Mycol Progress (2015) 14: 101 DOI 10.1007/s11557-015-1121-4
ORIGINAL ARTICLE
A morphological and molecular survey of Japanese Melampsora species on willows reveals a new species and two new records Peng Zhao 1 & Qing-Hong Wang 2 & Cheng-Ming Tian 3 & Qi Wang 4 & Yuichi Yamaoka 5 & Makoto Kakishima 4,5
Received: 23 August 2015 / Revised: 24 September 2015 / Accepted: 28 September 2015 / Published online: 14 October 2015 # German Mycological Society and Springer-Verlag Berlin Heidelberg 2015
Abstract During morphological and molecular phylogenetic studies of Melampsora species on willows, one novel species, Melampsora salicis-reinii sp. nov., and two new records, M. ribesii-viminalis and M. ribesii-purpureae, are described from Japan. Melampsora salicis-reinii on Salix reinii was characterized by its amphigenous telia and ellipsoid urediniospores with echinulate spines on a rugose surface. Phylogenetic results using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) supported morphological elucidation, and both morphological and molecular evidences supported M. salicis-reinii as a new species. In addition, M. ribesii-viminalis was identified on S. pet-susu and S. sachalinensis, and M. ribesii-purpureae was recognized on S. integra. These two Melampsora species were reported as new records in Japan for the first time. Keywords Molecular phylogeny . Pucciniales . Rust fungi . Species recognition . Willows Section Editor: Franz Oberwinkler * Peng Zhao
[email protected] 1
Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan
2
Beijing Key Laboratory of Oil and Gas Pollution Control, China University of Petroleum-Beijing, Beijing 102249, China
3
The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China
4
Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi, Jilin Agricultural University, Changchun, Jilin Province 130118, China
5
Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan
Introduction Leaf rusts, caused by species belonging to the genus Melampsora Castagne (Melampsoraceae, Pucciniales), are some of the most widespread and frequent diseases affecting short rotation coppice (SRC) willow (Royle and Hubbes 1992; Pei 2005). To date, at least 90 species of Melampsora have been described after this genus was established with M. euphorbiae (Ficinus & C. Schub.) Castagne on Euphorbia exigua L. as type species (Castagne 1843; Cummins and Hiratsuka 2003). Among them, about 50 species have been reported to cause diseases on willows in temperate and some tropic regions, including Asia, Europe, North America, Australasia and New Zealand (Wilson and Henderson 1966; Azbukina 1974; Ziller 1974; Spiers and Hopcroft 1996; Hiratsuka et al. 1992; Zhuang 2005). These Melampsora species are mainly heterecious macrocyclic fungi and are characterized by uredinia with intermixed and conspicuous paraphyses and crust-like telia with adherent one-celled teliospores (Cummins and Hiratsuka 2003; Pei 2005). Taxonomy of Melampsora species is still in a state of confusion, especially that of the rust fungi on willows. Due to the lack of comprehensive taxonomic studies, inconsistencies still remain in the descriptions of certain morphological characteristics. Although studies have been conducted in Europe, North America and Japan for many years, the taxonomic criteria for species delimitation are varied between taxonomists (Wilson and Henderson 1966; Ziller 1974; Hiratsuka and Kaneko 1982). In Japan, the earliest report on Melampsora species on willows was started in 1902 by Dietel and many species have been reported since then (Matsumoto 1915; Ito 1938). In 1982 a taxonomic revision of Melampsora species on willows in Japan was conducted by Hiratsuka and Kaneko (1982), and a total of 13 Melampsora species were recognized in their study. Thereafter, Hiratsuka et al. (1992) summarized the rust
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flora in Japan and confirmed the existence of these 13 Melampsora species on willows in Japan. During our taxonomic studies of Melampsora species on willows, one new species was recognized on S. reinii Franch. Table 1
& Sav. ex Seemen. In addition, two new Japanese records were also discovered. One was recognized on S. pet-susu Kimura and S. sachalinensis F. Schmidt, and another one was found on S. purpurea L. The new species and two new
Sequenced specimens of Melampsora and their GenBank accession numbers of rDNA ITS regions obtained from this study
Host plants
Specimen noa.
Localityb
GenBank accession no. of rDNA ITS regions
Species
Salix futura
TSH-R9618
Japan, Niigata
KF780794
M. epitea
S. gilgiana
TSH-R9831
Japan, Nagano
KC631841
M. epitea
TSH-R7492
Japan, Nagano
KF780768
M. epitea
S. japonica
TSH-R3885
Japan, Shizuoka
KF780780
M. epitea
S. koriyanagi
HH-53278 (isotype)
Japan, Tokyo
ND
M. humilis
TSH-R7550
Japan, Miyagi
KF780812
M. humilis
TSH-R7613
Japan, Miyagi
KF780811
M. humilis
S. miyabeana
TSH-R7731
Japan, Hokkaido
KF780828
M. epitea
TSH-R7681
Japan, Hokkaido
KF780804
M. epitea
S. pet-susu
TSH-R7643
Japan, Hokkaido
KC631861
M. ribesii-viminalis
TSH-R7661
Japan, Hokkaido
KC631863
M. ribesii-viminalis
S. purpurea
NWC-06843
England, Rothamsted
KF780830
M. ribesii-purpureae
TNS-F-186369
Germany
KF780831
M. ribesii-purpureae
BPI23007
Italy, Tudent
KF780839
M. ribesii-purpureae
BPI23008
Latvia
ND
M. ribesii-purpureae
BPI23009
Switzerland, Zurich
ND
M. ribesii-purpureae
TSH-R7549
Japan, Miyagi
KF780993
M. ribesii-purpureae
S. reinii
TSH-R7617
Japan, Miyagi
ND
M. ribesii-purpureae
TSH-R10306
Japan, Tochigi
KF780777
M. salicis-reinii
HH-53248
Russia, Kurile Islands
KF780840
M. salicis-reinii
TSH-R12023
Japan, Yamanashi
KF780781
M. epitea
TSH-R7487
Japan, Nagano
KF780826
M. epitea
TSH-R18314
Japan, Yamagata
ND
M. epitea
S. rorida
TSH-R7654
Japan, Hokkaido
KF780815
M. epitea
TSH-R7689
Japan, Hokkaido
KF780806
M. epitea
S.sachalinensis
TSH-R9836
Japan, Tochigi
KC631865
M. ribesii-viminalis
TSH-R9837
Japan, Nagano
KC631866
M. ribesii-viminalis
TSH-R3884
Japan, Shizuoka
KF780787
M. epiphylla
TSH-R10186
Japan, Shizuoka
KF780788
M. epiphylla
TSH-R12280
Japan, Yamagata
KF780789
M. epiphylla
HH-77578 (isotype)
Japan, Gifu
ND
M. epiphylla
HNMAS82389
China, Inner Mongolia
KF780773
M. ribesii-viminalis
HNMAP3058
China, Inner Mongolia
KF780800
M. ribesii-viminalis
HNMAP3218
China, Inner Mongolia
KF780796
M. ribesii-viminalis
HNMAP1698
China, Inner Mongolia
KF780795
M. ribesii-viminalis
S. viminalis
a
BPI: Systematic Mycology and Microbiology Laboratory, Agricultural Research Service, USDA, USA; HH: Hiratsuka Herbarium, Tokyo, Japan; HNMAP: Herbarium of the College of Forestry, Inner Mongolia Agricultural University; TSH: Mycological Herbarium of the Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan; TNS: National Museum of Nature and Science, Tsukuba, Japan
b
Locality with name of country followed by the name of provinces or prefectures
c
ND: Not analyzed
Mycol Progress (2015) 14: 101
records are described, based on morphological characteristics, and the phylogenetic positions of these Melampsora taxa were inferred from rDNA ITS region. In terms of morphological differences and phylogenetic results, the novel species and new Japanese records are described and illustrated in this paper.
Materials and methods Specimens Dried herbarium specimens for this study were loaned from the Mycological Herbarium of the Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan (TSH), Hiratsuka Herbarium, Tokyo, Japan (HH), the National Museum of Nature and Science, Tsukuba, Japan (TNS), the Systematic Mycology, Microbiology Laboratory, Agricultural Research Service, USDA, USA (BPI) and the Herbarium of the College of Forestry, Inner Mongolia Agricultural University, China (HNMAP). Several specimens were kindly supplied by Dr. Ming-Hao Pei from Rothamsted Research, Harpenden, Hertfordshire, UK. Voucher number, host species and geographic origins of these loaned herbarium specimens are listed in Table 1.
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Biosystems). The sequence data were manually aligned using BioEdit ver. 7.0.9 (Hall 1999). To make molecular comparison, a total of 79 sequences were retrieved from GenBank based on host species and localities (Appendix 1). Those sequences were deposited mainly by Smith et al. (2004), Pei et al. (2005), Feau et al. (2009), Yamaoka et al. (2010), Damadi et al. (2011), Samils et al. (2011), Milne et al. (2012), Zhao et al. (2014) and Zhao et al. (2015) during their phylogenetic studies of Melampsora species on willows. Multiple alignments were performed using Clustal X ver. 1.8 (Thompson et al. 1997). The final data set contained sequences from 107 specimens with a length of 567 bp, which included 127 parsimony-informative characters. Phylogenetic trees were constructed with two sequences of M. laricis-populina Kleb. as the outgroups. Topologies were constructed based on maximum likelihood (ML) analyses using Garli v. 0.95 (Zwickl 2006). Bayesian Markov chain Monte Carlo (MCMC) analyses were performed using MrBayes ver. 3.1.2 (Huelsenbeck and Ronquist 2001). In ML and Bayesian analyses, the best-fit substitution models were estimated using Modeltest ver. 3.7 (Posada and Crandall 1998), and K81uf+I+G was selected as the best evolutionary model.
Results Morphological observations Morphological characteristics Morphological characteristics in uredinial and telial stages were examined under the dissecting microscope (DM), the light microscope (LM) and the scanning electron microscope (SEM). The detailed methods for morphological analysis as outlined by Zhao et al. (2013), (2014) and (2015) were followed. Fifty measurements of sori and spores from each specimen were recorded, and the measurements obtained from all specimens were compared with the original descriptions, and/or other published descriptions of species involved (e.g. Thümen 1879; Sydow and Sydow 1915; Ito 1938; Kuprevich and Tranzschel 1957; Wilson and Henderson 1966; Azbukina 1974; Ziller 1974; Hiratsuka and Kaneko 1982; Bagyanarayana 2005; Pei 2005). Phylogenetic analysis For each specimen, genomic DNA was extracted from a single uredinium followed by the method from Virtudazo et al. (2001). The full length of the ITS region (complete ITS1, 5.8S rRNA gene and ITS2) was amplified with primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990). DNA was amplified using a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA), following Tian et al. (2004). After amplification, PCR products were sequenced on a 3130 Automated DNA Sequencer (PE Applied
Rust specimens on Salix reinii During our morphological examination of specimens on willows, we found unique morphological characteristics among two specimens on S. reinii (TSH-R10306 and HH53248) from Japan. The rust fungus on these specimens had hypophyllous uredinia (Fig. 1a), and its uredinia had capitate, peripheral and intermixed paraphyses (Fig. 1c, e). The urediniospores were mainly ellipsoid and had scattered germ pores (Fig. 1d, g). There were echinulate spines on the surface of urediniopsores, which were found as rugose based on ultrastructural observation (Fig. 1f, h). The telia were amphigenous, and the subepidermal teliospores did not have apparently thickened apical walls (Fig. 1i). According to Hiratsuka and Kaneko (1982), M. epitea Thüm. and its synonym, M. arctica Rostr., were reported on S. reinii. Thus, we included Japanese specimens on S. reinii (TSH-R12023, TSH-R18314 and TSH-R7487), on S. futura Seemen (TSHR9618), on S. gilgiana Seemen (TSH-R9831 and TSHR7492), on S. japonica Thunb. (TSH-R3885) and on S. miyabeana Seemen (TSH-R7731 and TSH-R7681) for comparative analysis. Specimens on these five willow species showed similar morphology in uredinial and telial stages, and their morphology fit well with the description of M. epitea
101 Page 4 of 13 Fig. 1 Morphology of Melampsora salicis-reinii (TSHR10306). a Uredinia (U) and telia (T) on the hypophyllous surface. b Telia (T) on the epiphyllous surface. c Uredinia observed by SEM, abundant peripheral paraphyses. d Ellipsoid urediniospores observed by LM. e Capitate paraphyses with evenly thickened apex. f Echinulate spines on rugose surface of urediniospores. G: Scattered germ pores (black arrows). g Urediniospores with echinulate spines observed by SEM. h Subepidermal teliospores with thickened apical wall. Bars: a, b 20 mm; c 50 μm; d, e 20 μm; f 1 μm; g 10 μm; h 15 μm; i 30 μm
Mycol Progress (2015) 14: 101
A
B
U T T
C
D
E
F G
H
according to Hiratsuka and Kaneko (1982). They had hypophyllous uredinia and telia, and echinulate spines on the globoid urediniospores with smooth surface, which were clearly different from TSH-R10306 and HH-53248. Morphological comparison of all these specimens indicated that the wall thickness from those specimens (especially these on S. reinii) ranged from 1 to 4 μm, and no clear differences were recognized among specimens. Besides M. epitea, we also compared TSH-R10306 and HH-53248 with morphologically similar species, such as M. arctica, M. epitea from Japan, M. humilis, M. iranica, M. ribesii-purpureae and M. salicispurpureae. These two specimens clearly differed from the Melampsora species in the shape of urediniospores, position
I
of uredinia and telia and surface structure of urediniospores (Table 2). Rust specimens on Salix pet-susu and S. sachalinensis Specimens on S. pet-susu (TSH-R7643 and TSH-R7661) and S. sachalinensis (TSH-R9836 and TSH-R9837) from Japan had hypophyllous uredinia (Fig. 2a) and globoid or ellipsoid urediniospores with echinulate spines on the surface (Fig. 2d, f). Their urediniospores had scattered germ pores and they were intermixed with capitate paraphyses in the uredinia (Fig. 2c, e, g, h). Telia were epiphyllous (Fig. 2b), and contained subepidermal or subcuticular teliospores with
Subepidermal
ND
slightly thickened apex up to 4.57 μm (Fig. 2i). These specimens resembled M. epiphylla Dietel and M. ribesiiviminalis Kleb. in their uredinial and telial morphology according to Wilson and Henderson (1966) and Hiratsuka and Kaneko (1982). We selected several specimens of M. epiphylla Dietel on S. sachalinensis (TSH-R3884, TSH-R10186, TSH-R12280 and HH-77578) and M. ribesii-viminalis Kleb. on S. viminalis L. from China for comparison. TSH-R3884, TSH-R10186, TSH-R12280 and HH-77578 (isotype) from Japan fitted well morphologically with the description of M. epipphylla because of subepidermal or subcuticular teliospores with slightly thickened apex. However, detailed morphological comparison indicated that HH-77578 (isotype) and the other three specimens on S. sachalinensis owned amphigenous telia, which clearly differed from TSH-R7643, TSHR7661, TSH-R9836 and TSH-R9837. When comparing the specimens of M. ribesii-viminalis on S. viminalis from China, TSH-R7643, TSH-R7661, TSH-R9836 and TSHR9837 showed no clear morphological differences in uredinial and telial stages, except slight variations in the apex thickness of teliospores. According to Hiratsuka and K a n e k o (1 9 8 2 ) , Z h ao e t a l ( 2 0 1 4 ) a n d ( 2 01 5 ) , M. salicis-argyraceae P. Zhao, C. M. Tian, Y. J. Yao & M. Kakishima, M. capraearum Thüm. and M. salicissinicae P. Zhao, C. M. Tian & Y. J. Yao also had slightly thickened apex in teliospores. However, our detailed comparison showed that TSH-R7643, TSH-R7661, TSHR9836 and TSH-R9837 were clearly distinguishable from these three species in the telia position, the teliospores position, dimension and apex thickness (Table 3).
ND ND
Hypophyllous
20–40×5–11 Subepidermal Smooth 1.55–1.63
Amphigenous
16–32×5–16 Subepidermal Smooth 1.28–1.33
Hypophyllous
16–31×4–11 Subepidermal Smooth 1.05–1.15
Amphigenous
17–45×5–14 Subepidermal Smooth 1.05–1.44
Amphigenous
Smooth 0.90–1.10
Hypophyllous
Subepidermal
18–46×6–12
Evenly thickened at apex and sides, wall 0.8–2.8 μm Evenly thickened at apex and sides, wall 0.8–2.5 μm Evenly thickened at apex and sides, wall 0.8–1.7 μm Evenly thickened at apex and sides, wall 0.7–1.8 μm Evenly thickened at apex and sides, wall 1–2.3 μm Evenly thickened at apex and sides, wall 0.6–1.5 μm ND 21–37×5–11 Subepidermal Rugose 0.90–1.18
Amphigenous
Surface structure of urediniospores
Japanese specimens on S. purpurea (TSH-R7549, TSHR7617) had amphigenous uredinia (Fig. 3a), and capitate paraphyses were intermixed with urediniospores in the uredinia (Fig. 3c, e). The urediniospores were globoid to ellipsoid with scattered germ pores (Fig. 3d, g), and evenly echinulate spines on the smooth surface (Fig. 3f, h). The telia were amphigenous (Fig. 3b), and the subepidermal teliospores did not have thickened apical walls (Fig. 3h). Specimens on the same host from Europe were examined and they had a similar morphology as TSH-R7549 and TSH-R7617. Besides, these specimens clearly differed from M. salicis-purpureae on S. purpurea from China in their dimension of teliospores according to Zhao et al. (2015) (Table 2).
ND Globoid or ellipsoid Hypophyllous
12-22×11–19 Globoid or ellipsoid Amphigenous
M. iranica (Zhao et al. 2015) M. arctica (Rostrup 1888)
12-27×10–23 Globoid or ellipsoid Hypophyllous M. humilis
16–21×14–19 Globoid or ellipsoid Amphigenous M. salicis-purpureae
15–26×10–20 Globoid or ellipsoid Amphigenous M. ribesii-purpureae
12–25×9–24 Globoid or ellipsoid Hypophyllous M. epitea from Japan
Ellipsoid Hypophyllous M. salicis-reinii
17–24×10–21
Rust specimens on Salix purpurea
ND: No data
Position
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Distance between spines (μm) Dimension of urediniospores (μm) Shape of urediniospores Position
Uredinia Species
Table 2
Morphological characteristics of Melampsora salicis-reinii and M. ribesii-purpureae with related species
Telia
Position of teliospores
Dimension of teliospores (μm)
Apex of teliospores
Mycol Progress (2015) 14: 101
Molecular phylogeny Molecular phylogenetic results of rDNA ITS regions based on MP and Bayesian inference resulted in consistent topologies
101 Page 6 of 13 Fig. 2 Morphology of M. ribesiiviminalis (TSH-R7643). a Uredinia (U) on the hypophyllous surface. b Telia (T) on the epiphyllous surface. c Uredinia observed by SEM, abundant paraphyses intermixed with urediniospores. d Globoid or ellipsoid urediniospores observed by LM. e Scattered germ pores (black arrows). f Capitate paraphyses with evenly thickened apex. g Urediniospores with echinulate spines observed by SEM. h Subepidermal teliospores with thickened apical wall. Bars: a, b 10 mm; c 50 μm; d, g, h 10 μm; e, i 30 μm; f 1 μm
Mycol Progress (2015) 14: 101
A
B
T
U
C
H
(Fig. 4). The topology revealed that two specimens on S. reinii (TSH-R10306 and HH-53248) located in a distinct lineage, which was phylogenetically distinct from specimens of other Melampsora species based on the sequence data retrieved from GenBank. TSH-R10306 and HH-53248 are also genetically distant from M. epitea on S. reinii, which were located in a group together with specimens on S. futura, S. gilgiana and S. japonica. The rust fungus on TSH-R10306 and HH53248 was a distinct species, and it is proposed as a new species. Several specimens on S. pet-susu (TSH-R7643 and TSHR7661) and S. sachalinensis (TSH-R9836 and TSH-R9837) were placed in a phylogenetic group with a sequence data from S. viminalis L. (AY652947.1) and several specimens of M. ribesii-viminalis on S. viminalis from China in the ITS phylogeny. However, this phylogenetic group was genetically distinct from other Melampsora species including M. epiphylla
D
E
F
G
I
on TSH-R3884, TSH-R10186 and TSH-R12280, M. salicisargyraceae, M. capraearum and M. salicis-sinicae. Thus, both morphological differences and molecular phylogenetic results revealed the rust fungus from TSH-R7643, TSHR7661, TSH-R9836 and TSH-R9837 as M. ribesii-viminalis, which is a new record for Japan. Japanese specimen on S. purpurea (TSH-R7549) was located in a phylogenetic group together with the sequence data of M. ribesii-purpureae Kleb. on S. purpurea (AY444770.1 and GQ479275.1). Thus, several specimens of M. ribesiipurpureae on S. purpurea (NWC-06843, TNS-F-186369 and BPI23007) from Europe were used for comparison. All these specimens formed a well-supported phylogenetic group together with TSH-R7549, and they were distinct from other Melampsora species on willows. Thus, the rust fungus on S. purpurea from Japan was recognized as M. ribesiipurpureae, which is a new record for Japan.
1.0–2 μm thick at sides, thickened at apex, up to 10.1 μm. 17–49×6–15 Amphigenous Subcuticular Smooth Hypophyllous Globoid, ellipsoid M. salicis-sinicae (Zhao et al. 2014)
14–25×11–22 1.00–1.10
1.5–2.5 μm thick at sides, thickened at apex, up to 8.5 μm. 16–37×6–14 Subcuticular Epiphyllous Smooth 13–27×10–25 0.81–0.89 Hypophyllous Globoid, ellipsoid M. capraearum
Epiphyllous Smooth 12–23×7–19
1.41–1.55
Smooth 13–19×10–15 1.11–1.16
Ampigenous
Smooth 10–20×9–19
1.02–1.26
Epiphyllous
Subepidermal or 14–45×5–13 subcuticular Subepidermal or 13–40×5–17 subcuticular Subepidermal 16–56×4–20
1.0–2.85 μm thick at sides, thickened at apex, up to 4.57 μm 1.0–2 μm thick at sides, thickened at apex, up to 5.1 μm 0.8–1.6 μm thick at sides, thickened at apex, up to 4.33 μm
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Hypophyllous Globoid, ellipsoid M. epiphylla Hypophyllous Globoid, ellipsoid M. salicis-argyraceae Hypophyllous Globoid or (Zhao et al. 2015) ellipsoid M. ribesii-viminalis
Surface structure Position Shape of Dimension of Distance urediniospores urediniospores between spines of urediniospores (μm) (μm) Position
Uredinia Species
Table 3
Morphological characteristics of Melampsora ribesii-viminalis and related species
Telia
Position of teliospores
Dimension of Apex of teliospores teliospores (μm)
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Taxonomy Melampsora salicis-reinii P. Zhao & M. Kakishima, sp. nov. Fig. 1 Mycobank no.: 814415 This species is characterized by ellipsoid urediniospores possessing echinulate spines on a rugose surface. This species had hypophyllous uredinia and amphigenous telia. It differed from M. epitea in its morphology of spines, shape of urediniospores and position of telia. Holotype: II, III on S. reinii, Japan, Tochigi prefecture, Nikko, Konsei Pass, 11 October 2000, Y. Yamaoka (TSHR10306). Sequence ex-holotype: KF780777 (ITS region), KF780660 (28S). Etymology: Named after the host plant of the type specimen. Spermogonia and aecia not found. Uredinia hypophyllous, scattered, 0.4–1 mm; urediniospores mainly ellipsoid, 17– 24×10–21 μm, wall 1.8–3.3 μm thick, echinulate spines on rugose urediniospores, the distance between spines 0.90– 1.18 μm, germ pores 4–7, scattered; paraphyses capitate, intermixed with urediniospores, 29–63×14–29 μm, with thickened membrane up to 7.2 μm; telia amphigenous, 0.4– 0.8 mm, scattered or aggregated, subepidermal; teliospores 21–37×5–11 μm, wall 0.8–2.8 μm, evenly thickened without thickened apical wall. Additional specimens examined in this study: II, III on S. reinii, Japan, Kuriles, Urup Isl., Russia, 9 September 1927, M. Tatewaki (HH-53248). Host and geographic distribution: II, III on S. reinii – Japan and Russia. Melampsora ribesii-viminalis Kleb., Pringsheims Jb. Wissenschaftl. Botanik 34: 363 (1900) Fig. 2 Spermogonia and aecia not found in Japan. Uredinia hypophyllous, scattered, rounded, 0.1–0.8 mm; urediniospores globoid, ellipsoid and ovoid, 10–20 × 9– 19 μm, wall 0.7–4.3 μm, evenly echinulate, the distance between spines 1.02–1.26 μm; germ pore scattered, 2–6; paraphyses capitate, intermixed with urediniospores, wall evenly thickened at apex; telia epiphyllous, 0.1–0.8 mm, scattered or grouped; teliospores subepidermal or subcuticular, 14– 45×5–13 μm, wall slightly thickened at apex, up to 4.57 μm. Specimens examined: II, III on S. pet-susu, Japan, Hokkaido prefecture, Sapporo, 11 September 995, H. Nakamura (TSH-R7643; TSH-R7661). II, III on S. sachalinensis, Japan, Tochigi prefecture, Nikko, Konsei Pass, 20 September 1995, H. Nakamura (TSH-R9836); Japan, Nagano prefecture, Kawakami-mura, Kawakami Forest, 12 August 1995, H. Nakamura (TSH-R9837). Host and geographic distribution: 0, I on Ribes alpinum L. – Denmark, Norway and Sweden (Sydow and Sydow 1915). 0, I on R. grossularia L. – United Kingdom (Wilson and
101 Page 8 of 13 Fig. 3 Morphology of M. ribesiipurpureae (TSH-R7549). a Uredinia (U) on the hypophyllous surface. b Telia (T) on the epiphyllous surface. c Uredinia observed by SEM, abundant paraphyses intermixed with urediniospores. d Globoid or ellipsoid urediniospores observed by LM. e Scattered germ pores (black arrows). f Capitate paraphyses with evenly thickened apex. g Urediniospores with echinulate spines observed by SEM. h Subepidermal teliospores with thickened apical wall. Bars: a, b 20 mm; c 60 μm; d, g 10 μm; e 30 μm; f 1 μm; h 20 μm; i 40 μm
Mycol Progress (2015) 14: 101
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U T T C
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G
H I
Henderson 1966). 0, I on R. nigrum L. – Denmark, Norway, Russia, Sweden (Sydow and Sydow 1915), United Kingdom (Wilson and Henderson 1966). 0, I on R. reclinatum L. – USSR (Azbukina 1974). 0, I on R. rubrum L. – Denmark, Norway, Sweden (Sydow and Sydow 1915), United Kingdom (Wilson and Henderson 1966), USSR (Azbukina 1974). 0, I on R. uva-crispa L. – Denmark, Norway, Sweden (Sydow and Sydow 1915). II, III on S. cinerea L. – Finland (Liro 1908). II, III on S. viminalis – China (Zhuang 2005), Denmark, Germany (Klebahn 1914), Italy, Norway, Poland, Romania, Scotland, Spain, Sweden (Sydow and Sydow 1915), United Kingdom (Wilson and Henderson 1966), USSR (Azbukina 1974). II, III on S. pet-susu – Japan. II, III on S. sachalinensis – Japan. Melampsora ribesii-purpureae Kleb., Pringsheims Jb. Wissenschaftl. Botanik 35: 667 (1901) Fig. 3 Spermogonia and aecia not found in Japan. Uredinia amphigenous, scattered, 0.2–1.4 mm; urediniospores globoid
or ellipsoid , 15–26×10–20 μm, wall 1.2–3.4 μm thick, echinulate, the distance between spines 1.05–1.44 μm, germ pores 3–7, scattered; paraphyses capitate, intermixed with urediniospores, 28–102×10–32 μm, with uniformly thickened membrane, up to 8.4 μm; telia amphigenous, 0.2–1.2 mm, scattered or aggregated, subepidermal; teliospores 17–45×5– 14 μm, wall 0.8–1.7 μm, not thickened at apical wall. Specimens examined: II, III on S. purpurea, Japan, Miyagi Prefecture, Sendai, Botanical Garden of Tohoku University, 08 September 2005, Y. Yamaoka (TSH-R7549, TSH-R7617). Host and geographic distribution: 0, I on R. alpinum – Finland, Norway, Poland, Romania, Sweden (Sydow and Sydow 1915), United Kingdom (Wilson and Henderson 1966). 0, I on R. aureum Pursh – Romania, USA (Ziller 1974). 0, I on R. diacanthum Pall – China (Zhuang 2005), Canada (Ziller 1974). 0, I on R. grossularia – United Kingdom (Klebahn 1914). 0, I on R. inerme Rydb. – USA (Ziller
Mycol Progress (2015) 14: 101
Page 9 of 13 101 M. epitea [S. arctica] AY471627.1
Fig. 4 Bayesian 50 % majority-rule consensus tree based on the internal transcribed spacer (ITS1 and ITS2) regions, including 5.8S of ribosomal RNA gene. Melampsora laricis-populina was used as outgroup. Values on the branches indicate maximum likelihood bootstrap values / Bayesian posterior probabilities. Asterisk indicates that bootstrap values were less than 75 % and Bayesian posterior probabilities were less than 0.75
M. paradoxa GQ479271.1 M. epitea [S. arctica] AY471628.1 78/94 M. epitea [S. arctica] AY471629.1 M. epitea [S. arctica] AY471630.1 M. arctica GQ479204.1 M. epitea [S. bebbiana] AY471646.1 M. epitea [S. bebbiana] AY471645.1 */98
M. epitea [S. bebbiana] AY471647.1 M. epitea [S. arctica] AY471635.1 M. epitea [S. arctica] AY471634.1 M. epitea [S. arctica] AY471633.1 M. abietis-caprearum GQ479202.1
1974). 0, I on R. lacustre Poir. – Canada (Ziller 1974). 0, I on R. leptanthum A. Gray – USA (Ziller 1974). 0, I on R. nigrum – Finland, Norway. 0, I on R. rubrum – Finland, Norway, Sweden (Wilson and Henderson 1966). 0, I on R. spicatum Schult. – Finland, Norway, Sweden. 0, I on R. triste Pall. – USA (Ziller 1974). 0, I on R. uva-crispa – Norway, Sweden. 0, I on R. wolfii Rothr. – USA (Ziller 1974). II, III on S. purpurea – China (Zhuang 2005), Germany (Klebahn 1914), Poland, Romania, Switzerland, United Kingdom (Wilson and Henderson 1966) and Japan.
M. salicis-sinicae [S. sinica] KC631845.1 M. salicis-sinicae [S. sinica] KC631839.1 M. epiphylla [S. sachalinensis] TSH-R3884
75/96
M. epiphylla [S. sachalinensis] TSH-R10186 M. epiphylla [S. sachalinensis] TSH-R12280 M. epitea [S. gilgiana] TSH-R9831 M. epitea [S. gilgiana] TSH-R7492 M. epitea [S. japonica] TSH-R3885 M. epitea [S. miyabeaba] TSH-R7681 M. epitea [S. miyabeaba] TSH-R7731 */91
M. epitea [S. futura] TSH-R9618 M. epitea [S. reinii] TSH-R7487 M. epitea [S. reinii] TSH-R12023 M. capraearum [S. bakko] KC631851.1
80/95
M. capraearum [S. bakko] KC631852.1 M. capraearum [S. bakko] KC631853.1 M. capraearum [S. bakko] KC631855.1 M. humilis [S. koriyanagi] TSH-R7613
92/96
M. humilis [S. koriyanagi] TSH-R7550 M. epitea [S. rorida] TSH-R7654
*/86
M. capraearum GQ479208.1 Melampsora sp. [S. lanata] JN646173.1 95/100 Melampsora sp. [S. lanata] JN646174.1
*/99
M. capraearum GQ479209.1 79/96 M. bigelowii GQ479206.1 M. capraearum GQ479207.1 M. epitea [S. viminalis] AY444778.2
Discussion
M. epitea [S. acutifolia] AY444777.2 M. laricis-epitea [S. viminalis] JF825970.1 M. laricis-epitea [S. viminalis] JF825969.1
Based on morphological observations and molecular phylogenetic studies of Japanese specimens, we recognized a new species, M. salicis-reinii on S. reinii. Although two species, M. epitea and its synonym, M. arctica, were previously reported on S. reinii in Japan (Hiratsuka and Kaneko 1982), our newly recognized species on S. reinii is morphologically distinguishable from M. epitea in its position of telia, shape of urediniospores and rugose surface on urediniospores. We compared the morphology of M. epitea on S. reinii, S. futura, S. gilgiana, S. japonica and S. miyabeana from Japan and found that it clearly differed from this new species. Besides, the urediniospores of this new species had a rugose surface, which have never been reported in Melampsora epitea and its related species (Spiers and Hopcroft 1996; Smith et al. 2004; Zhao et al. 2013, 2014, 2015). Molecular data also supported that this new species was distant from M. epitea and other Melampsora species from China, Japan, Europe and North America, which have a smooth spore matrix on the urediniospores. This morphological characteristic was effective to differentiate this new species from M. epitea and related species on willows. According to Hiratsuka and Kaneko (1982), M. arctica was occasionally reported on S. reinii in Japan, and this species was distinguishable from M. epitea in its relatively thickened urediniospore walls (Ito 1938; Hiratsuka and Kaneko 1982). Our observation showed that the wall thickness of most specimens on S. reinii from two distinct phylogenetic groups showed no clear difference. Similar morphological variations were recognized among specimens of M. epitea from China, Japan and North America (Zhao et al. 2014, 2015). Thus, the urediniospore wall thickness seemed to be unsuitable to differentiate this species. Melampsora arctica was first reported
M. laricis-epitea [S. viminalis] JF825968.1 84/94
Melampsora sp. [S. aurita] JN646169.1 Melampsora sp. [S. myrsinites] JN646193.1
*/79
Melampsora sp. [S. myrsinites] JN646199.1 Melampsora sp. [S. lapponum] JN646205.1 Melampsora sp. [S. lapponum] JN646206.1 Melampsora sp. [S. myrsinifolia] JN646233.1 M. epitea [S. burjatica] KF164455.1 M. epitea [S. stipularis] KF164456.1 M. epitea [S. viminalis] KF164457.1 M. ribesii-viminalis [S. sachalinensis] TSH-R9836 M. ribesii-viminalis [S. sachalinensis] TSH-R9837 M. ribesii-viminalis [S. pet-susu] TSH-R7643 M. ribesii-viminalis [S. viminalis] HNMAP1698 M. epiphylla [S. viminalis] AY652947.1 M. ribesii-viminalis [S. viminalis] HNMAP3058 M. ribesii-viminalis [S. viminalis] HNMAP3218
*/76
M. capraearum [S. caprea] AY444779.2 */100 M. paradoxa GQ479273.1 M. paradoxa GQ479272.1 M. epitea [S. nigra] AY471643.1 M. ribesii-purpureae GQ479900.1 M. epitea [S. nigra] AY471642.1 M. epitea [S. nigra] AY471641.1 92/95 M. epitea [S. nigra] AY471640.1 M. paradoxa GQ479269.1
95/100
*/77 93/100 78/100 */87
M. paradoxa GQ479270.1 M. ribesii-purpureae GQ479274.1 M. arctica GQ479203.1
M. bigelowii GQ479205.1 M. laricis-pentandrae AY444771.2
*/95 100/100
Melampsora sp. [S. herbacea] JN646119.1 Melampsora sp. [S. herbacea] JN646120.1 Melampsora sp. [S. herbacea] JN646121.1
75/97 M. coleosporioides [Salix sp.] AY652949.1 85/100
M. amygdalinae [S. triandra] AY444776.2 M. coleosporioides [Salix sp.] AY652948.1
95/97 Melampsora sp. [S. arbuscula] JN646136.1 86/95 Melampsora sp. [S. arbuscula] JN646138.1 Melampsora sp. [S. reticulata] JN646153.1 Melampsora sp. [S. reticulata] JN646154.1 M. chelidonii-pierotii [S. pierotii] AB646769.1 M. salicis-albae [S. alba] AY444775.2 M. salicis-albae [S. alba] FJ455127.1 100/100 M. salicis-albae [S. alba] KF780757.1 M. ribesii-purpureae [S. purpurea] NWC-06843 M. ribesii-purpureae [S. purpurea] TNS-F-186369 M. ribesii-purpureae [S. purpurea] TSH-R7549 100/100M. ribesii-purpureae [S. purpurea] AY444770.2 M. ribesii-purpureae GQ479275.1 M. yezoensis AB646768.1 */80
M. salicis-argyraceae [S. argyracea] KF780803.1 100/100 M. salicis-argyraceae [S. argyracea] KF780838.1
99/100
M. iranica [S. elbursensis] FJ386432.1
*/90
100/100
M. iranica [S. alba] KF780741.1 M. salicis-purpureae [S. pupurea] KF780766.1 [S. reinii] i TSH-R10306 100/100 M. salicis-reiniii [S.
100/100
M. salicis-reiniii [S. [S. reinii] i HH-53248 M. epitea [S. interior] AY471639.1 100/100 M. epitea [S. interior] AY471638.1 M. epitea [S. interior] AY471637.1 M. epitea [S. interior] AY471636.1
Melampsora laricis-populina TSH-R16979 AB116839.1 M. laricis-populina TSH-R16981 AB116841.1 10.0
101 Page 10 of 13
Mycol Progress (2015) 14: 101
by Rostrup (1888), and it was described with hypophyllous uredinia, globoid urediniospores and hypophyllous telia on S. glauca L., S. groenlandica Lundstr. and S. herbacea Schrenk in Europe based on the original description. Thus, it is clearly differentiated from our new species in the shape of urediniospores and the position of telia. Meanwhile, a rust fungus on S. herbacea L. (JN646119.1, JN646120.1 and JN646121.1) from Europe was clearly distinct from our new species. All this morphological and molecular evidence suggests that M. salicis-reinii on S. reinii in Japan is a new species. Previously, the rust fungus on S. pet-susu and S. sachalinensis was recognized as M. epiphylla because the teliospores on these two willow species had slightly thickened apices (Hiratsuka et al. 1992). In this study, we recognized specimens on S. pet-susu and S. sachalinensis as having a slightly thickened apex in the teliospores, but we also recognized morphological differences among specimens. Some specimens on S. sachalinensis had amphigenous telia, but some specimens on S. sachalinensis and S. pet-susu had epiphyllous telia. Molecular phylogeny revealed that specimens on S. sachalinensis with amphigenous telia were distinct from other Melampsora species, especially those on S. sachalinensis with epiphyllous telia. Although we failed to extract DNA from the type specimen of M. epiphylla (HH-77578), detailed morphological examination showed the type specimen had amphigenous telia. Thus, we could confirm specimens on S. sachalinensis with amphigenous telia as M. epiphylla. Specimens on S. sachalinensis and S. pet-
susu showed similar morphology with M. ribesii-viminalis on S. viminalis. Besides, they were phylogenetically grouped together; thus, the rust fungus on S. sachalinensis and S. petsusu from Japan was recognized as M. ribesii-viminalis. This species was reported in Japan for the first time, and host alternation is still unknown, although Ribes species have already been shown to be alternate hosts in Europe (Klebahn 1914; Wilson and Henderson 1966; Pei et al. 1993). A rust fungus on S. purpurea from Europe was recognized as M. ribesii-purpureae, which has its aecial host range on Ribes species (Klebahn 1914). The identity and host alteration of this species was further proved by Pei et al. (1993), and the monophyly of this species was also supported in several phylogenetic studies (Pei et al. 2005; Bennett et al. 2011; Milne et al. 2012; Zhao et al. 2015). In this study, we recognized that Japanese specimens on S. purpurea had similar morphology and sequence data, also supporting that they were M. ribesiipurpureae. Thus, we confirmed a Japanese rust fungus on S. purpurea as M. ribesii-purpureae, and it was reported on willows in Japan for the first time. Acknowledgments We express our gratitude to Dr. Ming-Hao Pei (Rothamsted Research, Harpenden, Hertfordshire, UK), Dr. Zhi-Min Cao (Forestry College, Northwest A & F University, Yangling, China) and Dr. Tsuyoshi Hosoya (National Museum of Nature and Science, Tsukuba, Japan) for providing dried specimens for this study. Compliance with ethical standards Disclosure The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.
Appendix Table 4
Details of reference sequences of Melampsora species on willows that were retrieved from GenBank database
Species
Host plants*
GenBank accession no. of rDNA ITS regions
Reference
M. amygdalinae
Salix triandra
AY444776.2
Pei et al. (2005)
M. abietis-caprearum M. arctica M. arctica M. bigelowii M. capraearum
— — — — S. caprea
GQ479202.1 GQ479203.1 GQ479204.1 GQ479206.1 AY444779.2
Vialle (unpublished) Vialle (unpublished) Vialle (unpublished) Vialle (unpublished) Pei et al. (2005)
M. capraearum M. capraearum M. capraearum M. capraearum
— — — S. bakko
GQ479207.1 GQ479208.1 GQ479209.1 KC631851.1
Vialle (unpublished) Vialle (unpublished) Vialle (unpublished) Zhao et al. (2015)
M. capraearum
S. bakko
KC631852.1
Zhao et al. (2015)
M. capraearum
S. bakko
KC631853.1
Zhao et al. (2015)
M. capraearum
S. bakko
KC631855.1
Zhao et al. (2015)
M. coleosporioides
S. pierotii
AY652948.1
Pei et al. (2005)
Mycol Progress (2015) 14: 101
Page 11 of 13 101
Table 4 (continued) Species
Host plants*
GenBank accession no. of rDNA ITS regions
Reference
M. coleosporioides
Salix sp.
AY652949.1
Pei et al. (2005)
M. chelidonii-pierotii
S. pierotii
AB646769.1
Yamaoka et al. (2010)
M. epiphylla
S. viminalis
AY652947.1
Pei et al. (2005)
M. epitea
S. viminalis
AY444778.2
Pei et al. (2005)
M. epitea
S. viminalis
KF164457.1
Zhao et al. (2015)
M. epitea
S. acutifolia
AY444777.2
Pei et al. (2005)
M. epitea
S. arctica
AY471627.1
Smith et al. (2004)
M. epitea
S. arctica
AY471628.1
Smith et al. (2004)
M. epitea
S. arctica
AY471629.1
Smith et al. (2004)
M. epitea
S. arctica
AY471630.1
Smith et al. (2004)
M. epitea
S. arctica
AY471633.1
Smith et al. (2004)
M. epitea
S. arctica
AY471634.1
Smith et al. (2004)
M. epitea
S. arctica
AY471635.1
Smith et al. (2004)
M. epitea
S. bebbiana
AY471645.1
Smith et al. (2004)
M. epitea
S. bebbiana
AY471646.1
Smith et al. (2004)
M. epitea
S. bebbiana
AY471647.1
Smith et al. (2004)
M. epitea
S. burjatica
KF164455.1
Zhao et al. (2015)
M. epitea
S. interior
AY471636.1
Smith et al. (2004)
M. epitea
S. interior
AY471637.1
Smith et al. (2004)
M. epitea
S. interior
AY471638.1
Smith et al. (2004)
M. epitea
S. interior
AY471639.1
Smith et al. (2004)
M. epitea
S. nigra
AY471640.1
Smith et al. (2004)
M. epitea
S. nigra
AY471641.1
Smith et al. (2004)
M. epitea
S. nigra
AY471642.1
Smith et al. (2004)
M. epitea
S. nigra
AY471643.1
Smith et al. (2004)
M. epitea
S. stipularis
KF164456.1
Zhao et al. (2015)
M. iranica
S. elbursensis
FJ386432.1
Damadi et al. (2011)
M. iranica
S. alba
KF780741.1
Zhao et al. (2015)
M. laricis-epitea
S. viminalis
JF825968.1
Samils et al. (2011)
M. laricis-epitea
S. viminalis
JF825969.1
Samils et al. (2011)
M. laricis-epitea
S. viminalis
JF825970.1
Samils et al. (2011)
M. laricis-pentandrae
S. pentandra
AY444771.2
Pei et al. (2005)
M. paradoxa M. paradoxa M. paradoxa M. paradoxa M. paradoxa
— — — — —
GQ479269.1 GQ479270.1 GQ479271.1 GQ479272.1 GQ479273.1
Vialle (unpublished) Vialle (unpublished) Vialle (unpublished) Vialle (unpublished) Vialle (unpublished)
M. ribesii-purpureae
S. purpurea
AY444770.2
Pei et al. (2005)
M. ribesii-purpureae M. ribesii-purpureae M. ribesii-purpureae M. salicis-albae
— — — S. alba
GQ479275.1 GQ479274.1 GQ479900.1 AY444775.2
Vialle (unpublished) Vialle (unpublished) Vialle (unpublished) Pei et al. (2005) Eslami et al. (unpublished)
M. salicis-albae
S. alba
FJ455127.1
M. salicis-albae
S. alba
KF780757.1
Zhao et al. (2015)
M. salicis-argyraceae
S. argyracea
KF780803.1
Zhao et al. (2015)
101 Page 12 of 13
Mycol Progress (2015) 14: 101
Table 4 (continued) Species
Host plants*
GenBank accession no. of rDNA ITS regions
Reference
M. salicis-argyraceae
S. argyracea
KF780838.1
Zhao et al. (2015)
M. salicis-purpureae
S. purpurea
KF780766.1
Zhao et al. (2015)
M. salicis-sinicae
S. sinica
KC631839.1
Zhao et al. (2014)
M. salicis-sinicae
S. sinica
KC631845.1
Zhao et al. (2014)
M. yezoensis
S. jessoensis
AB646768.1
Yamaoka et al. (2010)
Melampsora sp.
S. arbuscula
JN646136.1
Milne et al. (2012)
Melampsora sp.
S. arbuscula
JN646138.1
Milne et al. (2012)
Melampsora sp.
S. aurita
JN646169.1
Milne et al. (2012)
Melampsora sp.
S. herbacea
JN646119.1
Milne et al. (2012)
Melampsora sp.
S. herbacea
JN646120.1
Milne et al. (2012)
Melampsora sp.
S. herbacea
JN646121.1
Milne et al. (2012)
Melampsora sp.
S. lanata
JN646173.1
Milne et al. (2012)
Melampsora sp.
S. lanata
JN646174.1
Milne et al. (2012)
Melampsora sp.
S. lapponum
JN646205.1
Milne et al. (2012)
Melampsora sp.
S. lapponum
JN646206.1
Milne et al. (2012)
Melampsora sp.
S. myrsinites
JN646193.1
Milne et al. (2012)
Melampsora sp.
S. myrsinites
JN646199.1
Milne et al. (2012)
Melampsora sp.
S. myrsinifolia
JN646233.1
Milne et al. (2012)
Melampsora sp.
S. reticulata
JN646253.1
Milne et al. (2012)
Melampsora sp.
S. reticulata
JN646254.1
Milne et al. (2012)
M. laricis-populina
Populus simonii
AB116839.1
Tian et al. (2004)
M. laricis-populina
Populus simonii
AB116841.1
Tian et al. (2004)
*(—): No information from GenBank
Appendix
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