See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/274715720 A new species of Lophodermium on needles of mountain pine (Pinus mugo) from the Giant Mountains in Poland ARTICLE in MYCOLOGICAL PROGRESS · APRIL 2015 Impact Factor: 1.91 · DOI: 10.1007/s11557-015-1038-y CITATION READS 1 102 3 AUTHORS, INCLUDING: Ondřej Koukol Wojciech Pusz 31 PUBLICATIONS 207 CITATIONS 53 PUBLICATIONS 62 CITATIONS Charles University in Prague SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Wrocław University of Environmental and L… SEE PROFILE Available from: Wojciech Pusz Retrieved on: 05 February 2016 Mycol Progress (2015) 14:23 DOI 10.1007/s11557-015-1038-y ORIGINAL ARTICLE A new species of Lophodermium on needles of mountain pine (Pinus mugo) from the Giant Mountains in Poland Ondřej Koukol & Wojciech Pusz & David Minter Received: 19 August 2014 / Revised: 3 December 2014 / Accepted: 10 February 2015 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2015 Abstract A fungus tentatively identified as Lophodermium sp. was repeatedly isolated from living secondary needles of mountain pine (Pinus mugo) in the Giant Mountains of Poland. Needles showed symptoms of yellow spots, gradual discolouration and premature falling in July. Thin black zone lines, subepidermal conidiomata and partially subepidermal ascomata morphologically similar to L. pinastri occurred on fallen needles in litter below the same trees. Evidence from internal transcribed spacers of rDNA and the gene for actin showed that strains isolated from symptom-bearing needles and fruiting structures were identical, and differed from other Lophodermium species known from pine, including L. pinastri. The fungus differs subtly from L. pinastri, for example, in the lengths of its conidiomata, ascomata and asci, but can be reliably distinguished only by molecular data. Known only on mountain pine from the Giant Mountains, it is described here as L. corconticum sp. nov. Keywords ITS rDNA . Actin . Yellow needle spots . Potential parasite . Leptostroma O. Koukol (*) Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, CZ-128 44 Prague, Czech Republic e-mail: ondrej.koukol@natur.cuni.cz W. Pusz Department of Plant Protection, Division of Plant Pathology and Mycology, The Wroclaw University of Environmental and Life Sciences, Grunwaldzki Sq. 24a, Wroclaw 50-363, Poland D. Minter CAB International, Bakeham Lane, Egham, Surrey TW20 9TY, UK Introduction Species of the genus Lophodermium Chevall. (Rhytismatales, Leotiomycetes) are frequent and cosmopolitan colonisers of pine needles. The geographical distribution and range of associated pines varies substantially. Although there are exceptions, individual species tend to occur on only haploxylon (5needle) or diploxylon (2- to 3-needle) pines, not both. Lophodermium pinastri (Schrad.) Chevall., as currently understood, occurs widely on many pines. Others are on only a few pines and/or in a limited geographical area, like L. macci Sokolski & Bérubé, known only from haploxylon pines in North America (Sokolski et al. 2004). Some apparently colonise only a single pine species in a single locality, like L. pinimugonis C.L. Hou & M. Piepenbr., known exclusively on mountain pine (Pinus mugo Turra) from one site in Germany’s Bavarian Alps (Hou et al. 2009). Pinus mugo Turra is native to Europe, with a disjunctive distribution covering several mountain ranges. In the Giant Mountains, straddling Poland and the Czech Republic, it occurs up into the subalpine zone (1,250–1,450 m above sea level). Individual stands originate partly from relict populations and partly from artificial plantations (Przewoźnik 2008). To date, little attention has been paid to its mycobiota there. Some records of microfungi may be found in Cypers (1896), Schröter (1908), Baudyš (1924–25), Minter (1981b) and Minter and Holubová-Jechová (1981). Příhoda (1965) reported six parasitic ascomycetes and one rust observed or collected in 1948–1949. Among them, L. pinastri was the most frequent. This species was blamed for massive needle casts in the area in 1894 and 1895 (Cypers 1896), but Příhoda (1965), a forest pathologist, observed no visible damage to pines. Pusz et al. (2013) surveyed the incidence of a mountain pine disease manifesting as yellow needle spots in this area. 23 Page 2 of 13 This disease was first reported in Poland in 1978 in the mountain pine population of the Tatra Mountains (Lutyk 1978). Historical records from the area (Cypers 1896; Příhoda 1965) and a recent study of the health status of mountain pine in Montenegro (Andjelič 2000) suggested that L. pinastri might cause the disease. Various ecological studies, however, for example in Scotland on P. sylvestris L. (Minter and Millar 1980), have shown that L. pinastri typically occurs as a symptomless endobiont within apparently healthy needles, fruiting in litter after normal senescence and leaf fall. That is very different from the Lophodermium on P. mugo in the Giant Mountains. The present study was undertaken to resolve that apparent inconsistency. Material and methods Fungal sampling and isolation Living pine needles one to two years old and showing symptoms of disease, i.e., yellow spots, were collected in July and October 2012 from trees at 1,370 m above sea level near the mountain shelter BDom Ślaski^ in the Karkonosze National Park (Giant Mountains, Poland). Individual needle pairs were cut with scissors and a 2-centimetre-long fragment from the middle part of each needle was surface-sterilised in 30 % hydrogen peroxide for 90 s on a horizontal shaker, then placed in a Petri dish (ten fragments per dish) with wort agar prepared from brewers wort (Staropramen Brewery, Prague, Czech Republic) with a final sucrose content of 2 % w/v (2BA, Fassatiová 1986). Outgrowing colonies were isolated into pure cultures on 2BA. Strains were also cultivated on potato dextrose agar (PDA), cornmeal agar (CMA) and potato carrot agar (PCA), all prepared from fresh ingredients following Fassatiová (1986). Fallen needles were collected from the uppermost litter layer beneath the same trees in October 2013, and in April and May 2014, and placed in damp chambers composed of glass Petri dishes (18 cm diam.) each with a layer of cellulose tissue and filter paper. Damp chambers were autoclave sterilised prior to use. Pure cultures of fungi fruiting on needles were obtained by picking out internal tissue of conidiomata or ascomata with a sterile needle and inoculating on 2BA. Though conidia of Lophodermium function as spermatia and do not germinate in culture (Jones 1935), colonies grew out of other parts of the conidiomatal tissue. To produce sections of freshly collected needles and those kept in damp chambers with fungal fructifications, they were hand-cut with a razor blade. Microscopic preparations were made in Melzer’s reagent and lactic acid. Measurements (20 replicates for asci, ascospores Mycol Progress (2015) 14:23 and conidia) were made using the sectioned needles and fructifications from agar plates, with material mounted in Melzer’s reagent or lactic acid. DNA extraction and analyses Ten strains isolated from symptomatic living needles, three strains from ascomata and conidiomata, and three strains from surface-sterilised litter needles (without any sporulating structures) were used for DNA analysis (Table 1). Genomic DNA was isolated from 14-day-old cultures and also directly from ascomatal tissue using a Zymo Research Fungal/Bacterial kit (Zymo Research, Orange, USA). Nuclear rDNA containing internal transcribed spacers (ITS1 and ITS2), 5.8S and partial 28S regions (hereafter referred to as ITS rDNA) were amplified with a primer pair ITS1F/NL4 (O’Donnell 1993; White et al. 1990). The gene for actin was amplified with a primer pair ACT512F/ACT783R (Carbone and Kohn 1999). PCR products were purified with a Gel/ Polymerase Chain Reaction (PCR) DNA Fragments Extraction Kit (Geneaid Biotech Ltd., Taipei, Taiwan). Both strands of PCR fragments were sequenced with ITS1F/NL4 and ACT512F/ACT783R primers in the sequencing laboratory of the Faculty of Science, Charles University in Prague, Czech Republic. A preliminary search for strain identity was performed in the GenBank database using the BLAST algorithm (Altschul et al. 1990). Two datasets with consensual sequences of ITS rDNA and actin (Table 1) were aligned by the MAFFT algorithm implemented into the Geneious 6.1.5 program (Biomatters, Auckland, New Zealand) and edited manually in the same program to increase homology. The final ITS rDNA dataset involved 65 sequences, a total of 540 positions with 186 parsimony informative sites. The actin dataset was composed of 37 sequences, a total of 260 positions with 119 parsimony informative sites. The best-fit model and parameters were selected by jModelTest2.1.3 (Darriba et al. 2012), and the most adequate models for ITS rDNA and actin were the TrNef+G and TPM2+G models, respectively. Bayesian analysis was conducted with MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). Two independent runs of four million generations sampled every 100th generation with the first 25 % of samples discarded as burn-in. Posterior probabilities were used as a Bayesian branch support on the consensus trees. The average standard deviation (SD) of split frequencies estimating convergence reached 0.004 at the end of the analysis. A maximum likelihood analysis implemented in RAxML 7.6.3 and employing GTRCAT approximation (Stamatakis et al. 2008) was conducted on the CIPRES Science Gateway (Miller et al. 2009). Support for branching was calculated using a bootstrap test with 1,000 replicates. Mycol Progress (2015) 14:23 Table 1 Page 3 of 13 23 Species used in the molecular analyses of ITS rDNA and the actin gene and their respective GenBank accession numbers Species Host Genus Voucher Specimen GenBank Accession No. ITS rDNA Colpoma quercinum (Pers.) Wallr. Elytroderma deformans (Weir) Dark Darkera parca H.S. Whitney, J. Reid & Piroz. Hymenoscyphus epiphyllus (Pers.) Rehm ex Kauffman Leptostroma decipiens Petr. Lophodermium agathidis Minter & Hettige Lophodermium actinothyrium (Fuckel) Sacc. Lophodermium australe Dearn. Lophodermium baculiferum Mayr Lophodermium cathayae Y.R. Lin, H.Y. Huang & C.L. Hou Lophodermium conigenum (Brunaud) Hilitzer Lophodermium corconticum Lophodermium indianum Suj. Singh & Minter Lophodermium macci Sokolski & Bérubé Lophodermium molitoris Minter Lophodermium nitens Darker Actin U92306 AF203469 U92302 Quercus Pinus Picea Quercus Pinus Pinus Agathis Agathis Molinia Pinus Pinus Pinus Pinus Cathaya FJ005192 AY100653 AY100654 AY100661 AY100662 AY100663 AY100647 U92308 AY100656 AY100657 HQ992810 HQ992811 HM060648 Cathaya Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus 227-S1, litter needles CCF4868, litter needles,ascoma NK386, litter needles,ascoma DS155, litter needles,ascoma CCF4781, litter needles, conidioma CCF4782, litter needles 227-T1, living needles CCF4780, living needles 227-DS1, living needles Pinus L178, living needles Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus L204, living needles L210, living needles L280, living needles L297, living needles L288, living needles L318, living needles HM060650 FJ861974 FJ861975 AY100645 AY100646 HG939560 LM654179* LM654180 LM654178 HG939563 HG939561 HG939565 HG939566 HG939567 HG939570 HG939568 HG939564 HG939562 HG939569 AY100641 AY100642 AF540560 AF540561 AY100659 AF426061 AF426057 AY100640 HM060669 HM060670 HM060671 HM060672 HG965441 LM654182* LM654181 HG965439 HG965448 HG965443 HG965445 HG965446 HG965447 HG965440 HG965450 HG965444 HG965442 HG965449 23 Page 4 of 13 Mycol Progress (2015) 14:23 Table 1 (continued) Species Host Genus Voucher Specimen GenBank Accession No. ITS rDNA Lophodermium petrakii Durrieu Lophodermium piceae (Fuckel) Höhn. Lophodermium pinastri (Schrad.) Chevall. Lophodermium pini-mugonis C.L. Hou & M. Piepenbr. Lophodermium pini-excelsae Ahmad Lophodermium seditiosum Minter, Staley & Millar Meloderma desmazieri (Duby) Darker Cunninghamia Picea Picea Picea Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus FJ861984 AY775725 HQ902160 AF203471 FJ861985 AB511819 GU367906 GU367908 AY100649 HM060666 HM060664 HM060661 HM060659 HM060657 HM060655 Actin HM060681 HM060684 HM060680 HM060677 HM060673 HM060676 HM060674 HM060675 HM060678 HM060679 HM060682 HM060683 Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus Pinus JF332165 FJ861988 FJ861987 HM060654 AF462435 AF203468 HM060652 HM060667 HM060668 AF426056 Sequences in bold were generated in this study. The sequences from the ex-type strain are marked with an asterisk Results Morphology One- and two-year-old green secondary needles attached to the tree developed yellow spots with a diameter of 0.2– 0.5 mm in spring just after snow melt; affected needles subsequently turned straw-yellow, died and fell prematurely as early as July (Fig. 1a). No fungal structures were seen on the attached needles. On fallen needles, the first fungal structures to appear were conidiomata, which could be observed in autumn. These increased in number over winter, when zone lines began to appear. Zone lines were persistent, thin, black, transverse, clearly defined, and varied from abundant to almost absent (Fig. 1b, c). The colour of fallen needles colonized by the fungus varied, but tended to be different from that of fallen needles colonized by other fungi. Ascomatal initials were seen in spring after needle fall, and rapidly developed into fully formed ascomata (Fig. 1b,c). Conidiomata were formed immersed in the abaxial (rarely adaxial) side of needles, and were the same colour as the needle surface or pale grey, not raising the surface of the needles, and difficult to discern. They were 350–600 × 150–180 μm, oval to elliptical, and opened by a split at one side (Fig. 1d). In the vertical section, the upper wall was composed of a single layer of pigmented fungal cells underneath the needle cuticle and epidermal cells, with a distinct accumulation of additional dark brown to black fungal cells near the split; the basal wall was 6–9 μm thick, and consisted of Mycol Progress (2015) 14:23 Page 5 of 13 23 Fig. 1 Lophodermium corconticum on Pinus mugo. a. Symptoms of infection on needles, from yellow spots (left) to premature death of needle (right); b–c. Ascomata and conidiomata (arrow) formed on needles from the litter; d. Conidioma in a vertical section. Scale bar = 50 μm; e. Ascoma in a vertical section. Scale bar = 100 μm; f. Asci with ascospores and paraphyses. Scale bar = 20 μm; g. Conidiogenous cells. Scale bar = 20 μm; h. Conidia. Scale bars =10 μm; i. Ascospores. Scale bar = 20 μm; j. Interconnecting paraphyses in immature hymenium. Scale bar = 20 μm colourless, angular cells. Conidiogenous cells were colourless, 11–17×1–2.5 μm, tapering to the apex. Conidia were colourless, bacilliform, 3.5–6.5 × 1–1.5 μm, and produced sympodially from the conidiogenous cell apex (Fig. 1g, h, 2d). Ascomata were formed immersed in the abaxial side (or rarely on the edge or the adaxial side) of needles. When dry, they were black in the centre, surrounded by a paler margin lined by an elliptical black perimeter line, rounded or slightly acute at each end, and did not significantly raise the substratum 23 Page 6 of 13 surface; when wet, they were uniformly black, shiny and raised the substratum surface. They were (815) 960–1,460 (1,680) μm long and (405) 465–665 (800) μm wide (perimeter line), opening by a longitudinal split with colourless lips. In the median vertical section, they were partly subepidermal with eight or more host epidermal cells lying in a group above the basal wall and more than two epidermal cells in the upper wall on each side (Fig. 1e, 2a). The upper wall was composed of melanised cells becoming paler towards the interior, and thicker near the split, with lip cells up to 22 μm long. The basal wall was 28–35 μm thick. Paraphyses were septate, interconnected in younger ascomata, as long as the asci and 1.5– 2.5 μm wide, irregularly clavate and up to 6 μm wide at the tip (Fig. 1j, 2b). Asci were 94–142 (156) μm long and 8.5– 11 μm wide (Fig 1f, 2c). Ascospores were filiform, parallel or spirally coiled in the ascus, covered by a gelatinous sheath (2.5–3 μm thick) while they were in the ascus and for a short time after discharge, and (61) 72.5–82.5 (88) μm long and 1.5– 2 μm wide with rounded ends (Fig. 1i, 2c). Isolations from attached symptomatic needles repeatedly yielded colonies that were irregular, flat with undulate margins, ranging from almost completely white or white with thin Mycol Progress (2015) 14:23 black margins to half white with thick black margins and irregular black patches (Fig. 3). Conidiomata were sometimes produced in culture after at least two months on 2BA, and were irregular, erumpent, producing masses of conidia as grey droplets. Conidia were larger than in vivo, up to 3.5–8 μm long. Colonies growing out from symptomatic needles varied in both morphology and growth rate. Furthermore, the proportion of black margins and patches differed between strains and sometimes also changed with culture age (Fig. 3d–f). Production of conidiomata in pure cultures also differed between strains. At room temperature, some strains readily sporulated after several months (Fig. 3f); others were stimulated by cold (8 °C for at least one month) or by a sterile pine needle placed on the colony surface; others remained sterile. Molecular data Sequences obtained from the 16 cultured strains formed a well supported clade in the ITS rDNA analysis (Fig. 4) and the actin analysis (Fig. 5). In both analyses, several subsidiary clades with various bootstrap supports were formed within this well-supported clade (nucleotide difference 0.2–0.5 %). This variability could not be correlated with colony morphology. The relationship between these strains and other species of Lophodermium was not well resolved. ITS rDNA analyses suggested a higher affinity to the clade harbouring L. conigenum (Brunaud) Hilitzer and L. seditiosum Minter, Staley & Millar, but this received only moderate support from Bayesian analysis. Analysis of the ITS rDNA dataset with elimination of divergent regions curated by GBlocks (Castresana 2000, data not shown) suggested that these strains were more related to the L. pinastri species complex. This was partly supported by results of analyses of the actin dataset (Fig. 5), though relationships between these strains, L. pinastri and L. nitens Darker could not be resolved. Discussion Fig. 2 Lophodermium corconticum. a. Ascoma in vertical section. Scale bar = 100 μm; b. Interconnecting paraphyses in immature hymenium; c. Asci and ascospores. Scale bar = 20 μm; d. Conidiogenous cells producing conidia. Scale bar = 20 μm Reignoux et al. (2014), in their molecular study of Lophodermium on pines, found good congruence between phylogeny derived from ITS rDNA and the actin gene. Our analysis of actin, however, while producing broadly similar results to those using ITS rDNA, was less informative and did not permit us to distinguish relationships between the present fungus and other species (Fig. 5). This may be because few actin sequences of Lophodermium are available in the GenBank. Discussion of molecular aspects therefore focuses on ITS rDNA results. The habitat in which this fungus was found (semi-natural relict woodland and associated plantations of native trees) suggests it is indigenous, not an introduced exotic. Based on macroscopic and microscopic appearance in vivo and in culture, the fungus is a species of Lophodermium. Six members Mycol Progress (2015) 14:23 Page 7 of 13 23 Fig. 3 Lophodermium corconticum in culture. a. Multiple colonies outgrowing from surface sterilised needles collected from litter; b–c. Morphology of the ex-type strain (CCF4781) on 2BA/PDA and CMA/ PCA; d–f. Variability of colony morphology on 2BA after two months (CCF4780, L227-S1 and CCF4782), arrow shows conidia exuded in slimy drops from a conidioma of this genus have been recorded from pines in Europe: L. conigenum, L. pinastri, L. pini-excelsae Ahmad, L. pinimughonis, L. seditiosum and L. staleyi Minter (Hou et al. 2009; Minter 1981a). Of these, L. conigenum and L. seditiosum produce a few zone lines that are brown and diffuse rather than black and clearly defined. Compared with the present fungus, ascomata of L. conigenum are substantially larger, and ascomata of L. seditiosum are totally subepidermal rather than partially subepidermal. Lophodermium piniexcelsae is known only as an exotic on introduced five-needle pines. Lophodermium pini-mughonis has subcuticular ascomata, and ascomata of L. staleyi are mostly less than 800 μm long. The present fungus can, therefore, be easily distinguished visually, perhaps even by the unaided eye, from all five. The remaining species, L. pinastri, as currently understood, is very widely distributed. It occurs on many different pines, including records on P. mugo. The present fungus looks like L. pinastri, and colonies in pure culture are very similar in appearance to those of L. pinastri (Minter 1981a). Molecular results were, therefore, a surprise. Analysis of the ITS rDNA region showed that the well-supported clade containing all strains of the present Lophodermium is distinct from other clades containing strains identified as L. pinastri and other pine-inhabiting species of the genus (Fig. 4). The level of difference between these clades strongly suggests that the well-supported clade represents a distinct species. We interpret the several subsidiary clades within that well-supported clade to indicate infraspecific variability. The genetic difference between L. pinastri and the present fungus is as great as the difference between L. pinastri and L. conigenum or L. seditiosum, indicating that the present fungus is a different species from the fungi that provided the strains identified as L. pinastri. Given morphological similarities, they might be described as cryptic species. The picture is further complicated because results in Fig. 4 also show that strains identified as L. pinastri do not form a single clade by themselves. There are five separate clades all exclusively containing strains identified as L. pinastri. They too seem to be cryptic species. Two (marked as I and II in Fig. 4) were isolated from pine needles in China and Japan (Osono and Hirose 2011; Wang et al. 2010) and formed a monophyletic lineage with the L. cathayae Y.R. Lin, H.Y. Huang & C.L. Hou parasite on Cathaya argyrophylla Chun & K.Z. Kuang (Gao et al. 2013). The other three (III, IVand V in Fig. 4) resulted from work by Reignoux et al. (2014), who found differences in ITS rDNA, actin genes, RFLP fingerprints and colony morphology. These three cryptic species co-occurred in needles within a single population of P. sylvestris in Scotland. There are other reports of cryptic variations within L. pinastri as currently circumscribed. Osono and Hirose 23 Page 8 of 13 Mycol Progress (2015) 14:23 Mycol Progress (2015) 14:23 A phylogenetic tree generated from Bayesian analysis of the ITS rDNA. Thick branches represent posterior probabilities higher than 0.95. Numbers at internodes represent the bootstrap values from the maximum likelihood analysis that are above 90 %. The fungus of the present study is labelled as Lophodermium corconticum, the name given to it in this publication. Colpoma quercinum was used as the outgroup ƒFig. 4 (2011) recorded high nucleotide differences between two groups of L. pinastri isolates obtained from P. thunbergii Parl. and P. densiflora Sieb. et Zucc. needles which, however, did not correlate with phenotypic characteristics (lignin decomposition). Two cryptic species differing in latitudinal distribution in Sweden were also recognised based on ITS rDNA in the study of Millberg et al. (2013, sequences not included in our dataset). Ganley et al. (2004, sequences not included in our dataset) suggested North American pines may harbour further cryptic species. Similar variation was observed for L. conigenum. Sequences of ITS rDNA identified as this species and included in our dataset formed two distinct groups, tentatively marked I and II in Fig. 4. Wang et al. (2010) found that sequences obtained from L. conigenum in China formed two largely unrelated species. In our analyses, Chinese and New Zealand sequences formed one group distinct from Scottish sequences referred to by Reignoux et al. (2014). Results, therefore, show that (in addition to the present fungus) at least five species can be recognized on the basis of molecular evidence, all of which have been identified as L. pinastri on the basis of non-molecular characters. Is it possible to say which, if any, represents the real L. pinastri? It is necessary to consult type material. The identity of the type of L. pinastri was discussed by several authors (Terrier 1942; Staley 1975; Minter 1977; Minter et al. 1978). They agreed that exsiccatum no. 76 of Mougeot and Nestler (1810) is the best candidate to be type. This exsiccatum can be found in many major fungal reference collections, and all examples seen by Minter (1977) were morphologically similar. The example in the collection of the University of California, Berkeley, was designated lectotype by Staley (1975), with isolectotypes in Edinburgh and Kew cited by Minter et al. (1978). The fungus in the Mougeot & Nestler exsiccatum is a Lophodermium on dead secondary needles of P. sylvestris, collected somewhere in the Vosges region of France. The ascomata are partially subepidermal, and the lips lining the ascomatal split are at least sometimes red. Ascomata are accompanied by a conidial state and numerous thin black zone lines. Staley (1975), Minter (1977), Minter et al. (1978) and Minter (1981a) and most if not all subsequent people working on this topic have used this species concept for L. pinastri. There are many sequences of L. pinastri on P. sylvestris in the GenBank, and at least some from native pine forests of Scotland where Minter worked are likely to reflect the same Page 9 of 13 23 species concept. At the time of this writing, however, there seems to be no publicly available sequences from France identified as L. pinastri [GenBank, www.ncbi.nlm.nih.gov/ genbank, accessed 26 July 2014]. To establish the molecular identity of typical L. pinastri, sequences are needed from fresh collections fitting the current species concept of L. pinastri on dead fallen secondary needles of P. sylvestris in the litter from the Vosges region of France. It will also be important to establish the molecular profile of typical L. staleyi, which like the present fungus is similar in appearance to L. pinastri. Lophodermium staleyi differs subtly in length of ascomata, conidia and perhaps other dimensions. Its ascomata tend to have grey lips, and like L. pinastri, it is found on P. sylvestris. Reignoux et al. (2014) considered one cryptic species within L. pinastri (clade IV in Fig. 4) a possible candidate for L. staleyi. There has been a tendency to take sequences from samples where little or nothing is recorded of the exact habitat or needle history. Such samples may reveal cryptic species, but are otherwise uninformative. The habitat occupied by the source of each sequence should be noted carefully. Lophodermium on pine can be found in many different habitats. Secondary needles, primary needles, cones and even bare wood have all been recorded as harbouring these fungi. Several Lophodermium species are known to occur, often side by side, as endobionts in apparently healthy needles, and the Lophodermium that finally fruits may be determined by the genotype of the tree (for example, the internal pH regulation within the needle) and by what happens to that needle during its life. The age of the needle at time of death may be significant. Needles that senesce, die and fall into the litter provide a very different habitat from needles that die prematurely as a result of attack by a pathogen, or because the branch on which they are attached has broken off the tree. A branch broken off in June, for example, may provide a very different habitat from a branch broken off in November. An attached twig that has died because it is suppressed by those around could be different again. These are habitats that are obvious to the human eye. There may be many others to which the fungi themselves are sensitive. Lophodermium species on pine are superb examples of sympatric evolution exploiting those subtly different habitats (Burnett 1983). Linking particular molecular profiles to particular needle habitats will greatly assist efforts to find diagnostic non-molecular characteristics. Only thorough examination of the specimens on which such sequences are based can show whether they represent cryptic species or distinct species differing also in morphology and previously misidentified as L. pinastri (Sokolski et al. 2004). Is the present fungus on P. mugo really cryptic? Knowing that molecular evidence shows it to be separate, could meticulous examination reveal morphological differences? To do that, reliable information about the appearance of L. pinastri on different pines including P. mugo is needed. 23 Page 10 of 13 Fig. 5 A phylogenetic tree generated from Bayesian analysis of the actin. Thick branches represent posterior probabilities higher than 0.95. Numbers at internodes represent the bootstrap values from the maximum likelihood analysis that are above 90 %. The fungus of the Mycol Progress (2015) 14:23 present study is labelled as Lophodermium corconticum, the name given to it in this publication. Hymenoscyphus epiphyllus was used as the outgroup Mycol Progress (2015) 14:23 Minter (1977) analysed the appearance of L. pinastri on naturally senesced, dead, fallen secondary needles of P. sylvestris from native Scottish pine forests. Means are cited here, but sample sizes and standard errors were provided in the source. It produced thin black zone lines, conidiomata with a mean length of 337 μm, partially subepidermal ascomata with a mean length of 945 μm and with six or more displaced epidermal cells grouped along the ascomatal basal wall and usually red lips lining the ascomatal split, asci with a mean length of 134 μm, and ascospores with a mean length of 90 μm. The appearance of Lophodermium on naturally senesced, dead, fallen secondary needles of 30 other diploxylon pine species was also studied using samples of Bedgebury Pinetum in southern England (Minter 1977). The same qualitative and quantitative details were noted. In all cases, the Lophodermium looked like L. pinastri, and had dimensions that did not differ significantly from those of L. pinastri on P. sylvestris from Scotland. Pinus mugo was one of the species sampled. The Lophodermium observed on its needles collected from Bedgebury had thin black zone lines, conidiomata with a mean length of 340 μm, partially subepidermal ascomata with nine epidermal cells grouped along the ascomatal basal wall, with red lips, and with a mean length of 960 μm. On this basis, the fungus on P. mugo from Bedgebury and on all other 29 diploxylon pine species sampled at the same time was identified as L. pinastri. The Lophodermium on P. mugo from Poland and L. pinastri on P. mugo from Bedgbury both produce thin black zone lines, but conidiomata and ascomata of the Polish fungus are both substantially longer, and lips lining the ascomatal split are gray. There was not enough material from Bedgebury to obtain measurements of the ascus and ascospore lengths for L. pinastri on P. mugo, but given the strong evidence that these did not vary significantly over a wide range of diploxylon pines, statistics from P. sylvestris provide meaningful material for comparison. Asci of the Polish fungus tend to be slightly shorter, and its ascospores are much shorter than those of L. pinastri on P. sylvestris. There are also other slight differences. The ascomatal upper wall in the Polish fungus is markedly thicker near the split (Figs. 1e and 2a), where that of L. pinastri is only slightly thickened, and hyphal bridges interconnecting adjacent paraphyses were observed in the Polish fungus (Figs. 1j and 2b), but have not been reported from L. pinastri. Hyphal bridges between paraphyses are easily overlooked but have been observed in several members of the Rhytismataceae: examples include Hypoderma labiorum-aurantiorum Minter, L. durilabrum Darker and Ploioderma hedgcockii (Dearn.) Darker (Minter 1986). These anatomical differences may be evidence that ascomata of the Polish fungus differ in development from those of L. pinastri. Page 11 of 13 23 Thus, there is clear molecular evidence supported by morphological evidence that the Polish fungus is different from L. pinastri. In addition, molecular, morphological and ecological evidence of associated organisms and geographical distribution indicate that the Polish fungus is different from all other described species of Lophodermium on pines. It is, therefore, formally described as a new species with the name Lophodermium corconticum. Taxonomy Lophodermium corconticum Koukol, Pusz & Minter, sp. nov., Figs. 1, 2 and 3. Mycobank number: MB 808185 Diagnosis: Morphologically very similar to L. pinastri, but with longer conidiomata and ascomata, shorter ascospores, with only gray lips lining the ascomatal split and paraphyses with hyphal bridges; sequences of ITS rDNA (ex-type sequence LM654179) and actin (ex-type sequence LM654182) distinct from all available sequences of L. pinastri. Holotype Poland, Karkonosze National Park, Karpacz, 500 m W from BDom Ślaski^ mountain shelter, 50°44′ 28.624″ N, 15°43′19.638″ E, 1,370 m above sea level, on needles of Pinus mugo lying on the ground, leg. W. Pusz 9 May 2014 (PRM924322, ex-type strain CCF4868 = NK387). Etymology The epithet refers to an ancient people, Corconti, who resided in the Giant Mountains. Additional material Poland, Karkonosze National Park, Karpacz, 500 m W from BDom Ślaski^ mountain shelter, 50°44′28.624″ N, 15°43′19.638″ E, 1,370 m above sea level, on needles of Pinus mugo lying on the ground, leg. W. Pusz 22 October 2013 (PRM923786, living strain CCF4781 = NK366). BMały Śnieżny Kocioł,^ 50°46′50.46″ N, 15°33′ 51.181″ E, 1,250 m above sea level, on needles of Pinus mugo lying on the ground, leg. W. Pusz, 6 May 2014 (PRC2820, living strain NK386). Comment Lophodermium corconticum was recorded by Pusz et al. (2013) as the dominant species isolated from needles still attached to the tree and showing disease symptoms such as yellow spots and premature falling. In subsequent isolations, it was the only species outgrowing on 2BA from symptomatic needles after surface sterilisation (Fig. 3a). Sydowia polyspora (Bref. & Tavel) E. Müll., also recorded in several symptomatic needles, is a weak parasite previously known from the study area (Příhoda 1965). The high incidence of L. corconticum may indicate that it causes the disease, but synergistic effects with other known parasites may also be possible. Not enough 23 Page 12 of 13 is known about its relationship with mountain pine to dismiss it as simply parasitic. It may, for example, also be the dominant species inside symptomless and apparently healthy attached needles (on P. sylvestris, several Lophodermium species including L. seditiosum, often regarded as a serious pathogen, are common in such needles). The time of needle colonization is not known. Does this fungus colonize needles at a particular stage of development, for example as they flush, or can infection occur at any time? Nothing is known about any role the fungus may play as, for example, a regulator in living needles, or as a deterrent to other more serious threats to the tree. After falling into the litter, L. corconticum plays an important role in needle decomposition, but the exact nature of that role is not known. Mitchell and Millar (1978) and Koukol and Baldrian (2012) showed only limited abilities of L. pinastri to degrade cellulose. Osono and Hirose (2011), however, demonstrated that several isolates of L. pinastri can cause mass loss of lignin. Something similar may occur with the current species. Lophodermium corconticum seems to be rare and endemic on mountain pine in the Giant Mountains. It may, therefore, be of conservation concern. At present, using IUCN Categories & Criteria [IUCN, www.iucnredlist.org/technical-documents/ categories-and-criteria, accessed 27 July 2014], its conservation status can only be assessed as "Data Deficient." Pinus mugo is the only pine found in the studied area associated with this fungus, and isolations from needles of P. mugo outside this area yielded only other species of Lophodermium. Hou et al. (2009) described a new species, L. pini-mugonis, from the same pine species in similar climatic conditions in the Bavarian Alps, but it is phylogenetically unrelated to L. corconticum (Fig. 4). The disjunctive distribution of P. mugo and specific climate conditions suggest there may have been a repeated speciation event leading to two unrelated species occupying a similar niche. Andjelič (2000) identified L. pinastri as the main cause of mountain pine disease in mountain areas of Montenegro, but, in the absence of molecular data, this may have been a misidentification for L. corconticum, and the Lophodermium giving rise to that report should be re-examined. Similarly, the potential occurrence of L. corconticum in Bedgebury, Scotland, should be verified by fresh sampling. With recognition of several other apparently cryptic species in L. conigenum and L. pinastri, description of L. corconticum as a new and distinct species is unlikely to be the end of the story. Other Bisland^ populations of pines may reveal further endemic Lophodermium species with highly restricted geographical distributions, and several parts of the world important for pine diversity, including Central America and Mexico, the home of pines, have not yet been explored for needle fungi. Recent studies in Asia, particularly in China, contain many reports of species previously thought to be European or North American (Luo et al. 2010). Some of these are also Mycol Progress (2015) 14:23 likely, on closer examination, to represent undescribed native taxa. Staley (1975) interpreted Lophodermium species fruiting on pine foliage as a multiplicity of unnamed species. He wrote, Bthe task of outlining the taxonomy of this group… is …analogous to attempting a taxonomy of the genus Pinus based on a few pine species chosen from the more than 90 species recognized.^ It is increasingly looking as though he was correct. Acknowledgments The project was supported by Institutional Support for Science and Research of the Ministry of Education, Youth and Sports of the Czech Republic and by the National Science Centre of Poland (grant number N304/069940). We wish to thank Agata Kaczmarek from the Division of Phytopathology and Mycology of the Plant Protection Department at WUELS for her help in preparation of the mycological analyses, and to Wlodzimierz Kita from the Division of Phytopathology and Mycology of the Plant Protection Department at WUELS for his help in collection of needle samples. References Altschul S, Gish W, Miller W, Myers E, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Andjelič M (2000) Disease of the mountain pine (Pinus mugo Turra) in the area of BLovcen^ National Park (Montenegro, Yugoslavia). Zaštita Prirode 52:69–78 Baudyš E (1924–1925) Příspěvek k rozšíření mikromycetů u nás [Contribution to knowledge of distribution of our micromycetes]. Čas Morav Mus Zem, Brno 22–23:1–31 (In Czech) Burnett JH (1983) Presidential address. Speciation in fungi. Trans Br Mycol Soc 81:1–14 Carbone I, Kohn LM (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553– 556 Castresana J (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17:540– 552 Cypers V (1896) Beiträge zur Kryptogamenflora des Riesengebirges und seiner Vorlagen. Pilze. II. Verh Zool-Bot Ver Wien 46:310–320 Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Meth 9:772 Fassatiová O (1986) Moulds and filamentous fungi in technical microbiology. Elsevier, Amsterdam Ganley RJ, Brunsfeld SJ, Newcombe G (2004) A community of unknown, endophytic fungi in western white pine. Proc Natl Acad Sci U S A 101:10107–10112 Gao X-M, Lin Y-R, Huang H-Y, Hou C-L (2013) A new species of Lophodermium associated with the needle cast of Cathay silver fir. Mycol Progr 12:141–149 Hou C-L, Li L, Piepenbring M (2009) Lophodermium pini-mugonis sp. nov. on needles of Pinus mugo from the Alps based on morphological and molecular data. Mycol Progr 8:29–33 Jones SG (1935) The structure of Lophodermium pinastri (Schrad.) Chev. Ann Bot 49:699–728 Koukol O, Baldrian P (2012) Intergeneric variability in enzyme production of microfungi from pine litter. Soil Biol Biochem 49:1–3 Luo J-T, Lin Y-R, Shi G-K, Hou C-L (2010) Lophodermium on needles of conifers from Yunnan Province, China. Mycol Progr 9:235–244 Lutyk P (1978) The healthiness of mountain pine (Pinus mughus Scop.) in Tatra National Park. Sylwan 122:51–57 Mycol Progress (2015) 14:23 Millberg H, Boberg JB, Stenlid J (2013) Fungal community composition of Scots pine needles – geographic patterns and variation with needle age and between healthy and diseased needles. IUFRO working party 7.02.02, Brno, Czech Republic, 20–25 May 2013, Book of Abstracts, p 34 Miller M, Holder M, Vos R, Midford P, Liebowitz T et al (2009) The CIPRES Portals. CIPRES. 2009-08-04. URL:http://www.phylo. org/sub_sections/portal. Accessed: 2014-01-17. (Archived by WebCite(r) at http://www.webcitation.org/5imQlJeQa) Minter DW (1977) Lophodermium on Pines with Special Reference to Species Occurring on Pinus sylvestris in North-East Scotland. PhD Thesis. 401 pp. UK, Aberdeen; University of Aberdeen, Department of Forestry Minter DW (1981a) Lophodermium on pines. Mycol Pap 147:1–54 Minter DW (1981b) Microfungi on needles, twigs and cones of pinus in ČSSR. Čes Mykol 35:90–101 Minter DW (1986) Some members of the Rhytismataceae (Ascomycetes) on conifer needles from Central and North America. In: Petersen GW (ed) Recent Research on Conifer Needle Diseases. Proceedings of the IUFRO S2.06.04 Working Party Conference on Needle Diseases, Gulfport, Mississippi, October 14–18, 1984. USDA Forest Service General Technical Report GTR-WO, vol 50. US Department of Agriculture Forest Service, Washington, DC, pp 71–106 Minter DW, Holubová-Jechová V (1981) New or interesting Hyphomycetes on decaying pine litter from Czechoslovakia. Folia Geobot Phytotax 16:195–217 Minter DW, Millar CS (1980) Ecology and biology of three Lophodermium species on secondary needles of Pinus sylvestris. Eur J For Pathol 10(2–3):169–180 Minter DW, Staley JM, Millar CS (1978) Four species of Lophodermium on Pinus sylvestris. Trans Br Mycol Soc 71:295–301 Mitchell CP, Millar CS (1978) Mycofloral successions on Corsican pine needles colonized on the tree by three different fungi. Trans Br Mycol Soc 71:303–317 Mougeot JB, Nestler CG (1810) Stirpes Criptogamae Vogeso-Rhenanae etc. Fasc 1 O’Donnell K (1993) Fusarium and its near relatives. In: Reynolds DR, Taylor JW (eds) The fungal holomorph: mitotic, meiotis and Page 13 of 13 23 pleomorphic speciation in fungal systematics. CAB International, Wallingford, pp 225–233 Osono T, Hirose D (2011) Colonization and lignin decomposition of pine needle litter by Lophodermium pinastri. For Pathol 41:156–162 Příhoda A (1965) Houby na kosodřevině v Krkonoších. [Fungi on mountain pine in Giant Mts.]. Oper Corcont 2:61–70, In Czech Przewoźnik L (2008) Plants of Karkonosze National Park. Karkonosze National Park Press, Jelenia Góra (in Polish) Pusz W, Kita W, Kaczmarek A, Nowosad K, Koukol O (2013) The mountain pine’s needles diseases (Pinus mugo) on subalpine zone of Karkonosze Mts. Sylwan 157:761–769 Reignoux SNA, Green S, Ennos RA (2014) Molecular identification and relative abundance of cryptic Lophodermium species in natural populations of Scots pine, Pinus sylvestris L. Fungal Biol 118:835–845 Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574 Schröter J (1908) Die Pilze Schlesiens. - In: Krypt.-Fl. Schlesien, 3–2:1– 597 Sokolski S, Piche Y, Berube J (2004) Lophodermium macci sp. nov., a new species on senesced foliage of five-needle pines. Mycologia 96: 1261–1267 Staley JM (1975) The taxonomy of lophodermia on pines with special reference to problems in North American christmas tree plantations. Mitt Bundesforschungsanstalt Forst Holtzwirtschaft 108:79–85 Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol 75:758–771 Terrier CA (1942) Essai sur la systématique des Phacidiaceae (Fr.) sensu Nannfeldt (1932). Beitr Kryptogamenflora Schweiz 9:1–99 Wang S-J, Lin Y-R, Hou C-L, Gao X-M, Yang Z-Z, Liu F (2010) A preliminary study on phylogeny of Lophodermium based on rDNA-ITS sequences. Mycosystema 29:199–205 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: a guide to methods and applications. Academic, San Diego, pp 315– 322