TEF1-α and TUB2. MP and ML bootstrap support values above 50% are shown at the first and second positions. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 200 nucleotide substitutions. Ex-type strains are in bold. Strains in current study are in blue Taxonomy Sexual state: Undetermined. Asexual state: Conidiomata pycnidial, embedded in the bark, slightly erumpent through bark surface at maturity, with a single locule. Ectostromatic disc primrose to olivaceous buff, ovoid to circular. Locule undivided, (380–)430–550(−600) μm (av. = 490 μm, n = 20) diam. Conidiogenous cells hyaline, smooth, branched, phialidic, cylindrical, tapering towards apex, straight or slightly curved, (9–)10–14.5(−16) × (1−)1.5 μm (av. = 12 × 1.5 μm, n = 50). Alpha conidia abundant in twigs, hyaline, aseptate, ellipsoidal or oval, occasionally with one end cuspidal, 0–3guttulate, (7.5–)8–10(−11) × 2–2.5(−3) μm (av. = 9.5 × 2.5 μm, n = 50). Beta conidia not observed. Diaporthe tibetensis C.M. Tian, Q. Yang & Fan, sp. nov. Fig. 3. MycoBank: 824067. Etymology: tibetensis (Lat.): referring to the geographical origin of the type strain. Holotype: CHINA, Tibet Autonomous Region, Linzhi City, 29° 54′ 33.38″ N, 95° 37′ 07.66″ E, 2731 masl, on twigs and branches of Juglans regia, coll. X.L. Fan, July 2016 (holotype BJFC-CF2017601; living ex-type culture CFCC 51999). Mycol Progress Mycol Progress ƒFig. 2 Phylogram of the Diaporthe eres complex based on combined CAL, TEF1-α and TUB2. MP and ML bootstrap support values above 50% are shown at the first and second positions. Thickened branches represent posterior probabilities above 0.95 from BI. Scale bar = 6 nucleotide substitutions. Ex-type strains are in bold. Strains in current study are in blue Culture characters: Cultures on PDA incubated at 25 °C in darkness, colony originally flat with white felty aerial mycelium, becoming olivaceous to isabelline with smoke grey aerial mycelium, margin irregular, conidiomata sparse, irregularly distributed over agar surface. Other specimens examined: CHINA, Tibet Autonomous Region, Linzhi City, 29° 54′ 33.38″ N, 95° 37′ 07.66″ E, 2731 masl, on twigs and branches of Juglans regia, coll. X.L. Fan, July 2016 (paratype BJFC-CF2017602; living exparatype culture, CFCC 52000). Fig. 3 Morphology of Diaporthe tibetensis from Juglans regia (CFCC 51999). a, b Habit of conidiomata on branches. c Longitudinal section through conidioma. d, e Conidia. f Conidiophores. g Colonies on PDA at 30 days. Scale bars: c = 200 μm; d–f = 10 μm Notes: Diaporthe tibetensis clustered in a separate clade with high support values (MP/ML/BI = 100/100/1) in Fig. 1. Morphologically, it is characterised by ellipsoidal or oval, aseptate alpha conidia, which are conspicuously 0–3guttulate. Phylogenetic molecular data showed that it is close to D. citrichinensis and Diaporthe oraccinii (Fig. 1). However, Diaporthe tibetensis may be distinguished from D. citrichinensis in the size of the conidiogenous cells (10.0–14.5 × 1.5 μm in D. tibetensis vs. 9–19.5 × 1.5–3 μm in D. citrichinensis) and cultural colour in PDA (olivaceous to isabelline in D. tibetensis vs. light grey in D. oraccinii after 2 weeks) (Huang et al. 2015). Diaporthe tibetensis may also be distinguished from D. oraccinii by its shorter conidiogenous cells (10.0–14.5 × 1.5 μm in D. tibetensis vs. 10.5–22.5 × 1–2 μm in D. oraccinii) and larger alpha conidia (8–10 × 2–2.5 μm in D. tibetensis vs. 5.5–7.5 × 0.5–2.0 μm in D. oraccinii) (Gao et al. 2016). Mycol Progress Diaporthe eres Nitschke, Pyrenomyc. Germ. 2: 245 (1870). Fig. 4. = Diaporthe longicolla (Hobbs) J.M. Santos, Vrandečić & A.J.L. Phillips, in Santos, Vrandečić, Čosić, Duvnjak & Phillips, Persoonia 27: 13 (2011). = Diaporthe biguttusis Y.H. Gao & L. Cai, Fungal Biology 119: 305 (2015). = Diaporthe ellipicola Y.H. Gao & L. Cai, Fungal Biology 119: 303 (2015). = Diaporthe mahothocarpus (Y.H. Gao, W. Sun & L. Cai) Y.H. Gao & L. Cai. Australas. Lichenol. 119: 306 (2015). Sexual state: not found. Asexual state: Conidiomata pycnidial, immersed in the bark, erumpent through bark surface, with a single locule. Ectostromatic disc brown to black, with one ostiole per disc. Neck medium black, up to the level of the disc. Locule circular, undivided, (350–)450–600(−680) μm (av. = 550 μm, n = 20) diam. Conidiophores hyaline, unbranched, cylindrical, (5–)7–13(−16) × (1−)1.5 μm (av. = 11 × 1.5 μm, n = 50), straight or sinuous. Conidiogenous cells phialidic, cylindrical, terminal. Alpha conidia hyaline, Fig. 4 Morphology of Diaporthe eres from Juglans regia (CFCC 52009). a, b Habit of conidiomata on branches. c Transverse section of conidioma. d Longitudinal section through conidioma. e Conidia. f Conidiophores. g Colonies on PDA at 30 days. Scale bars: c–d = 200 μm; e–f = 10 μm aseptate, fusiform or fusoid, usually one guttule at each end, rarely three small guttules, (6–)6.5–8(−9) × (1.5–)2–2.5 μm (av. = 7.5 × 2 μm, n = 50). Beta conidia not observed. Culture characters: Cultures on PDA incubated at 25 °C in darkness, colony with white felty aerial mycelium, becoming white compact aerial mycelium at centre and sparse mycelium at margin, with one defined zone. Conidiomata sparse, irregularly distributed over agar surface. Other specimens examined: CHINA, Beijing, Changping District, 40° 12′ 22.46″ N, 115° 56′ 17.20″ E, 823 masl, on twigs and branches of Juglans regia, coll. X.L Fan and Q. Yang, April 2016 (BJFC-CF2016403; living culture CFCC 52009; ibid. BJFC-CF2016404; living culture CFCC 52010); Tongzhou District, 40° 00′ 00.41″ N, 116° 39′ 21.50″ E, 12 masl, on twigs and branches of Juglans regia, coll. X.L Fan and Q. Yang, May 2015 (BJFC-CF2015505; living culture CFCC 52005; ibid. BJFC-CF2015506; living culture CFCC 52006); Yanqing District, 40° 30′ 23.60″ N, 115° 47′ 36.17″ E, 765 masl, on twigs and branches of Juglans regia, coll. X.L Fan and Q. Yang, April 2016 Mycol Progress (BJFC-CF2016411; living culture CFCC 52015; ibid. BJFCCF2016412; living culture CFCC 52016); Henan Province, Shangqiu, 34° 29′ 00.40″ N, 114° 56′ 42.95″ E, 76 masl, on twigs and branches of Juglans regia, coll. X.L Fan, May 2014 (BJFC-CF2014505; living culture CFCC 52003); Gansu Province, Qingyang, 35° 38′ 17.08″ N, 107° 47′ 48.68″ E, 1253 masl, on twigs and branches of Juglans regia, coll. X.L Fan, July 2013 (BJFC-CF2013711; living culture CFCC 52007; ibid. BJFC-CF2013712; living culture CFCC 52008); Ningxia Province, Yinchuan, 38° 37′ 34.08″ N, 105° 56′ 56.61″ E, 1422 masl, on twigs and branches of Juglans regia, coll. X.L Fan, Aug 2015 (BJFC-CF2015804; living culture CFCC 52011; ibid. BJFC-CF2015805; living culture CFCC 52012); Sichuan Province, Guangyuan, 33° 56′ 35.33″ N, 104° 07′ 11.03″ E, 1400 masl, on twigs and branches of Juglans regia, coll. X.L Fan, April 2015 BJFCCF2015421; living culture CFCC 52013; ibid. BJFCCF2015422; living culture CFCC 52014); Notes: Diaporthe eres is the type species of the genus described by Nitschke (1870), from Ulmus sp. collected in Germany. The lack of an ex-type or ex-epitype culture for this generic type species has been a major issue, although a broad species concept has historically been associated with D. eres. Udayanga et al. (2014b) designed strain AR 5193 as the epitype of D. eres and provided the phylogram of this complex using seven genes. Phenotypic plasticity and extensive host associations have long complicated accurate identifications of species in the D. eres complex (Gomes et al. 2013; Udayanga et al. 2014b; Du et al. 2016; Gao et al. 2016; Tanney et al. 2016). The current results included 14 additional strains belonging to D. eres. The results suggest that Diaporthe eres is a good species that can be separated from other species in this complex (Fig. 2). Discussion The current study indicates that all strains related to the Diaporthe eres isolated from Juglans regia in China are Diaporthe eres. It provides a revised species description of the Diaporthe eres complex using CAL, TEF1-α and TUB dataset (Gomes et al. 2013; Udayanga et al. 2014b; Gao et al. 2014, 2015, 2016, 2017; Huang et al. 2015; Tanney et al. 2016). It presents evidence that D. biguttusis, D. ellipicola, D. longicolla and D. mahothocarpus were clustered in Diaporthe eres and should be treated as synonyms of D. eres. The current study also uncovered a novel species which is described and illustrated as D. tibetensis sp. nov. Previously, eight Diaporthe species (i.e. D. bicincta, D. eres, D. euonymi, D. juglandis, D. rostrata, D. rudis, Phomopsis albobestita, P. arnoldiae) were recorded from Juglans spp. in America, Asia and Europe (Uecker 1988; Anagnostakis 2007; Gomes et al. 2013; Udayanga et al. 2014a,b, 2015; Fan et al. 2015a). Therein, D. juglandis was reported as a synonym of Juglanconis oblonga (Voglmayr et al. 2017) from the host genus Juglans. Phomopsis albobestita Fairman. and P. arnoldiae Sutton were unascertainable due to the absence of fungal materials, such as the ex-type cultures or specimens, and no molecular data. Voglmayr et al. (2017) proposed Juglanconidaceae ( D i a p o r t h a l e s ) w i t h J u gl an c o ni s ap p en d ic u la ta , J. juglandina, J. oblonga and J. pterocaryae, resulting in walnut dieback disease. The current phylogenetic results indicate that most strains of Diaporthe species isolated from this study belong to the D. eres complex (Fig. 1). Although much progress has been made to restrict fungal species, several complexes are proposed referring to the confused clades. This is essential for determining patterns of speciation and potential hyper-diversity within a genus (Bickford et al. 2007; Udayanga et al. 2014b). Confusion may occur when a large number of Diaporthe species isolates are increasingly reported in the D. eres species complex based on diverse gene combinations (Gao et al. 2014, 2015, 2016, 2017; Tanney et al. 2016). For example, Gao et al. (2016) reported many isolates from Camellia sinensis belonging to the D. eres species complex; however, these fungi present intermediate morphology, and the phylogenetic tree also revealed clades with short branch and moderate support. The identification of this group is still confusing. Although the phylogeny is better when all five genes (CAL, HIS, ITS, TEF1-α, TUB) are simultaneously combined and analysed, as suggested from previous studies, defining the optimal set of genes that can be used for accurate Diaporthe species taxonomy (Gomes et al. 2013; Fan et al. 2015a; Santos et al. 2017). Santos et al. (2017) followed the five-gene data matrix and evaluated different combinations, which provided good tests to establish the boundaries for Diaporthe species. The recent maximum number of loci was used to establish the specific limits of Diaporthe eres, i.e. ACT, APN2, CAL, FG1093, HIS, TEF1-α and TUB (Udayanga et al. 2014b). The increasing number of genes used to build more informative phylogenetic trees is positively correlated to the sensitivity and accuracy in species separation. However, an increasing number of species in the alignment results in a decrease of an accurate separation, unless a higher number of appropriate loci is used to maintain the quality of that separation. It therefore illustrates the fact that better differentiation between Diaporthe species always relies on the number of the sequenced loci. A tougher issue is the potential over-separation or insufficient evidence to define the species boundaries due to the extremely indistinguishable morphological characters. In this study, we performed frequently used five genes (CAL, HIS, ITS, TEF1-α, TUB) to evaluate Diaporthe species, but it was found to be confusing in species separation of the Diaporthe eres complex (Fig. 1). To further investigate this complex, a second set of four (CAL, HIS, TEF1-α and TUB), three (CAL, TEF1-α and TUB), two Mycol Progress (TEF1-α and TUB) and one (TEF1-α) data matrix was performed following Santos et al. (2017). The results showed that the three genes analyses (CAL, TEF1-α and TUB) appeared to be better for species recognition (Fig. 2) (other trees are not showed), these results conform to the study of Udayanga et al. (2014b). However, we prefer to extend the species boundary of D. eres to the more reliable clade (MP/ML/BI = 89/85/1), including D. bicincta, D. celastrina, D. helicis, D. maritima and D. pulla. To perform a robust test to check it, we would need to include more type materials of the complex and analyse them in order to clarify the real species boundaries in this group. Acknowledgements This study is financed by Fundamental Research Funds for the Central Universities (Project No.: BLX201613) and National Natural Science Foundation of China (Project No.: 31670647). CM Tian and XL Fan thank Chungen Piao, Minwei Guo (China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing. 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