Mycol Progress (2015) 14:54 DOI 10.1007/s11557-015-1078-3
ORIGINAL ARTICLE
A new species and a new combination of Terriera based on morphological and molecular data Ting-Ting Zhang 1 & Xin Tong 1 & Ying-Ren Lin 2 & Cheng-Lin Hou 1
Received: 10 March 2015 / Revised: 10 June 2015 / Accepted: 16 June 2015 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2015
Abstract A new Terriera species, T. elliptica on twigs of Rhododendron sp., was reported from Yunnan Province, China. T. elliptica is similar to Lophodermium camelliicola in morphology and affiliation, but differs in having elliptic, non-curved ascomata and larger asci. In addition, ITS rDNA sequences of the two species differ significantly. Based on morphological and molecular analyses, L. camelliicola was transferred to Terriera. Keywords Lophodermium . Multigene analysis . Phylogeny . Rhytismatales . Taxonomy
Introduction The genus Terriera B. Erikss. is a member of Rhytismataceae (Rhytismatales, Leotiomycetes, Ascomycota) (Kirk et al. 2008). The type species is T. cladophila (Lév. in Moug. & Nestl.) B. Erikss (syn. Lophodermium cladophilum (Lév. in Moug. & Nestl.) Rehm) (Eriksson 1970). Johnston (1988, 1989) enumerated some important features for delimitation of this genus, such as oblong to sublinear ascomata, the lack of lip cells, covering stroma forming a platform in vertical section, and the triangular space in section between the covering stroma and basal stroma filled with vertically
* Cheng-Lin Hou
[email protected] 1
College of Life Science, Capital Normal University, Beijing 100048, People’s Republic of China
2
School of Forestry & Landscape Architecture, Anhui Agricultural University, Hefei 230036, People’s Republic of China
oriented cells. A phylogenetic analysis by Lantz et al. (2011) supported Terriera as monophyletic clade while Lophodermium was present in all three major clades of Rhytismatales. Many species in Lophodermium with the typical characteristics of Terriera have been transferred to Terriera (Johnston 2001). Up to now, 24 species of Terriera are recognized worldwide (www.speciesfungorum.org/); nine species of Terriera have been reported for China (Chen et al. 2011; Yang et al. 2011; Zheng et al. 2011; Gao et al. 2012; Song et al. 2012; Zhou et al. 2012; Chen et al. 2013). In the present study, a new Terriera species was identified and described based on morphological and molecular data and one new combination was proposed.
Materials and methods Morphological studies Dried material with mature ascomata was selected for morphological observation. External shape, size, color, mechanism of opening of ascomata, as well as the characteristics of zone lines, were observed under a Nikon SMZ-1000 dissecting microscope (Tokyo, Japan). Sections of different thickness of ascomata were prepared using a razor blade and mounted in water, Melzer’s reagent, 5 % KOH solution, or 0.1 % (w/v) cotton blue in lactic acid for observation of the internal structure under an Olympus EX51 microscope (Tokyo, Japan). Color of structures and ascospore contents were examined in water. Gelatinous sheaths surrounding ascospores and paraphyses were observed in water or 0.1 % (w/v) cotton blue in lactic acid. Measurements and drawing of asci, ascospores and paraphyses were made from 30 ascospores, asci, and paraphyses for each specimen using material
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mounted in 5 % KOH solution or Melzer’s reagent (Hou et al. 2009). The specimens are deposited in the Herbarium of the College of Life Science, Capital Normal University (BJTC). DNA extraction and sequencing Total genomic DNA was extracted from ascomata following the protocol of Hou et al. (2009). New sequences for the nuclear large subunit ribosomal DNA (LSU rDNA), internal transcriber spacer (ITS rDNA) and mitochondrial small subunit ribosomal DNA (mtSSU rDNA) were obtained from the new species (HOU327) and Lophodermium camelliicola (L0447). The ITS rDNA and LSU rDNA regions were amplified with PCR using the primers ITS1/ITS4 and LR0R/LR5, respectively (White et al. 1990; Gardes and Bruns 1993), and mrSSU1/mrSSU3R primers were used for mtSSU rDNA (Zoller et al. 1999). PCR procedure in 25-μl reactions was following Hou et al. (2009). The PCR products were purified, sequenced, and edited by Invitrogen Biotechnology Co. Ltd. (Beijing, China). The new sequences were submitted to the GenBank database. Other sequences of ITS rDNA, LSU rDNA, and mtSSU rDNA included in this study were downloaded from GenBank (Table 1). Phylogenetic analyses To reveal the affiliation of the new species within Terriera, a phylogenetic analysis was performed on the combined sequences of ITS rDNA, LSU rDNA, and mtSSU rDNA, covering all Terriera species that were available and of further representatives of Rhytismataceae. The ITS rDNA, LSU rDNA, and mtSSU rDNA sequence datasets were firstly aligned using Clustal X separately (Thompson et al. 1997), then manually corrected in Se-Al v.2.03a by eye (Rambaut 2000). Ambiguously aligned regions were excluded in the analysis. A partition homogeneity test was done to determine the congruence of the three datasets (Farris et al. 1995; Huelsenbeck et al. 1996). After a positive outcome, the datasets were analyzed together. The combined dataset of ITS rDNA, LSU rDNA, and mtSSU rDNA sequence data was prepared and analyzed with the maximum parsimony (MP) method using PAUP* 4.0b10 (Swofford 1998), Bisporella citrina and Neofabraea malicorticis were chosen as outgroup. For the Bayesian analysis, MrModeltest 3.7 with the Akaike information criterion was used to choose the substitution model for the separate dataset (Nylander 2004). The Bayesian analysis was performed with MrBayes 3.1.2 (Huelsenbeck et al. 2001; Ronquist and Huelsenbeck 2003). The detailed methods and parameters of MP and Bayesian analyses followed Lantz et al. (2011). Clades receiving both BP ≥ 70 % and PP ≥ 95 % were considered to be significantly supported.
Results Phylogenetic analyses The combined DNA matrix included 29 taxa, of which 15 lacked ITS markers and one lacked mtSSU markers. After removing regions impossible to align, the length of the combined sequence alignment corresponded to 3255 characters, with 1169 phylogenetically informative positions. The MP analysis of sequences resulted in one most parsimonious tree (Fig. 1) with a length (TL) of 5090 steps, consistency index (CI) of 0.5984, retention index (RI) of 0.5485, homoplasy index (HI) of 0.4016, and rescaled consistency index (RC) of 0.3282. The combined three-gene matrix and resulting maximum parsimony consensus tree (Fig. 1) are available at www.treebase.org with reference number 17,224. The phylogenetic result showed that the rhytismataceous species form a well-supported clade (BP=100, PP=1.00). Lophodermium piceae and Lirula macrospora formed a small clade as a sister group of the other species of Rhytismataceae. The new species Terriera elliptica together with Lophodermium camelliicola, T. minor and the type species T. cladophila of Terriera formed a strongly supported clade (BP=100, PP=1.00) and were sister to a clade composed of Lophodermium brunneolum P.R. Johnst., L. medium P.R. Johnst. and Hypohelion scirpinum (DC.) P.R. Johnst. with a low supported value. The remaining clades in our phylogenetic tree were similar to those of Wang et al. (2014).
Taxonomy Terriera elliptica T.-T. Zhang & C.-L. Hou, sp. nov. Figs. 2-7 MycoBank 811799 Diagnosis: Differs from Lophodermium camelliicola by non-curved ascomata, larger asci with obtuse to truncate apices, insertion type of ascomata in host tissues, and habit on living twigs of Rhododendron sp. Description: Ascomata developed on living twigs, associated with bleached, pale brown areas, and narrow, brown, incomplete zone lines. In surface view, ascomata varied in dimensions, 1000–1800×450–650 μm, scattered, entirety of ascomata dark brown to black, matt, elliptical, ends rounded to subacute, margins diffused, the central part of the ascomata strongly raising the surface of the substrate at maturity, opening by a longitudinal split, split extending almost the whole length of the ascoma. Immature ascomata brown, circular to slightly elongated, lips absent. In median vertical section, ascomata 230–260 μm deep, subhypodermal, but intraepidermal at base of covering stroma. Covering stroma 40–55 μm thick near the centre of the ascoma, slightly thinner towards the edges, extending to the basal stroma, but obvious thicker towards the ascomal opening, with a 20–30 μm thick
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GenBank accession numbers of sequences used for molecular analysis
Species
ITS accession number
LSU accession number
mtSSU accession number
Bisporella citrina (Batsch) Korf & S.E. Carp. Coccomyces crystalligerus Sherwood Coccomyces radiatus Sherwood Coccomyces tumidus (Fr.) De Not.
AF335454 —— —— ——
FJ176871 HM140501 HM140509 HM140510
FJ190632 HM143778 HM143786 HM143787
Colpoma juniperi (P. Karst. ex P. Karst.) Dennis Colpoma quercinum (Pers.) Wallr. Cryptomyces maximus (Fr.) Rehm Cudonia circinans (Pers.) Fr. Hypoderma rubi (Pers.) DC. Hypohelion scirpinum (DC.) P.R. Johnst. Lirula macrospora (R. Hartig) Darker Lophodermium arundinaceum (Schrad.) Chevall. Lophodermium brunneolum P.R. Johnst. Lophodermium medium P.R. Johnst. Lophodermium piceae (Fuckel) Höhn.
—— U92306
HM140511 HM140513
—— HM143789
—— EU784190 GU138750 —— HQ902159 —— —— —— AY775683
HM140514 HM140515 HM140526 HM140531 HQ902152 HM140535 HM140536 HM140545 HM140551
HM143790 HM143791 HM143801 HM143806 HM143807 HM143811 HM143812 HM143820 HM143825
Lophodermium pinastri (Schrad.) Chevall. Lophodermium platyplacum (Berk. & M.A. Curtis) Sacc. Lophodermium camelliicola Minter (L0447) Nematococcomyces rhododendri C.L. Hou, M. Piepenbr. & Oberw. Neofabraea malicorticis H.S. Jacks. Rhytisma acerinum (Pers.) Fr. Spathularia flavida Pers. Terriera cladophila (Le’v.) B. Erikss. Terriera elliptica T.T. Zhang & C. L. Hou (HOU 327) Terriera minor (Tehon) P.R. Johnst. (Lantz & Minter 418 UPS) Terriera minor (Tehon) P.R. Johnst. (ICMP13973) Terriera minor (Tehon) P.R. Johnst. (ICMP13974) Therrya pini (Alb. & Schwein.) Höhn. Tryblidiopsis pinastri (Pers.) P. Karst.
AY100649 —— FJ861977 —— AY787721 GQ253100 AF433153 —— KP878549 —— —— —— JF793677 JF793678
HM140552 HM140554 KP878552 KC312687 AY544662 FJ495190 AY433142 HM140568 KP878550 HM140569 HM140570 HM140571 KC312684 HM140573
HM143826 HM143827 KP878553 KC312691 AY544751 HM143837 AY575101 HM143840 KP878551 HM143841 HM143842 HM143843 KC312688 AF431963
B—^ means that no sequence was available.
extension comprising strongly carbonized tissue with no obvious cellular structure, covering stroma consisting of an outer layer of host cuticle, remains of epidermal and hypodermal cells filled with 3–5 μm diam, thick-walled, angular fungal cells, and an inner layer of textura angularis and globulosa with 3–5 μm diam, dark brown, thick-walled fungal cells. Excipulum moderately developed, closely adhering to the covering stroma and the extension, arising from the marginal paraphyses, becoming thinner towards the base, but thicker, multiseptate beneath the extension. Basal stroma concave, 10– 15 μm thick, dark brown, consisting of 2 rows of 3–5 μm diam, thick-walled angular cells. A triangular space in section between the covering stroma and basal stroma filled with 30–40 μm thick, vertically arranged rows of colorless, thin-walled cells. Subhymenium composed of textura intricata, 7–12 μm thick. Paraphyses filiform, 140–190×
2–3 μm, thin-walled, hyaline, branching 1–3 times, and not swollen near the apex, covered by a thin gelatinous sheath, forming a 5–10 μm thick epithecium at the tip. Asci maturing sequentially, 135–175×7–9 μm, cylindrical, but cylindrical-clavate at immaturity, somewhat longstalked, apex obtuse to truncate, thin-walled, without circumapical thickening, J-, 8-spored, arranged in a fascicle, discharging spores through a small apical pore. Ascospores, 60–85×1.5–2 μm, filiform, tapering slightly towards both ends, hyaline, aseptate, pluriguttulate, coverd by a 1–1.5 μm thick, gelatinous sheath. Conidiomata not observed. Etymology: elliptica, referring to the shape of the ascomata. Holotype: On living twigs of Rhododendron sp., China, Yunnan Province, Chuxiong, Zixishan, alt. ca. 2400 m, 5 August 2006, C.-L. Hou 327 (BJTC 201419).
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Fig. 1 Phylogenetic tree derived from maximum parsimony analysis of the combined ITS rDNA, LSU rDNA, and mtSSU rDNA sequences of Terriera species and related species, using Bisporella citrina and Neofabraea malicorticis as outgroup. Bootstrap values of more than 70 % from 1000 replications are shown above the respective branches. Bayesian posterior probabilities (PP) were estimated and clades with PP > 0.95 are marked under the branches. The position of Terriera elliptica on Rhododendron from China was bold in the tree
Additional specimen examined: On living twigs of Rhododendron sp., China, Yunnan Province, Lijiang, the hill Figs. 2-4 Terriera elliptica on twigs of Rhododendron sp. (holotype BJTC201419). 2. Mature ascomata observed under the dissecting microscope. 3. Immature ascomata. 4. Immature ascomata and a zone line
behind the Xinzhucun, alt. ca. 2400 m, 20 July 2013, C.-L. Hou 1121A (BJTC 201420).
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Figs. 5-7 Terriera elliptica on Rhododendron sp. (ex holotype BJTC201419). 5. Ascoma in vertical section. 6. Discharged ascospores. 7. Paraphyses, an empty ascus, a young ascus, and two mature asci with ascospores
Known distribution: Only known from Yunnan Province, China.
Discussion Terriera elliptica is assigned to Terriera because it is morphologically consistent with the type species of Terriera, T. cladophila. However, T. cladophila has subcuticular, shorter ascomata (350–900 μm), smaller asci (80–125×6–9 μm), ascospores with no gelatinous sheath, a black perimeter line, and also in the presence of conidiomata (Minter 1996). The insertion type of ascomata and the shape of asci and ascospores of T. elliptica are very similar to those of Lophodermium camelliicola Minter described by Lin et al. (2012). The phylogenetic analysis also revealed that T. elliptica is closely related to L. camelliicola (L0447) (BP = 100, PP = 1.00). However, L. camelliicola (L0447) has ascomata which are more or less curved, smaller asci (100–130×5–6 μm) with rounded apices, paraphyses with swollen apices, no epithecium, and observable conidiomata. Furthermore, the ITS sequence similarity in T. elliptica and L. camelliicola (L0447) was only 95 %. Therefore, based on morphological and sequence differences, this novel fungus was identified as a new species in Terriera. Lin et al. (2012) mentioned that the specimens of L. camelliicola (L0447 etc.) they observed were somewhat different from the type description by Minter and Sharma
(1982), such as by the insertion type of ascomata and host spectrum. The type specimen was only recorded on twigs of Camellia sinensis (L.) Kuntze, while specimens examined by Lin et al. occurred on a wider spectrum of hosts, such as Sabia japonica Maxim (L1975) (Sabiaceae) and Lindera reflexa Hemsl (L1624), (Lauraceae) as well as unknown hosts. Knowing the host species is important for some species identification and molecular data confirms that many species in Rhytismataceae are highly host specific. As for the insertion type of ascomata, the ascomata of the type specimen were partly subepidermal while those described by Lin et al. were irregular inner-cortical (Lin et al. 2012). Unfortunately, a DNA sequence of the type specimen L. camelliicola could not be obtained due to the age of the specimen. The relationships between the type specimen and specimens collected by Lin require further resolution based on molecular data from fresh samples in the future. In the present study, the topology of the phylogenetic tree is similar to the topologies of Lantz et al. (2011) and Wang et al. (2014). The phylogenetic analysis of the combined ITS, LSU, and mtSSU showed that species with typical characteristics of Terriera, including T. minor, T. cladophila, T. elliptica, and L. camelliicola, formed a monophyletic clade. Therefore, L. camelliicola should be transferred into genus Terriera. Terriera camelliicola (Minter) Y.-R. Lin & C.-L. Hou, comb. nov. MycoBank 811802 ≡ Lophodermium camelliicola Minter, in Minter & Sharma, Mycologia 74: 709, 1982.
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For descriptions and figures see Minter and Sharma (1982) and Lin et al. (2012). Holotype: On twigs of Camellia sinensis (L.) Kuntze, India, Assam, Tochlai Experimental Station, 9 August 1922, A. C. Tunstall (IMI 23122). Additional specimens examined: On dead stems and branches of an unidentified broad-leaf tree, China, Guangxi Province, Nanning, 18 June 1990, Y.-R. Lin 0447 (AAUF 66555) and China, Anhui Province, Tiantangzhai, alt. ca. 860 m, 14 September 2005, Y.-R. Lin et al. 1976 (AAUF68084); Lindera reflexa Hemsl., China, Anhui Province, Mount Huang, 12 August 1995, Y.-R. Lin & S.M. Yu 1624 (AAUF 67732) and 24 September 2004, Y.-R. Lin et al. 1879 (AAUF 67987); Rosa henryi Boulenger, China, Anhui Province, Mount Huang, 9 August 1995, Y.-R. Lin & S.-M. Yu 1589 (AAUF 67697); Sabia japonica Maxim., China, Anhui Province, Tiantangzhai, alt. ca. 860 m, 14 September 2005, Y.-R. Lin et al. 1975 (AAUF 68083). Known distribution: India: Assam; China: Anhui Province, Guangxi Province. Acknowledgments This study was supported by Beijing Natural Science Foundation (No. 5132009) and the National Natural Science Foundation of China (No. 31170019 and 31270065).
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