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j.funbio.2018.08.001

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Accepted Manuscript
Four new Ophiostoma species associated with hardwood-infesting bark beetles in
Norway and Poland
Truls Aas, Halvor Solheim, Robert Jankowiak, Piotr Bilański, Georg Hausner
PII:
S1878-6146(18)30211-3
DOI:
10.1016/j.funbio.2018.08.001
Reference:
FUNBIO 947
To appear in:
Fungal Biology
Received Date: 2 May 2018
Revised Date:
21 July 2018
Accepted Date: 1 August 2018
Please cite this article as: Aas, T., Solheim, H., Jankowiak, R., Bilański, P., Hausner, G., Four new
Ophiostoma species associated with hardwood-infesting bark beetles in Norway and Poland, Fungal
Biology (2018), doi: 10.1016/j.funbio.2018.08.001.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
TEF-1α Taxon 4 KFL80WRJTD Poland
CAL
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Taxon 2 2016-1623/1/1 Norway
Taxon 4 KFL9916RJTD Poland
*/94 Taxon 4 KFL46116RJTD Poland
99/100 Taxon 2 2016-1627/2/2 Norway
Taxon 2 2016-1631/2/3 Norway
Taxon 4 KFL46016RJTS Poland
Taxon 4 2016-0625/3/1 Norway
Taxon 2 2016-1622/2/2 Norway
88/83
99/97 Taxon 4 2016-1605/2/3 Norway
Ophiostoma bacillisporum MUCL 44874
96/99
100/97 Taxon 4 2016-0524/3/6/1 Norway
O. bacillisporum MUCL 44885
Taxon 4 2016-0618/2/2 Norway
bacillisporum CBS 771.71
100/100 O.
Taxon 4 2016-0665/2/1 Norway
Taxon
3 2016-1579/2/3 Norway
Taxon 4 KFL26816RJAD Poland
Taxon 3 2016-1693/2/1 Norway
100/100 Ophiostoma karelicum GU930821
Taxon 3 2016-1572/2/1 Norway
Tree # 1
*/100
O. karelicum GU930820
Taxon 3 2016-1684/3/1 Norway
Length 872 85/* O. karelicum KFL95216RJSR Poland
Taxon 1 2016-0018/2/5 Norway
O. karelicum 2016-1209/2/1 Norway
CI 0.711
Taxon 1 2016-0481/2/3 Norway
O. karelicum KFL96816RJSR Poland
O. karelicum 2016-1117/1/2 Norway
Taxon 1 2016-1298/3/1 Norway
RI 0.939
Taxon 1 KFL28015RJHC Poland
100/100 Taxon 1 2016-1456/3/1 Norway
RC 0.668
Taxon 1 KFL57616RJHC Poland
Taxon 1 2016-0021/2/5 Norway
Taxon 1 2016-0018/2/5 Norway
HI 0.289
Taxon 1 KFL28015RJHC Poland
82/81 Taxon
Taxon 1 2016-0481/2/3 Norway
1 KFL57616RJHC Poland
Taxon 1 2016-1298/3/1 Norway
85/100
Taxon 4 2016-0618/2/2 Norway
81/99
Taxon 1 2016-1456/3/1 Norway
Taxon 4 2016-0665/2/1 Norway
96/100
Taxon 1 2016-0021/2/5 Norway
Tree # 1
Taxon 4 2016-0524/3/6/1 Norway
O. catonianum AY466699
Taxon 4 2016-0625/3/1 Norway
Taxon 2 2016-1622/2/2 Norway
Length 546
100/100
Taxon 4 KFL26816RJAD Poland
Taxon 2 2016-1623/1/1 Norway
CI
0.745
82/87
Taxon 4 KFL46016RJTS Poland
Taxon 2 2016-1627/2/2 Norway
Taxon 2 2016-1631/2/3 Norway
Taxon 4 KFL46116RJTD Poland
RI 0.919
99/100
O. bacillisporum CBS 771.71
Taxon 4 KFL80WRJTD Poland
RC0.685
*/100
O. bacillisporum MUCL 44874
78/100
Taxon 4 KFL9916RJTD Poland
O. bacillisporum MUCL 44885
HI 0.255
Taxon 4 2016-1605/2/3 Norway
Taxon
3
2016-1572/2/1
Norway
100/100
O. karelicum 2016-1209/2/1 Norway
99/100
Taxon 3 2016-1693/2/1 Norway
97/98 O. karelicum 2016-1117/2/2 Norway
100/100
Taxon 3 2016-1579/2/3 Norway
84/100
O. karelicum KFL95216RJSR Poland
Taxon 3 2016-1684/3/1 Norway
O. karelicum KFL96816RJSR Poland
O. novo-ulmi KFL67716RJSM Poland 97/100
O. novo-ulmi 2016-1212/1/1 Norway
O. novo-ulmi FJ430490
O. novo-ulmi 2016-1516/2/1 Norway
O. novo-ulmi 2016-1212/1/1 Norway
83/96
O. novo-ulmi 2016-1516/2/1 Norway
O. novo-ulmi KFL18416RJSM Poland
100/100
O. novo-ulmi FJ430491
O. novo-ulmi KFL67716RJSM Poland
96/100
O. novo-ulmi FJ430493
O. borealis KFL10116aRJTD Poland
O. novo-ulmi FJ430494
79/94
O. quercus 2016-1570/3/1 Norway
100/100
O. novo-ulmi KF899885
O. quercus 2016-1288/1/2 Norway
O. novo-ulmi KFL18416RJSM Poland
O. quercus 2016-1198/3/3 Norway
O. novo-ulmi FJ430492
O. quercus KFL10116bRJTD Poland
O. himal-ulmi FJ430489
100/100
O. quercus KFL81WRJXM Poland
O. borealis KF899867
100/100
O. araucariae KU184332
O. quercus 2016-1198/3/3 Norway
97/100 O. quercus 2016-1288/1/2 Norway
O. distortum KU184371
O. quercus AY466688
100/100
O. quercus AY466689
0.09
O. quercus KFL81WRJXM Poland
95/99
O. quercus FJ441268
O. quercus 2016-1570/3/1 Norway
86/*
O. quercus KFL10116bRJTD Poland
100/100
O. denticiliatum KF899872
O. tasmaniense GU797226
O. tsotsi FJ441274
O. australiae KF899891
0.09
O.
undulatum
GU797233
97/100
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Four new Ophiostoma species associated with hardwood-infesting bark beetles in
Norway and Poland
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Truls Aasa,b, Halvor Solheima,b* Robert Jankowiakc, Piotr Bilańskid, Georg Hausnere
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*Corresponding author. Tlf +47 92033663
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E-mail: halvor.solheim@nibio.no.
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Norwegian Institute of Bioeconomy Research, P.O. Box 115, 1431 Ås, Norway
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Department of Ecology and Natural Resource Management, Norwegian University of Life Sciences, P.O. Box
5003, 1432 Ås, Norway
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Institute of Forest Ecosystem Protection; Department of Forest Pathology, Mycology and Tree Physiology;
University of Agriculture in Krakow, Al. 29 Listopada 46, 31-425 Krakow, Poland;
Department of Microbiology, Buller Building 213, University of Manitoba, Winnipeg, R3T 2N2, Canada
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Institute of Forest Ecosystem Protection; Department of Forest Protection, Entomology and Forest Climatology;
University of Agriculture in Krakow, Al. 29 Listopada 46, 31-425 Krakow, Poland
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Abstract
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Ophiostoma spp. (Ophiostomatales, Ascomycota) are well-known fungi associated with bark
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and ambrosia beetles (Curculionidae: Scolytinae, Platypodinae). Fungi in the Ophiostomatales
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include serious tree pathogens as well as agents of timber blue-stain. Although these fungi
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have been extensively studied in the northern hemisphere, very little is known regarding their
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occurrence on hardwoods in Europe. The aims of the present study were to identify and
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characterize new Ophiostoma spp. associated with bark and ambrosia beetles infesting
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hardwoods in Norway and Poland, and to resolve phylogenetic relationships of Ophiostoma
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spp. related to the Norwegian and Polish isolates, using multigene phylogenetic analyses.
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Results obtained from five gene regions (ITS, LSU, ß-tubulin, calmodulin, translation
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elongation factor 1-α) revealed four new Ophiostoma spp. These include O. hylesinum sp.
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nov., O. signatum sp. nov., and O. villosum sp. nov. that phylogenetically are positioned
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within the O. ulmi complex. The other new species, O. pseudokarelicum sp. nov. reside along
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with O. karelicum in a discrete, well-supported phylogenetic group in Ophiostoma s. stricto.
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The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on
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hardwoods in Europe is poorly understood and that further studies are required to enrich our
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knowledge about these fungi.
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Keywords: Hardwoods, Ophiostoma ulmi complex, Phylogeny, Taxonomy
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1. Introduction
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The Ophiostomatales are ascomycetes fungi that currently include nine lineages of which
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seven are well-supported lineages represented by the following genera Aureovirgo,
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Ceratocystiopsis, Fragosphaeria, Graphilbum, Hawksworthiomyces, Raffaelea s. str., and
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Sporothrix (De Beer et al. 2016). In addition, two major groups do not form well-supported
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monophyletic lineages and are treated as Leptographium sensu lato and Ophiostoma sensu
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lato (De Beer & Wingfield 2013; De Beer et al. 2013; De Beer et al. 2016). Ophiostoma s. l.
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comprises the Ophiostoma s. str. that includes the O. clavatum Math., O. ips (Rumbold/
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Nannf., O. pluriannulatum (Hedgc.) Syd., O. piceae (Münch) Syd., and O. ulmi (Buisman)
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Nannf. complexes, and several smaller lineages (De Beer & Wingfield 2003; Linnakoski et al.
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2016; Yin et al. 2016).
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The members of the genus Ophiostoma (Sydow & Sydow 1919) produce ascomata
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with short to long necks, crescent to allantoid shaped ascospores produced in short-lived
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deliquescent asci, and pesotum-, hyalorhinocladiella- or sporothrix-like asexual morphs (De
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Beer & Wingfield 2013). Sexual and asexual states are adapted to produce spores in slimy
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droplets optimized for dispersal by various arthropods, mainly bark beetles (Coleoptera:
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Scolytinae) (Malloch & Blackwell 1993; Six 2003; Kirisits 2004; Harrington 2005). Most
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Ophiostoma spp. are considered causative agents of blue-stain on wood, causing significantly
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reduction in the economic value of timber. However, some members are serious tree
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pathogens, such as O. ulmi, O. novo-ulmi Brasier and O. himal-ulmi Brasier & M.D. Mehrotra
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that cause Dutch elm disease (Schwarz 1928; Brasier 1991; Brasier & Mehrotra 1995).
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According to recent reports (De Errsati et al. 2016; Yin et al. 2016), the O. ulmi
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complex includes 13 species. In a previous proposed classification of De Beer & Wingfield
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(2013), this complex also included O. karelicum Linnakoski, Z.W. de Beer & M.J. Wingf. and
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O. triangulosporum Butin, although morphological features (reniform ascospores with unique
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triangular sheaths) and preference to conifers as hosts suggested that the latter species could
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reside outside of this group. Generally, the members of the O. ulmi species complex are
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isolated from hardwood hosts and are characterized by long necked ascomata, allantoid to
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orange-section shaped ascospores, and pesotum- and sporothrix-like anamorphs (De Beer &
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Wingfield 2013). In addition, O. bacillisporum and O. triangulosporum produce
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hylorhinocladiella-like anamorphs (Butin & Zimmermann 1972; Butin 1978). The ascospores
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of O. basillisporum (Butin & G. Zimm.) de Hoog & Scheffer are bacilliform (Butin &
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Zimmermann 1972).
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Over the years, the majority of ecological and taxonomic studies on the fungal
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associates of bark beetles belonging to Ophiostoma s. str. in the Ophiostomatales have
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focussed on members associated with conifers, especially in Europe (Kirisits 2004;
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Linnakoski et al. 2012). The small number of Ophiostoma spp. reported so far from European
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hardwoods might leave the impression that the continent does not harbour a large diversity of
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these fungi, although it may also indicate that Europe has been poorly explored in this regard.
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We are inclined towards the second option. The occurrence of Ophiostomatales on hardwoods
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has been studied in the past, but usually at a time when massive dieback of trees was
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observed. For example, several ophiostomatoid species were isolated from dying tissues of
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Quercus robur L. (Kowalski & Butin 1989; Kowalski 1991, 1996; Aghayeva et al. 2004,
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2005; Selochnik et al. 2015), while the occurrence of O. ulmi and O. novo-ulmi has been
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discovered during studies on elm dieback, caused by Dutch elm disease (Webber & Brasier
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1984; Webber 1990; Brasier 1991; Menkis et al. 2016).
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In Europe, there are few reports describing the associations between phloem- and
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wood-boring beetles on hardwood trees and fungi. In general, the mycobiota of
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xylomycetophagous bark beetles is well studied, but these surveys mainly focussed on
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ambrosia fungi (e.g. Hartig 1844; 1872; Francke Grosmann 1967; Batra 1967; Cassar &
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Blackwell 1996; Mayers et al. 2015). In recent years, Leptographium verrucosum (Gebhardt,
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R. Kirschner & Oberw.) Z.W. de Beer & M.J. Wingf. was described from Xyleborus
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dryographus (Ratz.) in Germany (Gebhardt et al. 2002), while O. arduennense F.X. Varlier,
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Decock, K. Jacobs & Maraite (currently, a synonym of O. distortum (R.W. Davidson) de
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Hoog & Sckeffer, Yin et al. 2016) was recoverd from Trypodendron domesticum (L.) and T.
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signatum (F.) in Belgium (Carlier et al. 2006). In Belgium, Carlier et al. (2006) reported that
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both Trypodendron species vectored O. bacillisporum, O. floccosum Math., and O. quercus
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(Georgev.) Nannf. Ophiostoma bacillisporum, was originally described from Fagus sylvatica
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L. infested by T. domesticum in Germany (Butin & Zimmermann 1972). In addition,
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Leptographium trypodendri R. Jankowiak, B. Strzałka & R. Linnakoski has been recognized
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in association with T. domesticum in Norway and Poland (Jankowiak et al. 2017).
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Relatively little is known about phloem-breeding bark beetle-associated fungi.
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Kirschner (2001) and Kirisits et al. (2000) described mycobiomes of Leperisinus varius (F.),
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Scolytus intricatus (Ratz.) and Taphrorychus bicolor (Hbst.). The fungal community of S.
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intricatus was also studied in the Czech Republic (Kubátová et al. 2002). However, these
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reports were based exclusively on morphological criteria. Recently, O. karelicum was
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described from Scolytus ratzeburgi (Jans.) infesting birch in Finland and Russia (Linnakoski
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et al. 2008). Ophiostoma karelicum and three other ophiostomatalean fungi, O. borealis
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Kamgan, H. Solheim & Z.W de Beer, O. denticiliatum Linnakoski, Z.W. de Beer & M.J.
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Wingf. and O. quercus were found in association with this beetle species in southern Norway
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(Linnakoski et al. 2009). The presence of O. karelicum on Betula sp. has also been confirmed
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in Polish investigations (Jankowiak 2011). In addition, Leptographium betulae R. Jankowiak,
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B. Strzałka & R. Linnakoski was discovered in association with S. ratzeburgi in Poland
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(Jankowiak et al. 2017).
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A survey of bark beetles and their associated fungi on hardwood trees in Norway and
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Poland was conducted from 2015 to 2017. Among others, four species belonging to
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Ophiostoma s. str. were isolated from various species of bark beetles, and their galleries. The
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aim of this study was to characterize the morphology, phylogenetic affinities, and taxonomy
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of these Ophiostoma s. str. species found in Norway and Poland and to resolve the
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phylogenetic relationships between these fungi and closely related known species within the
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Ophiostoma ulmi species complex.
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2. Materials and methods
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2.1. Isolates
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Fungal isolations were made from beetles recovered in Norway and Poland that can be
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assigned to the following beetle species: Anisandrus dispar (F.), Dryocoetes alni (Georg), D.
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villosus (F.), Hylesinus crenatus (F.), H. varius (F.), Scolytus laevis Chapuis, S. ratzeburgi, S.
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intricatus (Ratz.), T. domesticum and T. signatum. In addition, in Poland, fungi were isolated
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from Pteleobius vittatus (F.), Scolytus mali (Bechst. & Scharf.), S. multistratus (Marsh.), S.
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rugulosus (Ratz.), S. scolytus (F.), Taphrorychus bicolor, Xyleborinus saxsesenii (Ratz.), and
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Xyleborus monographus (F.). The adult beetles were excised from galleries established on
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decaying trees of Alnus incana (L.) Moench, Betula pubescens Ehrh., Corylus avellana L.,
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Fraxinus excelsior L., Ulmus glabra Huds. and Q. robur in Norway, and from A. incana,
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Betula pendula Roth., F. sylvatica, F. excelsior, Malus sylvestris Mall., Populus tremula L.,
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Q. robur, Tilia cordata Mill. and Ulmus leavis Pall. in Poland. Beetles were excised with
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sterilised tweezers and stored individually in sterile 1.5 ml Eppendorf tubes for later fungal
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isolations. Strains of various beetles were collected at four localities in Poland during April-
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October 2014-2016 and from 10 localities during September 2015 to September 2016 in
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Norway (Table 1).
In Poland, fungal isolations directly from beetles were done by crushing them onto the
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surface of 2% Malt Extract Agar [MEA: 20 g malt extract l-1 (BiocorpTM, Warszawa, Poland),
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20 g BiocorpTM agar-1and 1 L distilled water], containing cycloheximide (200 mg, Aldrich-
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Sigma, St. Louis, Co. LLC.) and tetracycline sulphate (200 mg, Polfa, Tarchomin SA). These
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plates were incubated at 22°C and later examined for fungal growth. In Norway, each bark
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beetle was divided into three parts, elytra, head and the rest, before placing the parts in three
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separte Petri dishes with 2% MEA [6.25 g malt Bactomalt extract (Beckton, Dickinson,
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Sparks, USA), 10 g agar (bacto agar powder from VWR International, Leuven, Belgium), 0.5
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l deionized water] without any cycloheximide.
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All fungal isolates used in this study are listed in Table 1, all together 42 isolates were
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examined. The Polish isolates are maintained in the culture collection of the Department of
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Forest Pathology, Mycology and Tree Physiology; University of Agriculture in Krakow,
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Poland. The Norwegian isolates are kept at the culture collection of Norwegian Institute of
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Bioeconomy. Ex-type isolates of new species described in this study were deposited in the
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Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, the Netherlands, and in the culture
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collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI),
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University of Pretoria, South Africa. Cultures of O. bacillisporum, originated from Belgium
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and Germany, were sourced from the culture collection of the BCCM/MUCL Agro-Food &
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Environmental Fungal Collection, Louvain-la-Neuve, Belgium and from the Westerdijk
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Fungal Biodiversity Institute (CBS). Taxonomic descriptions and nomenclatural data are
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registered in MycoBank (www.MycoBank.org) (Robert et al. 2013).
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2.2. DNA extraction, PCR, and sequencing
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Fungal isolates were grown on 2% malt extract agar [MEA: 6.25 g malt Bactomalt extract
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(Beckton, Dickinson, Sparks, USA), 10 g agar (bacto agar powder from VWR International,
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Leuven, Belgium), 0.5 l deionized water] in 90 mm plastic Petri dishes (Heger AS, Rjukan,
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Norway) for 1–2 weeks prior to DNA extraction. DNA was extracted using the Protocol #8
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Isolation of DNA from Mouse Tails kit (Easy-DNA™ Kit; Invitrogen, San Diego, USA)
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according to the manufacturer’s protocol.
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Five loci, including ITS1–5.8 S–ITS2 (ITS), ITS2–LSU (LSU), beta-tubulin (βT),
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calmodulin (CAL) and translation elongation factor 1-alpha (TEF1-α) were amplified for
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sequencing and phylogenetic analyses. Primers used in this study were: ITS 1-F (Gardes &
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Bruns 1993) and ITS4 (White et al. 1990) for ITS, ITS3 and LR3 (White et al. 1990) for
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LSU, Bt2a (Glass & Donaldson 1995) or T10 (O’Donnell & Cigelnik 1997) and Bt2b (Glass
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& Donaldson 1995) for βT, CL2F and CL2R (Duong et al. 2012) for CAL, and F-728F
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(Carbone & Kohn 1999) and EF2 (O’Donnell et al. 1998) for TEF 1-α.
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The PCR settings for ITS were: an initial denaturation step for 5 minutes at 95 ˚C,
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followed by 35 cycles of 95 ˚C for 30 seconds, 53 ˚C for 30 seconds and 72 ˚C for 1 minute,
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followed by a final elongation step at 72 ˚C for 10 minutes. The PCR conditions for ITS2-
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LSU were: an initial denaturation step for 3 minutes at 95 ˚C, followed by 35 cycles of 95 ˚C
203
for 30 seconds, 58 ˚C for 45 seconds and 72 ˚C for 1 minute, followed by a final elongation
204
step at 72˚C for 8 minutes. The PCR-settings for βT were: an initial denaturation step for 5
205
minutes at 95 ˚C, followed by 35 cycles of 95 ˚C for 30 seconds, 56 ˚C for 30 seconds and 72
206
˚C for 1 minute, followed by a final elongation step at 72 ˚C for 10 minutes. The PCR
207
conditions for calmodulin, TEF1-α and when using primer T10 for βT were: an initial
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denaturation step for 3 minutes at 95 ˚C, followed by 35 cycles of 95 ˚C for 30 seconds, 55 ˚C
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for 45 seconds and 72 ˚C for 1 minute, followed by a final chain elongation step at 72 ˚C for 8
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minutes.
Gene fragments were amplified in a 50 µL reaction mixture containing components of
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the HotStar Taq Plus DNA Polymerase kit (Qiagen, Hilden, Germany). The reaction mixture
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contained 1 µL of DNA-template, 5 µL 10x PCR buffer (containing 15 mM MgCL2), 2 µL
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25mM MgCL2, 1 µL dNTPs (10 mM of each), 5 µL BSA (0.4%), 5 µL TMACL (0.1 mM),
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0.4 µl Hot Star Taq+, 1 µL of each primer (10 mM) and 27.6 µL RNase-free water. PCR was
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performed using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).
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PCR products were resolved on 1% agarose gels stained with ethidium bromide and
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visualized under UV light. In preparation for sequencing, the Illustra ExoProStar clean-up kit
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(GE Healthcare Life Science, Buckinghamshire, UK) was used to remove excess dNTPs and
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primers from the PCR products, following the manufacturer’s protocol.
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Amplified products were sequenced by utilizing the LIGHTRUN sequencing service
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of GATC Biotech (GATC Biotech, Konstanz, Germany) using the same primers that were
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applied for the PCR. The newly obtained sequences (Table 1) were deposited in NCBI
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GenBank (see Table 1 for accession numbers) and compared with those in GenBank using the
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BLASTn algorithm.
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2.3. Phylogenetic analyses
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BLAST searches using the BLASTn algorithm were performed to retrieve similar sequences
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from GenBank (http://www.ncbi.nlm.nih.gov).. Accession numbers of these sequences are
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presented in the corresponding phylogenetic trees (Figs 2-5).
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Individual gene sequences, as well as concatenated constructs of multiple protein
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coding gene sequences were used. Datasets were compiled and edited with the Molecular
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Evolutionary Genetic Analysis (MEGA) v6.06 program (Tamura et al. 2013). The ITS and
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LSU datasets included all available sequences that could be extracted from GenBank for
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reference species in Ophiostoma s. stricto (Figs 2-3) to show the placement of Norwegian and
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Polish isolates within this genus. The outgroup taxa for the ITS dataset analysis were
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Sporothrix stenoceras (Robak) Nannf. and S. chilensis A.M. Rodrigues, R.C. Choappa, G.F.
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Fernandes, G.S. de Hoog, Z.P. de Camargo, while Hawksworthiomyces crousii Z.W. de Beer,
239
Marinc. & M.J. Wingf., H. lignivorus (De Mey., Z.W. de Beer & M.J. Wingf.) Z.W. de Beer,
240
Marinc. & M.J. Wingf., and Sporothrix schenckii Hektoen & C.F. Perkins were used as
241
outgroups in the LSU dataset analysis. The three protein coding gene regions (βT, CAL, and
242
TEF1-α) were sequenced for 42 isolates in order to delineate closely related species (Table 1).
243
Sequence alignments were performed using the online version of MAFFT v7 (Katoh &
244
Standley 2013). The ITS, LSU, βT, CAL and TEF-1α datasets were aligned using the E-INS-i
245
strategy with a 200PAM/κ=2 scoring matrix, a gap opening penalty of 1.53 and an offset
246
value of 0.00. The alignments were checked manually with BioEdit, as implemented in
247
MEGA v6.06 (Tamura et al. 2013), and compared with gene maps (Yin et al. 2015) to ensure
248
that introns and exons were aligned appropriately.
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Phylogenetic analyses were performed for each of the datasets using three different
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methods: maximum likelihood (ML), Maximum Parsimony (MP) and Bayesian inference
251
(BI). For ML and Bayesian analyses, the best-fit substitution models for each dataset were
252
established using the corrected Akaike Information Criterion (AICc) in jModelTest 2.1.10
253
(Guindon & Gascuel 2003; Darriba et al. 2012).
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Maximum likelihood (ML) analyses were conducted with PhyML 3.0 (Guindon et al.
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2010), via the Montpelier online server (http://www.atgc-montpellier.fr/phyml/) with 1000
256
bootstrap pseudoreplicates in order to assess node support values.
257
MP analyses were conducted with PAUP* 4.0b10 (Swofford 2003). Gaps were treated as
258
fifth state characters. One thousand bootstrap replicates were generated and analysed to
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determine the levels of confidence for the nodes within the inferred tree topologies. Tree
260
bisection and reconnection (TBR) was selected as the branch swapping option. The tree
261
length (TL), Consistency Index (CI), Retention Index (RI), Homoplasy Index (HI) and
262
Rescaled Consistency Index (RC) were recorded for each dataset analysed after the trees were
263
generated.
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BI analyses based on a Markov Chain Monte Carlo (MCMC) were carried out with
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MrBayes v3.1.2 (Ronquist & Huelsenbeck 2003). The MCMC chains were run for 10 million
266
generations using the best-fit model. Trees were sampled every 100 generations, resulting in
267
100,000 trees from both runs. The burn-in value for each dataset was determined in Tracer
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v1.4.1 (Rambaut & Drummond 2007). The remaining trees were utilized to generate a
269
majority rule consensus tree for determing the posterior probability values.
270
2.4. Morphology, growth studies, and mating tests
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Morphological characters were examined for selected isolates and herbarium specimens
273
chosen to represent the type specimens. Cultures were grown on 2% MEA [6.25 g malt
274
Bactomalt extract (Beckton, Dickinson, Sparks, USA), 10 g agar (bacto agar powder from
275
VWR International, Leuven, Belgium), 0.5 l deionized water] with or without host tree twigs
276
to induce potential ascocarp formation. The autoclaved twigs with bark were placed in the
277
middle of the agar plates. Fungal cultures were generated from single spores, and crossings
278
were made following the technique described by Grobbelaar et al. (2010). To obtain ascomata
279
for species description, single conidial isolates originating from Norway and Poland were
280
crossed in all possible combinations. Cultures were incubated at 25 oC and inspected regularly
281
for the appearance of fruiting structures.
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Morphological characteristics were examined by mounting the sexual and asexual
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fruiting structures in 80% lactic acid on glass slides, and these were observed using a Leica
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DMR microscope (Leica, Heerbrugg, Switzerland) equipped with a Leica camera that is
285
operated with the Leica application suite software (version 4.0). Fifty measurements were
286
made for each of the taxonomically relevant structures when possible, using the Leica
287
application software. Averages, ranges and standard deviations (SD) were computed for all
288
measurements and the measurements are presented in the following format: (min–)(mean-
289
SD)–(mean+SD)(–max).
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Growth characteristics for the four newly described species were determined by
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analysing the radial growth for four isolates in pure culture that represent each of the studied
292
species (Table 1). Cultures were prepared by placing agar disks (5 mm diam.), cut from
293
actively growing margins of colonies of each isolate tested at the centre of plates containing
294
2% MEA. Four plates for each of the isolates studied were incubated at the following
295
temperatures: 5, 10, 15, 20, 25, 30 and 35 °C. Colony diameters (two measurements per plate)
296
were determined 4 d after inoculation and growth rates were calculated as mm/d.
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3. Results
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Alignments for the ITS, LSU, βT, CAL, TEF1-α and the concatenated combined
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datasets contained 749, 434, 273, 591, 468 and 1348 characters (including gaps), respectively.
304
The exon/intron arrangement of the βT data included exons 4, 5, and 6, interrupted with
305
introns 3 and 4, but lacking intron 5. The aligned TEF1-α gene region consisted of exons 4
306
and 5, interrupted by intron 3, while lacking intron 4. The alignment of the CAL dataset
307
contained exons 4, 5 and 6, interrupted with introns 3, 4, and 6, while lacking intron 5. The
308
best evolutionary substitution models for ITS, LSU, βT, CAL, TEF1-α and the combined
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datasets were GTR+G, HKY+I, HKY+G, GTR+G, HKY+G and GTR+G, respectively. The
310
burn-in values in BI analyses for all data matrices were 25% of the trees.
The ITS tree (Fig. 2) shows the placement of the Norwegian and Polish isolates
312
(referred to as Taxon 1 to Taxon 4) within Ophiosotoma s. str. All newly proposed taxa
313
appear to have affinities towards the O. ulmi species complex. Taxon 1, 2 and 3 group among
314
members of the O. ulmi species complex and Taxon 4 is placed on a branch that is basal to the
315
branch that leads to O. ulmi and O. novo-ulmi. Taxon 1, 2 and 3 are part of a grouping that
316
includes O. ulmi and O. novo-ulmi along with 12 other known species. Only O. catonianum
317
(Goid.) Goid. and Taxon 1 had identical ITS sequences. Taxon 4 and O. karelicum based on
318
the ITS data are monophyletic (bootstrap based node support = 100%). Overall ITS sequences
319
for members of the O. ulmi species complex and allied taxa are very similar and thus many
320
nodes only achieved either no significant bootstrap support values or only moderate node
321
support values (Fig. 2). The LSU gene region (Fig. 3) provided better resolution concerning
322
separating Taxon 1 and Taxon 3. The LSU gene sequence for Taxon 2 was identical to those
323
of O. bacillisporum, O. borealis, O. novo-ulmi and O. ulmi. The LSU gene region did not
324
provide any resolution between Taxon 4 and O. karelicum (Fig. 3).
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The BI, MP, ML analyses of the protein-coding datasets (βT, CAL, TEF1-α and
326
combined) resulted in similar tree topologies (Figs 4-5) they all support the placement of the
327
newly proposed species to be allied with the O. ulmi species complex and they all indicate
328
that members of Taxon 4 and O. karelicum appear to form a well-supported distinct lineage
329
within the O. ulmi complex or possible within Ophiostoma s. str. The results of the analysis of
330
the combined data set (βT, CAL, TEF1-α) are presented in Fig. 5b and the tree topology
331
clearly distinguishes Taxon 1, 2, 3 and 4 into distinct monophyletic groupings that share a
332
node (node support values at 98 and 100% based on ML and MP analysis respectively, and
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>95% posterior probability based on BI analysis) that suggests a common ancestor with O.
334
novo-ulmi.
335
3.2. Morphology, growth studies, and mating tests
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Isolates of the four new taxa emerging from this study displayed differences with regards to
339
growth in culture, but they also shared some similarities with regards to colouration being
340
initially hyaline, turning white, grey or black with age. The new taxa displayed synnemata
341
that formed singly or in groups with white to brown spore drops, and these were abundant in
342
culture. Sporothrix-like synanamorphs were also present in culture. In addition, Taxon 4
343
produced chlamydospore-like and yeast-like cells. A sexual state was induced in Taxon 2, 3
344
and 4, the most distinct characteristics common in the herbarium specimens and the studied
345
isolates were the pale brown, straight ostiolar hyphae (Taxon 2 and Taxon 3), necks without
346
ostiolar hypahe (Taxon 4), and allantoid ascospores without visible gelatinous sheaths (Taxon
347
2, 3 and 4). Single ascospore derived isolates of Taxon 2 and Taxon 3 produced ascomata in
348
culture, suggesting that these species are homothallic. No sexual state was observed for Taxon
349
1 in crosses done between different isolates. The matings of the single conidium derived
350
cultures of Taxon 4 resulted in several successful crosses. The successful crosses occurred
351
only between some Norwegian and Polish isolates. None of the single spore derived cultures
352
formed ascomata when mated with themselves, indicating that this species is heterothallic. In
353
addition, Taxon 4 abundantly produced brown protoperithecia in culture. Morphological
354
differences that differentiate between these new taxa are discussed in the Notes under the new
355
species descriptions in the Taxonomy section. With regards to radial growth the optimal
356
temperature for all new species was 20 °C (Taxon 2 and Taxon 3) or 25 °C (Taxon 1 and
357
Taxon 4). All of the isolates grew at 30 °C, except Taxon 3. None of the isolates grew above
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35 °C. Growth was very slow at 5 °C for all isolates, except Taxon 2 for which no growth was
359
detected.
360
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3.3. Taxonomy
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Our phylogenetic analysis based on five different loci showed that four taxa associated with
364
hardwood-infesting bark beetles from Norway and Poland (Taxa 1 to 4) were distinct from
365
each other and any known taxa and therefore these are described here as new species.
366
According to the recommendations of De Beer & Wingfield (2013), we assign all new species
367
described herein to Ophiostoma s. str., even though a sexual state has not been observed for
368
Taxon 1.
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3.3.1. Taxon 1
371
Ophiostoma hylesinum T. Aas, H. Solheim & R. Jankowiak, sp. nov.
372
MycoBank No: 826926 (Fig. 6).
373
374
Etymology – The epithet reflects the genus name of the bark beetle vectors of this fungus,
Hylesinus spp.
375
Sexual state not observed.
376
Asexual states: pesotum-like and sporothrix-like.
377
Pesotum-like: Conidiophores macronematous, synnematous, abundant in culture, synnemata
378
occurring singly or in groups, expanding towards both the apex and the base, dark brown at
379
base, becoming paler toward apex, sometimes thickened at the base, (372–)478–771(–1054)
380
µm long including conidiogenous apparatus, (24.8–)26.4–70.7(–136.4) µm wide at base.
381
Conidiogenous cells (8–)12–19.6(–28) × (1–)1–1.2(–1.6) µm. Conidia hyaline, 1-celled,
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smooth, oblong to elliptical, often slightly curved, (2.7–)3.3–5.3(–7.7) × (1.2-)1.3–1.9(–2.5)
383
µm aggregating into a cream-white or later brownish-yellow mucilaginous spore drop.
384
Synnematal anamorph attached to substrate by brown rhizoid-like hyphae.
385
Sporothrix-like: Conidiophores mononematous, micronematous, hyaline, (10.9–)15.8–58.9(–
386
123.2) µm long and (1.2–)1.6–2.2(–2.8) µm wide at the base, denticulate forming up to 6
387
denticles, often giving rise to ramoconidia (primary conidia). Denticles terminated, but often
388
form below the apex and intercalary. Primary conidia 0-3 septate, hyaline, clavate, (7.4–)9.4–
389
20.3(–33.9) × (2–)2.5–3.3(–4) µm, producing distinct denticles and giving rise to secondary
390
conidia. Secondary conidia hyaline, smooth, 1-celled, fusiform or obovoid with a pointed
391
base, (3–)4.1–7.2(–9.6) × (1.2–)1.6-2.1(–2.4) µm.
392
Culture characteristics: Colonies on 2% MEA slow growing, hyaline at first, later becoming
393
white, floccose, sporothrix-like morph dominant initially, later pesotum-like morph
394
predominant. Optimal growth temperature on MEA is 25 °C with radial growth rate 1.4 (±
395
0.2) mm/d, while growth is reduced at 5 °C, and no growth occurred at 35 °C.
396
Type material: POLAND, Resko, from Hylesinus crenatus infesting Fraxinus excelsior, 15
397
September 2015, P. Wieczorek, holotype OF 305324, culture ex-holotype CBS 144296 =
398
CMW 51680; NORWAY, Ås from H. crenatus infesting F. excelsior, 2 January 2016, A.
399
Truls & M.E. Waalberg, paratype OF 305325, culture ex-paratype CBS 144263 = CMW
400
51682; NORWAY, Ås from H. crenatus infesting F. excelsior, 2 January 2016, T. Aas &
401
M.E. Waalberg, paratype OF 305326, culture ex-paratype CBS 144264 = CMW 51683.
402
Host tree: Fraxinus excelsior.
403
Insect vectors: Hylesinus crenatus, Hylesinus varius.
404
Distribution: Norway, Poland.
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Notes: This species is most closely related to O. catonianum (Goidánich 1935). However, O.
406
catonianum can be distinguished from this new species by a homothallic mating system.
407
Goidánich states that single conidial isolates produce perithecia and ascospores in contrast to
408
the heterothallic mating systems of O. hylesinum. In addition, this species has shorter
409
synnemata than O. hylesinum.
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3.3.2. Taxon 2
412
Ophiostoma signatum T. Aas, H. Solheim & R. Jankowiak, sp. nov.
413
MycoBank No: 826927 (Fig.7).
414
Etymology – The epithet reflects the specific epithet of the bark beetle vector of this fungus,
415
Trypodendron signatum.
416
Sexual state present. Ascomata abundantly produced on media, superficially or completely
417
embedded in the agar, bases dark brown, globose, (92–)119–148(–171) µm diam., sparsely
418
ornamented with straight olivaceous hypha (4.9–)8–11(69–) µm long, 2.0–3.8 µm wide. Necks
419
dark brown at the base, lighter brown at the apex, straight or curved, (173–)264–368(–439)
420
µm long. Diameter of the necks (10.8–)15–20.5(–28) µm at the apex and (25.6–)28–38.5(53)
421
µm at the base. Ostiolar hyphae present or not, hyaline, 0-1 septate, straight, cylindrical,
422
bluntly rounded at the apex (1–)1–11(–16) in number, (3.2–)8.6–21.5(–37) µm long, (1.6–
423
)1.7–1.8(–1.9) µm wide at the apex and (1.7–)1.8–2(2.1) µm wide at the base. Asci not
424
observed. Ascospores one-celled, hyaline, allantoid in side view, (3.7–)4–5(–6.2) × (1.2–)1.4–
425
1.7(–2) µm, elliptical in front view (3.7–)3.8–4.7(–5) × (1.6–)1.7–1.8(–1.9) µm and circular in
426
end view, without visible sheath, accumulated in a whitish to yellowish droplet at the apex of
427
the neck.
428
Asexual states: pesotum-like and sporothrix-like.
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Pesotum-like: Conidiophores macronematous, synnematous, seldom in culture, synnemata
430
loosely arranged and not compact, occurring singly or in small groups, not expanding towards
431
the apex or the base, light brown at base, becoming hyaline toward apex, (56–)79.9–109.9(–
432
129.4) µm long including conidiogenous apparatus, (12.3–)19.1–42.2(–70.8) µm wide at base.
433
Conidiogenous cells (18.5–)22.3–31(–33.9) × (1.4–)1.6–1.9(–2) µm. Conidia hyaline, 1-
434
celled, smooth, tear-drop shaped, (3.6–)3.8–5.8(–8) × (1.4–)1.6–3.1(–4) µm aggregating into a
435
cream-white or later mucilaginous spore drop.
436
Sporothrix-like: Conidiophores, mononematous, micronematous, hyaline, sometimes twisted,
437
mostly terminated, sometimes also intercalary, (2–)8–34(–60) µm long, (1.2–)1.5–1.9(–2) µm
438
wide, denticulate (forming up to 3–4 denticles) or non-denticulate. Conidia narrowly clavate,
439
aseptate, smooth, thin walled, (3.8–)4.2–6.3(–8) × (1.2–)1.4–2(–2.4) µm.
440
Culture characteristics: Colonies on 2% MEA slow growing, hyaline at first, later becoming
441
brown or pale brown in concentric rings of ascomata. Asexual states rarely occur, ascomata
442
dominant. Optimal growth temperature on MEA is 20 °C with radial growth rate 1.5 (± 0.3)
443
mm/d, while growth is slightly reduced at 25 °C, and no growth occurred at 5 and 35 °C.
444
Type material: NORWAY, Målselv, from Trypodendron signatum infesting Alnus incana, 29
445
August 2016, G. Kvammen, holotype OF 305327, culture ex-holotype CBS 144269 = CMW
446
51689; NORWAY, Målselv, from Trypodendron signatum infesting Alnus incana, 29 August
447
2016, G. Kvammen, paratype OF 305328, culture ex-paratype CBS 144267 = CMW 51687;
448
NORWAY, Målselv, from Trypodendron signatum infesting Alnus incana, 29 August 2016,
449
G. Kvammen, paratype OF 305329, culture ex-paratype CBS 144270 = CMW 51690.
450
Host tree: Alnus incana.
451
Insect vector: Trypodendron signatum.
452
Distribution: Norway.
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Notes: This species is most closely related to O. bacillisporum (Butin & Zimmerman 1972)
454
and O. borealis (Kamgan Nkuekam et al. 2010). However, O. bacillisporum can be
455
distinguished from this new species by bacilliform ascospores and production of
456
hyalorhinocladiella-like asexual state, while O. borealis has a heterothallic mating system,
457
shorter synnemata and larger ascospores. In contrast to these species, Taxon 2 forms colonies
458
with concentric rings of ascomata.
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3.3.3. Taxon 3
461
Ophiostoma villosum T. Aas, H. Solheim & R. Jankowiak, sp. nov.
462
MycoBank No: 826928 (Fig. 8).
463
Etymology – The epithet reflects the specific epithet of the bark beetle vector of this fungus,
464
Dryocoetes villosus.
465
Sexual state present. Ascomata abundantly produced on media, bases black, globose, (62–
466
)88–130(–169) µm diam., ornamented with brown hyphal hairs of variable length, (23–)37–
467
67(–86) µm long, 2.0–3.8 µm wide at the base, 1.3–2.5 µm wide at the apex. Necks black,
468
straight or slightly curved, often extended at the base, (347–)444–559(–632) µm long with
469
annuli absent or occasionally 1–2 present. Diameter of the necks (10.8–)11.8–14.5(–15.4) µm
470
at the apex and (24.6–)29–41.5(49.3) µm at the base. Ostiolar hyphae present, pigmented,
471
aseptate, straight, tapering towards the apex, (8–)8–12(–15) in number, (18.5–)27.7–36.6(–40)
472
µm long, (1.2–)1.4–1.7(–1.8) µm wide at the apex and (2.1–)2.4–3.2–(3.6) µm wide at the
473
base. Asci not observed. Ascospores one-celled, allantoid in side view, (3.6–)3.9–4.9(–5.2) ×
474
(1.2–)1.4–1.7(–1.8) µm, elliptical in front view (3.7–)4–5(–5.5) × (1.5–)1.6–1.7(–1.7) µm and
475
circular in end view, generally without visible sheath, occasionally with residual sheath up to
476
1 µm thick.
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Asexual states: pesotum-like and sporothrix-like.
478
Pesotum-like: Conidiophores macronematous, synnematous, abundant in culture, synnemata
479
occurring singly or in groups, expanding towards both the apex and the base, dark brown or
480
black at the base, becoming paler toward apex, (200–)244–296(–345) µm long including
481
conidiogenous apparatus, (15–)38–60(–82) µm wide at base. Conidiogenous cells (10–)15.2–
482
23.1(–30) × (1.8–)1.9–2.1(–2.2) µm. Conidia hyaline, 1-celled, oblong, often slightly curved
483
(3.5–)3.6–4.3(–5) × (1.2–)1.4–1.7(–1.9) µm aggregating into a cream-white mucilaginous
484
spore drop. Synnematal anamorph attached to substrate by brown rhizoid-like hypahae.
485
Sporothrix-like: Conidiophores mononematous, micronematous, hyaline, (16–)20.1–37.8(–
486
50) µm long and (1.8–)2.1–2.5(–2.6) µm wide at the base, denticulate forming up to 11
487
denticles giving rise to ramoconidia (primary conidia). Denticles terminated, but often form
488
below the apex and intercalary. Primary conidia non-septate, hyaline, clavate, (7–)8.1–14.1(–
489
20) × (2–)2.2–2.8(–3.2) µm, producing distinct denticles and giving rise to secondary conidia.
490
Secondary conidia hyaline, smooth, 1-celled, obovoid, with a truncated base, often curved,
491
(3.5–)4.6–6.8(–8.7) × (1.2–)1.4–1.9(–2.5).
492
Culture characteristics: Colonies on 2% MEA greenish to brown at first, later becoming dark
493
brown, sporothrix-like morph dominant initially, later pesotum-like morph predominant.
494
Optimal growth temperature on MEA is 20 °C with radial growth rate 1.9 (± 0.1) mm/d, while
495
growth is reduced at 5 °C, and no growth occurred at 30 and 35 °C.
496
Type material: NORWAY, Larvik, from Dryocoetes villosus infesting Quercus robur, 28
497
August 2016, T. Aas & K.D. Hansen, holotype OF 305330, culture ex-holotype CBS 144274
498
= CMW 51694; NORWAY, Larvik, from Dryocoetes villosus infesting Quercus robur, 28
499
August 2016, T. Aas & K.D. Hansen, paratype OF 305331, culture ex-paratype CBS 144272
500
= CMW 51692; NORWAY, Larvik, from Dryocoetes villosus infesting Quercus robur, 28
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August 2016, T. Aas & K.D. Hansen, paratype OF 305332, culture ex-paratype CBS 144271
502
= CMW 51691.
503
Host trees: Quercus robur.
504
Insect vector: Dryocoetes villosus.
505
Distribution: Norway.
506
Notes: This species is a member of the O. ulmi species complex, however it can be
507
distinguished from all its members by the ITS, LSU and the protein coding sequences (βT,
508
CAL, TEF1-α).
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3.3.4. Taxon 4
512
Ophiostoma pseudokarelicum T. Aas, H. Solheim & R. Jankowiak, sp. nov.
513
MycoBank No: 826929 (Fig. 9).
514
Etymology – The epithet reflects the morphological similarity with Ophiostoma karelicum
515
Linnakoski, Z.W. de Beer & M.J. Wingf.
516
Sexual state present. Ascomata developing after 30 d on sterilized Alnus twigs when two
517
mating types were paired: superficially or partly embedded in the agar, single or in groups.
518
Bases black, globose, (123–)153–210(–285) µm diam., ornamented with olivaceous, septate,
519
unbranched hyphal hairs of variable length, sometimes thickened at the base, (18.5–)34.5–
520
84.5(–135.5) µm long, 2–8 µm wide at base. Necks black, lighter brown at the apex, straight
521
or curved, (216–)332–537(–647) µm long. Diameter of the necks (8–)9–13(–15.5) µm at the
522
apex and (18.5–)30–41.5(52.5) µm at the base. Ostiolar hyphae not present. Asci not
523
observed. Ascospores one-celled, hyaline, allantoid in side view, (3.5–)3.8–4.5(–5) × (1.2–
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)1.4–1.7(–1.9) µm, cylindrical in frontal view (3.7–)3.6–4(–4.7) × (1.2–)1.5–1.7(–1.8) µm,
525
and circular in end view, without visible sheath, accumulated in a cream-coloured mass at the
526
tip of the neck.
527
Asexual states: pesotum-like, sporothrix-like.
528
Pesotum-like: Conidiophores macronematous, synnematous, (78–)117–165(–246) µm long
529
including conidiogenous apparatus, synnemata simple or in groups, stipe hyaline, light or dark
530
pigmented, (12.5–)24–54(–102) µm wide at base. Conidiogenous cells discrete, terminal,
531
hyaline, cylindrical, (11.5–)14.5–25.5(–31) × (1.2–)1.4–1.6(–1.6) µm. Conidia hyaline, one-
532
celled, variable in shape, usually obovoid with truncated base, rarely globose or ellipsoidal,
533
(2.6–)3.4–4.5(–5.9) × (1.2–)1.9–2.6(–3.6) µm, aggregating into a white with shades of light
534
brown mucilaginous spore drop. Synnemata rarely observed on malt extract agar and
535
abundant on sterilized beech twigs.
536
Sporothrix-like: Conidiophores, form sparsely on the agar, mononematous, micronematous,
537
hyaline, terminated, (10–)14.8–36.9(–88) µm long, (1.2–)1.6–3(–2.3) µm wide, denticulate
538
not well developed or non-denticulate. Conidia variable in shape obovoid to cylindrical,
539
aseptate, smooth, (2.7–) 3.3–5.4(–7.4) × (1–) 1.1–2(–2.6) µm.
540
Yeast-like cells present. Arising directly on the vegetative hyphae or short lateral branches
541
with dimensions (1–)1–7(–10) × (1–)1–2.1(–3), variable in shape, often in chains, (3–)3.8–
542
5.8(–8) × (1.8–)2.5–3.6(–4).
543
Protoperithecia on malt extract agar present, globose, with brown hyphae, brown, (27.7–
544
)36.7–45.9(–49.3) µm in diameter.
545
Chlamydospore-like cells terminal or intercalary, in short chains, often formed in synnemata,
546
3 to 8 µm in diameter.
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Cultural characteristics: Colonies on 2% MEA fast growing, more or less slimy, hyaline at
548
first, later becoming white with sparsely aerial mycelium or light brown to dark brown.
549
Optimal growth temperature on MEA is 25 °C with radial growth rate 3.2 (± 0.7) mm/d, while
550
growth is reduced at 5 °C, and no growth occurred at 35 °C.
551
Type material: NORWAY, Ås, from Trypodendron domesticum infesting Alnus incana, 7
552
February 2016, T. Aas, culture ex-holotype CBS 144281 = CMW 51704; NORWAY,
553
Målselv, from Trypodendron signatum infesting A. incana, 29 August 2016, G. Kvammen,
554
culture ex-paratype CBS 144278 = CMW 51701; POLAND, Rozpucie, from T. domesticum
555
infesting F. sylvatica, 4 May 2016, P. Bilański, paratype, culture ex-paratype CBS 144275 =
556
CMW 51696; POLAND, Resko, from T. domesticum infesting F. sylvatica, 24 June 2016, P.
557
Wieczorek, paratype, culture ex-paratype CBS 144276 = CMW 51697.
558
Fruitbodies were only obtained after crosses between a Norwegian and a Polish isolate.
559
Holotype
560
144281 and ex-paratype CBS 144276. Paratype OF 305334, a dried culture obtained after a
561
cross between ex-paratype CBS 144278 and ex-paratype CBS 144276.
562
Host trees: Alnus incana, Betula sp., Fagus sylvatica, Fraxinus excelsior, Quercus robur.
563
Insect vectors: Anisandrus dispar, Dryocoetes villosus, Scolytus intricatus, Trypodendron
564
domesticum, Taphrorychus bicolor, Trypodendron signatum, Xyleborus monographus,
565
Xyleborinus saxesenii.
566
Distribution: Norway, Poland.
567
Notes: This species is closely related to Ophiostoma karelicum (Linnakoski et al. 2008).
568
Ophiostoma pseudokarelicum can be distinguished from O. karelicum by much shorter
569
synnemata, smaller ascospores and the lack of ostiolar hyphae. In addition, this species
570
commonly produces protoperithecia, and chlamydospore-like and yeast-like cells in culture.
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OF 305333, a dried culture obtained by the cross between ex-holotype CBS
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572
4. Discussion
In this study, multigene phylogenies (ITS, LSU, ßT, CAL, TEF1-α) of 42 isolates
574
revealed four new species of Ophiostoma s. str. associated with eleven species of hardwood-
575
infesting bark beetles in Norway and Poland. In our analyses, three species from Norway and
576
Poland can be assigned to a larger lineage defined by de Beer & Wingfield (2013) as the
577
Ophiostoma ulmi complex based on ITS and LSU sequences. Twelve known species were
578
also included in this species complex: Ophiostoma australiae (Kamgan, K. Jacobs & M.J.
579
Wingf.) Z.W. de Beer & M.J. Wingf., O. bacillisporum, O. borealis, O. catonianum, O.
580
denticiliatum, O. himal-ulmi, O. novo-ulmi, O. patagonicum de Errasti & Z.W. de Beer, O.
581
tasmaniense Kamgam, Jol. Roux & Z.W. de Beer, O. tsotsi (Grobbelaar, Z.W. de Beer & M.J.
582
Wingf., O. ulmi, O. undulatum Kamgam, Jol. Roux & Z.W. de Beer, and O. quercus
583
(Georgevitch 1926, 1927). The monophyly of this species complex was not well supported in
584
our ITS and LSU sequence analysis. However, in previous studies, this lineage had good
585
support and was referred to as the “hardwood clade’ of the O. piceae complex (Grobbelaar et
586
al. 2009, 2010; Linnakoski et al. 2010), or the O. quercus complex (Kamgan Nkuekam et al.
587
2011). Among the isolates analysed in the present study, only O. pseudokarelicum together
588
with O. karelicum were placed on long branches suggesting that they might be a distinct
589
lineage. However, additional taxa are needed to support this new clade or species complex.
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All hardwood-inhabiting species, including the four new species described herein,
591
exhibit morphological affinities to the formation of type A ascospores (according to De Beer
592
& Wingfield 2013) and pesotum- and sporothrix-like anamorphs. However, the
593
morphological characteristics of O. bacillisporum and O. undulatum do not match those of
594
other species from this species complex. In contrast to other species that have synnematous
595
anamorphs, O. bacillisporum and O. undulatum tend to form, hyalorhinocladiella- and
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sporothrix-like anamorphs. In addition, the ascospores of O. bacillisporum are bacilliform
597
(Type B, De Beer & Wingfield 2013) although De Beer & Wingfield (2013) stated that these
598
ascospores can be interpreted as an elongated form of type A.
All four Ophiostoma species described here can easily be distinguished from each
600
other and from the other species that reside in Ophiostoma s. str. based on DNA sequence
601
comparisons. They are also morphologically distinct from each other and from other
602
Ophiostoma s. str. species. Analyses of ITS sequence data were insufficient to distinguish
603
between O. hylesinum and O. catonianum, and two other non-European species, O. australiae
604
and O. tasmaniense. This shows that these four species are very closely related. However,
605
analyses of protein-coding genes, including the ßT, CAL and TEF1-α sequence, support the
606
notion that O. hylesinum represents a distinct taxon. Morphologically, this new species is
607
similar to O. catonianum. The main morphological differences between O. hylesinum and O.
608
catonianum refer to the mating-type systems. According to Goidánich (1935) O. catonianum
609
is homothallic, while our mating test using several isolates from Norway and Poland have
610
demonstrated that O. hylesinum is heterothallic. In addition, the conidial mass from a
611
synnema of O. catonianum is white on MEA (Harrington et al. 2001), while our cultures of O.
612
hylesinum generally produce brown, gelatinous conidial mass at the apex of the synnema.
613
Ophiostoma hylesinum also has unique ecological characteristics in the forest ecosystems; this
614
fungus appears to be a common associate of H. crenatus and H. varius infesting F. excelsior
615
solely.
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The asexual morphs of O. villosum resembled those produced by O. catonianum and
617
O. quercus. However, this new fungus produced perithecia from single ascospore-derived
618
cultures indicating its homothallic system, in contrast to the heterothallic mating systems of
619
the latter two species. The colony colour of O. villosum is much darker compared to those of
620
O. catonianum and O. quercus. Moreover, in contrast to the majority of species from the O.
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621
ulmi complex, the ITS sequences of this new species are unique. In addition, Ophiostoma
622
villosum was only found in association with D. villosus infesting Q. robur in Norway
623
suggesting that this fungus has a specific relationship with this beetle.
Among the isolates analysed in the present study, O. signatum could also be
625
distinguished based on ITS sequences. This new species is characterized by short light brown
626
synnemata without expanding towards the apex and the base, and by brown-coloured colonies
627
with concentric rings of ascomata. Ophiostoma signatum was commonly found in association
628
with T. signatum infesting A. incana in Norway indicating that it could be a consistent fungal
629
associate of this ambrosia beetle species.
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Based on the molecular data generated in this study O. karelicum and O.
631
pseudokarelicum appear to reside on long branches in a discrete, well-supported phylogenetic
632
group within Ophiostoma s. stricto. However, these two species are closely related to the
633
species of the O. ulmi complex, based on morphology and DNA sequences. They have
634
allantoid to orange-section shaped ascospores and pesotum- and sporothrix-like anamorphs,
635
similar to other species in the O. ulmi complex. In contrast to members of the O. ulmi
636
complex O. karelicum and O. pseudokarelicum have synnemata with short stipes expanding
637
widely towards the apex and faster growth rate at 20 °C. Ophiostoma pseudokarelicum is
638
morphologically similar to O. karelicum. These two fungi produce synnemata with short
639
stipes generally only on wood, and have fast growing colonies with white to dark brown
640
colour. The sexual state of O. karelicum was generated only by crossing different strains
641
(Linnakoski et al. 2008). The teleomorph state of O. pseudokarelicum was also induced only
642
by crossing different strains of the fungus in different combinations on agar plates with Alnus
643
chips. This would suggest that both species are heterothallic. Based on morphology, O.
644
pseudokarelicum differs from O. karelicum in having substantially shorter synnemata and
645
smaller ascomata without ostiolar hyphae. Ophiostoma pseudokarelicum can also be
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distinguished from O. karelicum by smaller ascospores, more variable shapes of conidia and
647
the production of chlamydospore-like and yeast-like cells in culture.
The presence of a yeast-like state in the Ophiostomatales order has been previously
649
described for O. novo-ulmi (Brasier 1991), O. himal-ulmi (Brasier & Mehrotra 1995) and
650
Sporothrix inflata de Hoog (Halmschlager & Kowalski 2003). Further, we found that O.
651
pseudokarelicum abundantly formed protoperithecia in culture. Protoperithecia have been
652
observed in several species from the O. ulmi complex, e.g. O. quercus or O. novo-ulmi
653
(Harrington et al. 2001) but not in O. karelicum (Linnakoski et al. 2008). Ophiostoma
654
karelicum is known to mainly occur on Betula spp. in association with S. ratzeburgi in Europe
655
(Linnakoski et al. 2008; Linnakoski et al. 2009; Linnakoski 2010; Jankowiak 2011). In
656
contrast, O. pseudokarelicum was found on a wide range of hardwoods including A. incana,
657
Betula sp., F. sylvatica, F. excelsior and Q. robur in association with different beetle species,
658
especially ambrosia beetles.
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Some Ophiostoma species belonging to the O. ulmi complex, such as O. ulmi, O.
660
novo-ulmi, and O. himal-ulmi are dangerous tree pathogens (Schwarz 1928; Brasier 1991;
661
Brasier & Mehrotra 1995). Nothing is known regarding the pathogenicity of the new
662
Ophiostoma spp. described in the present study, whose potential pathogenic capabilities
663
should be further investigated.
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In conclusion, results of this study have shown that Ophiostoma spp. probably occur
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commonly on hardwoods in associations with bark and ambrosia beetles in the northern part
666
of Europe. Four new Ophiostoma spp. were identified in this study alone, the discovery of a
667
relatively large number of new taxa strongly reflects the fact that these fungi have been poorly
668
studied in the European hardwood forests. It is likely that similar studies on other hardwoods
669
and beetle species in other regions of Europe will yield additional new taxa that can be
670
assigned to the Ophiostomatales. The current and future studies will improve our knowledge
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of the ophiostomatalean fungi, as well as the fungal diversity associated with hardwood trees
672
in Europe.
673
Conflicts of interest
674
The authors declared no conflict of interest.
676
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Acknowledgements
677
This work was supported by the National Science Centre, Poland (contract No. UMO-
679
2014/15/NZ9/00560). The work in Norway was funded by the Norwegian biodiversity
680
information centre on the project “Ophiostomatoid fungi in Norway” and Norwegian Institute
681
of Bioeconomy Research, and is part of a Master thesis at Norwegian University of Life
682
Sciences (T.A). The foresters Leif Evje and Geir Kvammen collected beetles in northern
683
Norway.
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869
870
Legends to table and figures
871
Table 1 – Isolates used in the present study
SC
872
873
TE
D
Fig. 1 – Origin of isolates used in this study: 1 – Målselv, Norway (69°4'5.54''N,
18°28'46.01''E); 2 – Kvæfjord, Norway (68°42'14.36''N, 16°18'33.53''E); 3 – Bjørkelangen,
Norway (59°53'27.87''N, 11°34'50.63''E); 4 – Oslo, Ljan, Norway (59°50'41.15''N,
10°47'19.54''E); 5 – Ås, Norway (59°41'37.33''N, 10°44'51.92''E); 6 – Ås, Norway
(59°41'20.79''N, 10°45'10.18''E); 7 – Hurum, Norway (59°33' 8.06''N, 10°25'55.24''E), 8 –
Larvik Norway (59°11'49.05''N, 9°55'6.78''E); 9 – Larvik, Norway (59°3'36.99''N,
10°4'6.68''E); 10 – Larvik, Norway (58°58'33.79''N, 9°57'50.45''E); 11 – Resko, Poland
(53°43'34.82''N, 15°17'55.40''E); 12 – Myszyniec, Poland (53°21'52.61"N, 21°17'32.78"E);
13 – Rozpucie, Poland (49°35'33.89"N, 22°25'10.28"E); 14 – Prószków, Poland
(0°31'54.05''N, 17°52'53.93''E); 15 – Hannover-Münden, Germany (51°25'20.91"N,
9°38'57.67"E); 16 – Libin, Belgium (49°57'50.27"N, 5°16'38.55"E); 17 – Sugny, Belgium
(49°47'58.91"N, 4°54'38.91"E).
EP
887
M
AN
U
874
875
876
877
878
879
880
881
882
883
884
885
886
RI
PT
868
Fig. 2 – ML tree of Ophiostoma sensu stricto generated from the ITS DNA sequence data.
Sequences generated from this study are printed in bold type. The Bootstrap values ≥ 75% for
ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold
branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI)
analyses. * Bootstrap values <75%. The tree is drawn to scale with branch length measured in
the number of substitutions per site.
902
903
Fig. 4 – ML tree of species in the Ophiostoma ulmi complex generated from the DNA
sequences of TEF1-α (a) and CAL (b) gene regions. Sequences generated from this study are
AC
C
888
889
890
891
892
893
894
895
896
897
898
899
900
901
Fig 3. – ML tree of Ophiostoma sensu stricto generated from the LSU DNA sequence data.
Sequences generated from this study are printed in bold type. The Bootstrap values ≥ 75% for
ML and Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold
branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI)
analyses. * Bootstrap values <75%. The tree is drawn to scale with branch length measured in
the number of substitutions per site.
ACCEPTED MANUSCRIPT
printed in bold type. The Bootstrap values ≥ 75% for ML and Maximum Parsimony (MP)
analyses are presented at nodes as follows: ML/MP. Bold branches indicate posterior
probabilities values ≥ 0.95 obtained from Bayesian Inference (BI) analyses. * Bootstrap
values <75%. The tree is drawn to scale with branch length measured in the number of
substitutions per site.
910
911
912
913
914
915
916
Fig. 5 – ML tree of species in the Ophiostoma ulmi complex generated from the DNA
sequences of βT (a) and the combined datasets of three protein gene regions (b) . Sequences
generated from this study are printed in bold type. The Bootstrap values ≥ 75% for ML and
Maximum Parsimony (MP) analyses are presented at nodes as follows: ML/MP. Bold
branches indicate posterior probabilities values ≥ 0.95 obtained from Bayesian Inference (BI)
analyses. * Bootstrap values <75%. The tree is drawn to scale with branch length measured in
the number of substitutions per site.
917
918
919
920
921
922
Fig 6. – Morphological characters of Ophiostoma hylesinum sp. nov. (CBS 144296, Taxon 1)
(A). synnemata of pesotum-like asexual state; (B) conidiogeneous apparatus; (C) conidia; (D)
conidiogeneous cells; (E) conidiophore of sporothrix-like asexual state, arrow indicates
denticles of conidiogenous cell; (F) development of primary and secondary conidia (arrow);
(G) primary and secondary conidia; (H) Fourteen days old culture on MEA. Scale bars: (A) =
100 µm, (B) = 50 µm, (C) = 20 µm, (D) = 20 µm, (E) = 20 µm, (F) = 20 µm, (G) = 20 µm.
923
924
925
926
927
928
929
Fig 7. – Morphological characters of Ophiostoma signatum sp. nov. (CBS 144269, Taxon 2)
(A). ascoma; (B) ostiolar hyphae; (C) ascoma base; (D) ascospores; (E) synnemata of
pesotum-like asexual state; (F) conidiogeneous cells; (G) conidia; (H) conidiophore of
sporothrix-like asexual state, arrow indicates denticles of conidiogenous cell; (I) Fourteen
days old culture on MEA. Scale bars: (A) = 100 µm, (B) = 20 µm, (C) = 20 µm, (D) = 20 µm,
(E) = 20 µm, (F) = 10 µm, (G) = 20 µm, (H) = 20 µm.
930
931
932
933
934
935
936
937
938
939
940
Fig 8. – Morphological characters of Ophiostoma villosum sp. nov. (CBS 144274, Taxon 3)
(A). ascoma; (B) ascoma base; (C) ascospores; (D) ostiolar hyphae; (E) conidiogeneous
apparatus; (F) synnemata of pesotum-like asexual state; (G) conidia; (H); conidiogeneous
cells (I) conidiophore of sporothrix-like asexual state, arrow indicates denticles of
conidiogenous cell; (J) primary (arrow) and secondary conidia; (K-L) conidiophores of
sporothrix-like asexual state, arrow indicates development of primary conidia; (M) fourteen
days old culture on MEA. Scale bars: (A) = 100 µm, (B) = 20 µm, (C) = 20 µm, (D) = 20 µm,
(E) = 20 µm, (F) = 50 µm, (G) = 20 µm, (H) = 20 µm, (I) = 20 µm, (J) = 20 µm, (K) = 20 µm,
(L) = 20 µm.
941
942
943
944
945
946
947
948
949
950
Fig 9. – Morphological characters of Ophiostoma pseudokarelicum sp. nov. (CBS 144281,
Taxon 4) (A). ascoma; (B) ascoma base; (C) top of neck without ostiolar hyphae; (D)
ascospores; (E-G) synnemata of pesotum-like asexual state with different of stipe
pigmentation; (H) conidiogeneous cells; (I) conidia; (J) sporothrix-like asexual state; (K)
conidia of sporothrix-like asexual state; (L-M) yeast-like cells formed directly from hypahe
(black arrows) and on lateral branches (white arrows); (N) chains of yeast-like cells; (O)
chlamydospores-like cells; (P) protoperithecia; (R) fourteen days old culture on MEA
differing in colony colour. Scale bars: (A) = 100 µm, (B) = 20 µm, (C) = 20 µm, (D) = 10 µm,
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
904
905
906
907
908
909
ACCEPTED MANUSCRIPT
EP
TE
D
M
AN
U
SC
RI
PT
(E) = 50 µm, (F) = 50 µm, (G) = 50 µm, (H) = 20 µm, (I) = 20 µm, (J) = 20 µm, (K) = 20 µm,
(L) = 20 µm, (M) = 20 µm, (N) = 50 µm, (O) = 20 µm, (P) = 20 µm, (R) = 20 µm.
AC
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951
952
ACCEPTED MANUSCRIPT
Table 1
CBSA
GenBank accession noD
CMWB OthersC
Host
H,E
Insect
Origin
RI
PT
Species
ITS
LSU
βT
TEF-1α
CAL
MH055636
MH055675
MH055706
MH062835
MH062793
144296 51680
KFL28015RJHC
Fraxinus excelsior
Hylesinus crenatus
Resko, PL
O. hylesinum sp. nov.
144262 51681
KFL57616RJHC
Fraxinus excelsior
Hylesinus crenatus
Resko, PL
MH055637
MH055676
MH055707
MH062836
MH062794
144263 51682
N2016-0018/2/5P,E
Fraxinus excelsior
Hylesinus crenatus
Ås, NO
MH055638
MH055677
MH055708
MH062837
MH062795
144264 51683
N2016-0021/2/5P,E
Fraxinus excelsior
Hylesinus crenatus
Ås, NO
MH055639
MH055678
MH055709
MH062838
MH062796
144265 51684
N2016-0481/2/3
Fraxinus excelsior
Hylesinus crenatus
Ås, NO
MH055640
MH055679
MH055710
MH062839
MH062797
144297 51685
N2016-1298/3/1
Fraxinus excelsior
Hylesinus crenatus
Larvik, NO
MH055641
MH055711
MH062840
MH062798
144266 51686
N2016-1456/3/1
Fraxinus excelsior
Hylesinus crenatus
Taxon 2
144267 51687
N2016-1622/2/2P,E
Alnus incana
Trypodendron signatum
Målsev, NO
O. signatum sp. nov
144268 51688
N2016-1623/1/1
Alnus incana
Trypodendron signatum
Målsev, NO
MH055644
MH055681
MH055714
MH062843
MH062801
Alnus incana
Trypodendron signatum
Målsev, NO
MH055645
MH055682
MH055715
MH062844
MH062802
MH055716
MH062845
MH062803
Larvik, NO
M
AN
U
H,E
SC
Taxon 1
MH055642
MH055643
MH055680
MH055712
MH062841
MH062799
MH055713
MH062842
MH062800
N2016-1627/2/2
144270 51690
N2016-1631/2/3P,E
Alnus incana
Trypodendron signatum
Målsev, NO
MH055646
Taxon 3
144271 51691
N2016-1572/2/1
P,E
Quercus robur
Dryocoetes villosus
Larvik, NO
MH055647
MH055683
MH055717
MH062846
MH062804
O. villosum sp. nov.
144272 51692
N2016-1579/2/3P,E
Quercus robur
Dryocoetes villosus
Larvik, NO
MH055648
MH055684
MH055718
MH062847
MH062805
144273 51693
N2016-1684/3/1
Quercus robur
Dryocoetes villosus
Larvik, NO
MH055649
MH055719
MH062848
MH062806
144274 51694
N2016-1693/2/1H,E
Quercus robur
Dryocoetes villosus
Larvik, NO
MH055650
MH055685
MH055720
MH062849
MH062807
KFL80WRJTD
Quercus robur
Trypodendron domesticum
Prószków, PL
MH055651
MH055686
MH055721
MH062850
MH062808
Fagus sylvatica
Trypodendron domesticum
Rozpucie, PL
MH055652
MH055687
MH055722
MH062851
MH062809
Fagus sylvatica
Trypodendron domesticum
Resko, PL
43138
Taxon 4
144275 51696
KFL9916RJTD
144276 51697
KFL46116RJTDP,E
MH055688
MH055723
MH062852
MH062810
Fagus sylvatica
Trypodendron signatum
Resko, PL
MH055654
MH055689
MH055724
MH062853
MH062811
144277 51699
KFL26816RJADE
Fagus sylvatica
Anisandrus dispar
Rozpucie, PL
MH055655
MH055690
MH055725
MH062854
MH062812
144298 51700
N2016-0618/2/2
Alnus incana
Dryocoetes alni
Larvik, NO
MH055726
MH062855
MH062813
144278 51701
N2016-1605/2/3P,E
Alnus incana
Trypodendron signatum
Målsev, NO
MH055656
MH055691
MH055727
MH062856
MH062814
144279 51702
N2016-0625/3/1
Alnus incana
Trypodendron domesticum
Kvaefjord, NO
MH055657
MH055692
MH055728
MH062857
MH062815
144280 51703
N2016-0665/2/1
Alnus incana
Dryocoetes alni
Ås, NO
MH055658
MH055729
MH062858
MH062816
144281 51704
N2016-0524/3/6/1H,E
Alnus incana
Trypodendron domesticum
Ås, NO
Ophiostoma bacillisporum
MH055659
MH055693
MH055730
MH062859
MH062817
Fagus sylvatica
Trypodendron domesticum
Hannover-Münden, Germany
MH055660
MH055694
MH055731
MH062860
MH062818
MUCL 44874
Fagus sylvatica
Wood stain
Sugny, Belgium
MH055661
MH055695
MH055732
MH062861
MH062819
MUCL 44885
Fagus sylvatica
Wood stain
Libin, Belgium
MH055662
MH055696
MH055733
MH062862
MH062820
EP
MH055653
KFL46016RJTS
51698
E
AC
C
O. pseudokarelicum sp. nov
P,E
TE
D
144269 51689
771.7
ACCEPTED MANUSCRIPT
144282 51705
KFL10116aRJTD
Fagus sylvatica
Trypodendron domesticum
Rozpucie, PL
MH055663
MH055697
MH055734
Ophiostoma karelicum
144283 51706
KFL95216RJSR
Betula pendula
Scolytus ratzeburgi
Myszyniec, PL
MH055664
MH055698
MH055735
MH062863
MH062822
144284 51707
KFL96816RJSR
Betula pendula
Scolytus ratzeburgi
Myszyniec, PL
MH055665
MH055699
MH055736
MH062864
MH062823
144285 51708
N2016-1117/1/2
Betula pubescens
Scolytus ratzeburgi
Bjørkelangen, NO
MH055737
MH062865
MH062824
144286 51709
N2016-1209/2/1
Betula sp.
Scolytus ratzeburgi
Hurum, NO
MH055666
MH055700
MH055738
MH062866
MH062825
144287 51710
KFL18416RJSM
Ulmus leavis
Scolytus scolytus
Resko, PL
MH055667
MH055701
MH055739
MH062867
MH062826
144288 51711
KFL67716RJSM
Ulmus leavis
Scolytus multistratus
Las Mogilski, PL
MH055668
MH055740
MH062868
MH062827
144289 51712
N2016-1212/1/1
Ulmus glabra
Scolytus laevis
Oslo, Ljan, NO
MH055669
MH055702
MH055741
MH062869
MH062828
144290 51713
N2016-1516/2/1
Ulmus glabra
Scolytus laevis
MH055742
MH062870
MH062829
144291 51714
KFL81WRJXM
Quercus robur
Xyleborus monographus
Prószków, PL
MH055670
MH055703
MH055743
MH062871
MH062830
144292 51715
KFL10116bRJTD
Fagus sylvatica
Trypodendron domesticum
Rozpucie, PL
MH055671
MH055744
MH062872
MH062831
144293 51716
N2016-1570/3/1
Quercus robur
Dryocoetes villosus
Larvok, NO
MH055672
MH055704
MH055745
MH062873
MH062832
144294 51717
N2016-1198/3/3
Betula sp.
Scolytus ratzeburgi
Hurum, NO
MH055673
MH055705
MH055746
MH062874
MH062833
144295 51718
N2016-1288/1/2
Quercus robur
Anisandrus dispar
Ås, NO
MH055674
MH055747
MH062875
MH062834
A
SC
Ophiostoma quercus
Oslo, Ljan, NO
M
AN
U
Ophiostoma novo-ulmi
RI
PT
Ophiostoma borealis
-
AC
C
EP
TE
D
CBS Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.
B
CMW Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.
C
KFL Culture collection of the Department of Forest Pathology, Mycology and Tree Physiology; University of Agriculture in Krakow, Poland; N Culture Collection at
Norwegian Institute of Bioeconomy, Norway; MUCL BCMM/MUCL Agro-food & Environmental Fungal Collection, Croix du Sud, Belgium
D
ITS= internal transcribed spacer region of the nuclear ribosomal DNA gene; LSU= large subunit of the nuclear ribosomal DNA gene; βT= Beta-tubulin; CAL = Calmodulin;
TEF 1-α = Translation elongation factor 1-alpha.
E
Isolates used in growth and morphological studies; Pex-paratype; Hex-holotype.
MH062821
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig 1
Ophiostoma denticiliatum FJ804490
O. quercus AY466626
O. undulatum GU797218
O. australiae EF408603
O. tasmaniense GU797211
O. tsotsi FJ441287
Taxon 2 CBS144269 Norway
O. borealis GQ249325
O. catonianum AF198243
Taxon 1 CBS144296 Poland
Taxon 3 CBS144274 Norway
91/89
O. himal-ulmi AF198233
O. bacillisporum CBS 771.71
83/89
O. novo-ulmi FJ430476
O. novo-ulmi FJ430478
O. ulmi AF198232
100/100
Taxon 4 CBS144281 Norway
O. karelicum EU443759
O. patagonicum KT362247
O. tetropii AY934524
O. distortum AY924386
76/92
O. minus AF234834
O. minus AY542494
O. allantosporum AY934506
O. kryptum AY304436
O. ssiori AB096209
O. nikkoense AB506674
O. araucariae KU184418
O. triangulosporum AY934525
O. sugadairense LC090226
O. breviusculum AB200420
O. canum HM031489
O. flexuosum AY924387
O. micans KU184433
O. nitidum KU184436
O. qinghaiense KU184447
O. setosum AF128929
O. rachisporum HM031490
O. piceae AF198226
O. subalpinum AB096211
O. novae-zelandiae KT362249
*/81
O. pluriannulatum DQ539508
O. sparsiannulatum FJ906817
84/77
O. subannulatum AY934522
O. multiannulatum AY934512
100/100
O. conicolum AY924384
O. longiconidiatum EF408558
O. floccosum AF198231
O. brunneolum KU094684
95/92 O. pseudocatenulatum KU094686
84/* 92/100 O. brunneo-ciliatum KU094683
O. macroclavatum HM031499
O. clavatum
97/100
O. clavatum KU094685
complex
O.ainoae HM031495
O. poligraphi KU184443
O. shangrilae KU184453
O. tapionis HM031494
O. adjuncti AY546696
96/84
98/100
O. ips AY546704
100/100
O. pulvinisporum AY546714
96/86
O. ips
O. bicolor DQ268604
complex
O. fuscum HM031504
93/*
O. montium AY546711
O. japonicum GU134169
O. piliferum AF221070
Sporothrix chilensis KP711811
S. stenoceras AF484464
EP
AC
C
0.1
100/100
Fig 2
Ophiostoma sensu stricto
O. piceae complex
O. pluriannulatum
complex
TE
D
M
AN
U
SC
RI
PT
Tree # 1
Length 1528
CI 0.517
RI 0.739
RC 0.382
HI 0.483
O. ulmi complex
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
Taxon 4 KFL80WRJTD Poland
Taxon 4 CBS144275 Poland
Taxon 4 CBS144276 Poland
Tree # 1
Taxon 4 KFL46016RJTS Poland
Taxon 4 CBS144277 Poland
Length 191
Taxon
4 CBS144278 Norway
CI 0.440
Taxon 4 CBS144279 Norway
RI 0.720
Taxon 4 CBS144281 Norway
RC 0.317
Ophiostoma karelicum CBS144283 Poland
O. karelicum CBS144284 Poland
HI 0.560
84/95 O. karelicum CBS144286 Norway
O. karelicum EU443756
O. karelicum EU443757
Taxon 3 CBS144272 Norway
Taxon 3 CBS144274 Norway
Taxon 3 CBS144271 Norway
O. australiae EF408607
O. quercus CBS144291 Poland
O. quercus DQ294376
O. quercus CBS144293 Norway
O. quercus CBS144294 Norway
Taxon 1 CBS144296 Poland
Taxon 1 CBS144262 Poland
Taxon 1 CBS144263 Norway
Taxon 1 CBS144264 Norway
Taxon 1 CBS144265 Norway
Taxon 2 CBS144267 Norway
Taxon 2 CBS144268 Norway
Taxon 2 CBS144269 Norway
O. bacillisporum CBS771.71
O. bacillisporum MUCL 44874
O. bacillisporum MUCL 44885
O. borealis CBS144282 Poland
O. novo-ulmi CBS144287 Poland
O. novo-ulmi CBS144289 Norway
O. novo-ulmi DQ294375
O. ulmi DQ294374
O. patagonicum KT362224
76/*
O. canum DQ294372
O. distortum DQ294369
O. flexuosum DQ294370
O. piceae AF234837
0.04
O. araucariae DQ294373
90/84 O. carpenteri DQ294363
99/96
O. subannulatum DQ294364
99/100
O. pluriannulatum DQ294365
O. multiannulatum DQ294366
O. floccosum AF234836
99/99 O. macrospora AF282873
O. tingens AF282871
O. ainoae DQ294368
84/92
O. ponderosae KX590867
O. piliferum DQ294377
O. montium DQ294379
85/76
O. pulvinisporum DQ294380
O. ips DQ294381
95/81
Hawksworthiomyces crousii KX396548
84/92
H.lignivorus KX396545
Sporothrix schenckii DQ294352
Fig 3
ACCEPTED MANUSCRIPT
TEF-1α Taxon 4 KFL80WRJTD Poland
CAL
Taxon 2 CBS144268 Norway
Taxon 4 CBS144275 Poland
Taxon 4 CBS144276 Poland
99/100 Taxon 2 CBS144269 Norway
Taxon 2 CBS144270 Norway
Taxon 4 KFL46016RJTS Poland
Taxon 4 CBS144279 Norway
Taxon 2 CBS144267 Norway
88/83
99/97 Taxon 4 CBS144278 Norway
Ophiostoma bacillisporum MUCL 44874
96/99
100/97 Taxon 4 CBS144281 Norway
O. bacillisporum MUCL 44885
Taxon 4 CBS144298 Norway
bacillisporum CBS771.71
100/100 O.
Taxon 4 CBS144280 Norway
Taxon 3 CBS144272 Norway
Taxon 4 CBS144277 Poland
Taxon 3 CBS144274 Norway
100/100 Ophiostoma karelicum GU930821
Taxon 3 CBS144271 Norway
Tree # 1
*/100
O. karelicum GU930820
Taxon 3 CBS144273 Norway
Length 872 85/* O. karelicum CBS144283 Poland
Taxon 1 CBS144263 Norway
O. karelicum CBS144286 Norway
CI 0.711
Taxon 1 CBS144265 Norway
O. karelicum CBS144284 Poland
O. karelicum CBS144285 Norway
Taxon 1 CBS144297 Norway
RI 0.939
Taxon 1 CBS144296 Poland
100/100 Taxon 1 CBS144266 Norway
RC 0.668
Taxon 1 CBS144262 Poland
Taxon 1 CBS144264 Norway
Taxon 1 CBS144263 Norway
HI 0.289
Taxon 1 CBS144296 Poland
82/81 Taxon
Taxon 1 CBS144265 Norway
1 CBS144262 Poland
Taxon 1 CBS144297 Norway
85/100
Taxon 4 CBS144298 Norway
81/99
Taxon 1 CBS144266 Norway
Taxon 4 CBS144280 Norway
96/100
Taxon 1 CBS144264 Norway
Tree # 1
Taxon 4 CBS144281 Norway
O. catonianum AY466699
Taxon 4 CBS144279 Norway
Taxon 2 CBS144267 Norway
Length 546
100/100
Taxon 4 CBS144277 Poland
Taxon 2 CBS144268 Norway
CI 0.745 82/87
Taxon 4 KFL46016RJTS Poland
Taxon 2 CBS144269 Norway
Taxon 2 CBS144270 Norway
Taxon 4 CBS144276 Poland
RI 0.919
99/100
O. bacillisporum CBS 771.71
Taxon 4 KFL80WRJTD Poland
RC0.685
*/100
O. bacillisporum MUCL 44874
78/100
Taxon 4 CBS144275 Poland
HI 0.255
O. bacillisporum MUCL 44885
Taxon 4 CBS144278 Norway
Taxon 3 CBS144271 Norway
100/100
O. karelicum CBS144286 Norway
99/100
Taxon 3 CBS144274 Norway
97/98 O. karelicum CBS144285 Norway
100/100
Taxon 3 CBS144272 Norway
84/100
O. karelicum CBS144283 Poland
Taxon 3 CBS144273 Norway
O. karelicum CBS144284 Poland
O. novo-ulmi CBS144288 Poland
97/100
O. novo-ulmi CBS144289 Norway
O. novo-ulmi FJ430490
O. novo-ulmi CBS144290 Norway
O. novo-ulmi CBS144289 Norway
83/96
O. novo-ulmi CBS144290 Norway
O. novo-ulmi CBS144287 Poland
100/100
O. novo-ulmi FJ430491
O. novo-ulmi CBS144288 Poland
96/100
O. novo-ulmi FJ430493
O. borealis CBS144282 Poland
O. novo-ulmi FJ430494
79/94
O. quercus CBS144293 Norway
100/100
O. novo-ulmi KF899885
O. quercus CBS144295 Norway
O. novo-ulmi CBS144287 Poland
O. quercus CBS144294 Norway
O. novo-ulmi FJ430492
O. quercus CBS144292 Poland
O. himal-ulmi FJ430489
100/100
O. quercus CBS144291 Poland
O. borealis KF899867
100/100
O. araucariae KU184332
O. quercus CBS144294 Norway
97/100 O. quercus CBS144295 Norway
O. distortum KU184371
O. quercus AY466688
100/100
O. quercus AY466689
0.09
O. quercus CBS144291 Poland
95/99
O. quercus FJ441268
O. quercus CBS144293 Norway
86/*
O. quercus CBS144292 Poland
100/100
O. denticiliatum KF899872
O. tasmaniense GU797226
O. tsotsi FJ441274
O. australiae KF899891
0.09
O. undulatum GU797233
97/100
Fig 4
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
*/94
ACCEPTED MANUSCRIPT
Taxon 4 CBS144275 Poland
Taxon 4 CBS144276 Poland
βT+CAL+TEF-1α
Taxon 4 KFL46016RJTS Poland
Taxon 4 CBS144278 Norway
91/88
Taxon 4 CBS144279 Norway
Taxon 4 CBS144280 Norway
Tree # 1
Taxon 4 CBS144298 Norway
Tree # 1
79/88
Length 1548
Taxon 4 CBS144281 Norway
Length 252 86/*
Taxon 4 KFL80WRJTD Poland
CI 0.765
Taxon 4 CBS144277 Poland
CI 0.556
RI 0.926
Ophiostoma karelicum CBS144285 Norway
92/99
O. karelicum EU443769
RI 0.898
RC 0.708
O. karelicum CBS144286 Norway
RC 0.499 82/85
O. karelicum EU443771
HI 0.235
O. karelicum EU443773
HI 0.444
O. karelicum CBS144284 Poland
O. karelicum EU443776
Taxon 2 CBS144268 Norway
100/100 Taxon 2 CBS144269 Norway
O. karelicum EU443772
*/85
O. karelicum EU443774
Taxon 2 CBS144270 Norway
O. karelicum CBS144283 Poland
Taxon 2 CBS144267 Norway
Taxon 1 CBS144296 Poland
89/78
*/81
Taxon 1 CBS144262 Poland
Ophiostoma bacillisporum MUCL 44874
Taxon 1 CBS144263 Norway
85/*
O. bacillisporum MUCL 44885
Taxon 1 CBS144264 Norway
O. bacillisporum CBS771.71
100/100
Taxon 1 CBS144265 Norway
Taxon 3 CBS144272 Norway
Taxon 1 CBS144297 Norway
100/100
Taxon 1 CBS144266 Norway
81/*
Taxon 3 CBS144274 Norway
O. catonianum AY466653
Taxon 3 CBS144271 Norway
Taxon 2 CBS144267 Norway
Taxon 3 CBS144273 Norway
89/100
Taxon 2 CBS144268 Norway
Taxon 1 CBS144266 Norway
Taxon 2 CBS144269 Norway
*/81
Taxon 2 CBS144270 Norway
Taxon 1 CBS144265 Norway
O. bacillisporum CBS771.71
Taxon 1 CBS144263 Norway
O. bacillisporum MUCL 44874
Taxon 1 CBS144297 Norway
100/100
O. bacillisporum MUCL 44885
Taxon 1 CBS144264 Norway
Taxon 3 CBS144271 Norway
Taxon 3 CBS144272 Norway
Taxon 1 CBS144262 Poland
Taxon 3 CBS144273 Norway
Taxon 1 CBS144296 Poland
Taxon 3 CBS144274 Norway
novo-ulmi CBS144287 Poland
97/100 */100 Taxon 4 CBS144275 Poland
97/95 O.
Taxon 4 CBS144276 Poland
O. novo-ulmi FJ430509
*/100 Taxon 4 KFL46016RJTS Poland
O. novo-ulmi CBS144288 Poland
O. novo-ulmi CBS144289 Norway
*/100 Taxon 4 CBS144280 Norway
O. novo-ulmi CBS144290 Norway
98/100 Taxon 4 CBS144279 Norway
97/90 O. novo-ulmi DQ296095
O. novo-ulmi FJ430505
*/85 Taxon 4 CBS144278 Norway
75/*
O. novo-ulmi FJ430507
Taxon 4 CBS144298 Norway
*/100
100/100
O. ulmi DQ296094
Taxon 4 CBS144281 Norway
*/91
O. borealis CBS144282 Poland
100/
98/100
Taxon 4 KFL80WRJTD Poland
O. borealis GQ249317
100
Taxon 4 CBS144277 Poland
O.
borealis
GQ249318
98/98
O. himal-ulmi FJ430504
O. karelicum CBS144283 Poland
97/98 O.
O. quercus CBS144291 Poland
karelicum CBS144286 Norway
O. quercus CBS144292 Poland
90/100 O. karelicum CBS144284 Poland
O. quercus AY466631
99/100
O. karelicum CBS144285 Norway
O. quercus AY466643
O. quercus AY466652
O. novo-ulmi CBS144289 Norway
100/100
O. quercus CBS144294 Norway
O. novo-ulmi CBS144290 Norway
O. quercus CBS144295 Norway
O. novo-ulmi CBS144288 Poland
O. quercus AY466634
O. novo-ulmi CBS144287 Poland
O. quercus CBS144293 Norway
*/92
O. quercus AY466648
*/81
O. quercus CBS144293 Norway
O. quercus AY466637
95/99
O. quercus CBS144295 Norway
O. quercus AY466632
O. quercus CBS144294 Norway
86/93
O. tsotsi KF143801
O. quercus CBS144291 Poland
tasmaniense GU797190
93/100100/100O.O.
australiae EF408605
94/97 100/100 O. quercus CBS144292 Poland
O. australiae GU797184
86/81
O. araucariae KU184375
0.1
O. undulatum GU797185
O. distortum KU184414
O. denticiliatum FJ804502
100/100
O. patagonicum KT381281
O. patagonicum KT381285
0.08
85/86
Fig 5
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
βT
EP
AC
C
Fig. 6
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig. 7
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
Fig 8
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
Fig 9
ACCEPTED MANUSCRIPT
Highlights
Four new Ophiostoma species were isolated from hardwoods in Norway and Poland.
•
New species were described based on morphological and molecular characters.
•
Ophiostoma hylesinum, O. signatum, and O. villosum are part of the O. ulmi complex.
•
Ophiostoma pseudokarelicum resides in a separate lineage in Ophiostoma s. stricto.
•
Many undescribed ophiostomatoid fungi could be associated with hardwoods in Europe.
AC
C
EP
TE
D
M
AN
U
SC
RI
PT
•
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