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Phytotaxa 309 (1): 055–065
http://www.mapress.com/j/pt/
Copyright © 2017 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Samantha Karunarathna: 14 May 2017; published: 9 Jun. 2017
https://doi.org/10.11646/phytotaxa.309.1.5
55
Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0
A new rust species of Diaphanopellis on Rhododendron oreodoxa from Southern
China
JING CAO1, CHENG-MING TIAN1, YING-MEI LIANG2 & CHONG-JUAN YOU1*
1The Key Laboratory for Silviculture and Conservation of Ministry of Education, Beijing Forestry University, Beijing 100083, China
2Museum of Beijing Forestry University, Beijing 100083, China
*Corresponding author: chongjuanyou@bjfu.edu.cn
Abstract
A novel rust species Diaphanopellis purpurea on Rhododendron oreodoxa collected in Southern China was identified and
described. Light and scanning electron microscopy observations indicated that this rust species was morphologically distinct
from other known Diaphanopellis species and Chrysomyxa species in teliospore morphology and urediniospore surface
structure. Diaphanopellis purpurea can be phylogenetically separated from other Chrysomyxa species based on analysis of
internal transcribed spacer (ITS) partial gene sequences. The aecial stage of the new species was also confirmed.
Keywords: Molecular phylogeny, phylogeny, Pucciniales, taxonomy
Introduction
Rust genus Diaphanopellis was established by Crane with the type species Diaphanopellis forrestii P. E. Crane
occurring on Rhododendron selense Franch (Crane 2005, Kirk et al. 2008). Diaphanopellis is characterized by the
teliospores enclosed in hyaline sheaths, and the uredinia surrounded by a peridium with ornamented cells (Barclay
1891, Balfour-Browne 1955, Crane 2005). Most rusts infecting Rhododendron belong to the genus Chrysomyxa,
which are morphologically different from Diaphanopellis species in having uredinial peridium and distinct teliospores.
Chrysomyxa species produce catenulate teliospores without gelatineous layers and uredinia covered by an inconspicuous
peridium (Berndt 1999).
During an investigation of rust fungi in China, we collected a rust species on Rhododendron oreodoxa Franch.,
producing urediniospores and teliospores that were different from these of Diaphanopellis forrestii and other known
Chrysomyxa species. In this paper, we described and illustrated this rust as a new species. We also performed
phylogenetic analysis of rDNA to confirm the aecial stage of this new taxon discovered on Picea purpurea.
Materials and methods
Specimen collection and herbaria
Fresh specimens were collected in Sichuan and Yunnan Province in China and deposited at the Mycological Herbarium,
Museum of Beijing Forestry University (BJFC) in China. Herbaria were borrowed from the Mycological Herbarium,
Chinese Academy of Sciences (HMAS). Host plants, locality of collection and accession numbers for sequence data
from GenBank and Barcode of Life Database (BOLD, www.barcodinglife.org) were listed in Table 1.
Morphological characteristics observation
Spores and hand sections of telia were mounted in lactophenol or lactophenol-cotton blue solution on the microscopic
slides. For each specimen, approximately 30 spores were randomly selected and measured using a DM2500 upright
microscope (Leica, Germany). The surface macro-structures of samples were examined by using scanning electron
microscopy (SEM). Aeciospores and urediniospores were adhered onto aluminum stubs covered with double-sided
CAO ET AL.
56 • Phytotaxa 309 (1) © 2017 Magnolia Press
adhesive tape, coated with gold using the SCD-005 Sputter Coater (Hitachi, Tokyo, Japan), and then observed using a
S-3400N scanning electron microscope (Hitachi, Tokyo, Japan) operated at 10–15 kV.
DNA extraction, PCR and sequencing
Genomic DNA was extracted using the modified method of Tian et al. (2004). The internal transcribed spacer (ITS)
and 5.8S region of rDNA was amplified with primers ITS1F (5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS4
(5-TCCTCCGCTTATTGATATGC-3) (White et al. 1990, Gardes & Bruns 1993). Amplifications were performed in
25 L of PCR solution containing 1 L of DNA template, 2.5 L of sense primer (2 M), 2.5 L of antisense primer
(2 M), 12.5 L of 2×Es Taq MasterMix (Cwbio, Beijing, China), and 6.5 L of dd H2O.The PCR conditions were as
follows: 94°C for 3 min, 35 cycles of 94°C for 30 s, 50°C for 1 min, and 72°C for 1 min, and a final step of 72°C for
10 min. PCR products were purified and cloned for sequencing (TSINGKE, Beijing, China).
Phylogenetic analysis
The raw sequences obtained were aligned using ClustalX1.83 and MAFFT v.7 (Thomson et al. 1997, Katoh & Standley
2013). We compiled two datasets for phylogenetic analyses: (A) an rDNA ITS dataset that consisted of sequence data
obtained from this study, and rust species of Coleosporiaceae from BOLD (Feau et al. 2011) and GenBank (Table 1),
and (B) a rDNA ITS dataset, which included the related rust genera with Chrysomyxa, representing major lineages of
Pucciniaceae, Pucciniastraceae, Coleosporiaceae, Cronartiaceae and Melampsoraceae (Table 3). Phylogenetic trees
obtained from analyses (A) and (B) were rooted with Melampsora epitea (AY471646) and Puccinia melampodii
(EU659697) respectively. Maximum parsimony (MP) analysis was carried out using the heuristic search option with
1,000 random-addition sequences and tree bisection and reconnection as the branch-swapping algorithm implemented
in PAUP v.4.0b10 (Swofford 2002). In the MP analyses, gaps were treated as missing data, and all characters were
equally weighted. Clade stability was assessed using a bootstrap analysis with 1,000 replicates (Felsenstein 1985).
Tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC) were also calculated.
Bayesian analysis was performed with MrBayes 3.1.2 (Ronquist & Huelsenbeck 2003) using the Markov Chain
Monte Carlo (MCMC) method, the best-fit substitution models were estimated using Modeltest ver. 3.7 based on the
implementation of the Akaike information criterion (AIC) (Posada and Crandall 1998). GTR + I + G was selected as
the best evolutionary model for the rDNA ITS datasets, and the Markov chains were run for 1,000,000 generations.
The trees were sampled every 100 generations, resulting in 10,000 total trees. Sequence alignments were deposited at
TreeBase (http://www.treebase.org/) under the accession number 19266.
TABLE 1. Sequence data analyzed in this study or obtained from GenBank and BOLD. (new species in bold).
Fungal taxon Host plant Specimen no Date of
collection
Geograpphic origin GenBank or
BOLD
accession no.(ITS)
Diaphanopellis
purpurea
Picea purpurea BJFC-R02299* 2014-07-21 Sichuan,China KX225401a
Picea purpurea BJFC-R02300* 2014-07-21 Sichuan,China KX225402a
Rhododendron oreodoxa HMAS-55188* 1987-05-01 Sichuan,China KX225403a
Rhododendron oreodoxa BJFC-R01698* 2014-05-21 Sichuan,China KX225404a
Rhododendron oreodoxa BJFC-R01699* 2014-05-21 Sichuan,China KX225405a
Chrysomyxa
arctostaphyli
Picea mariana DAOM 229628 1986-06-30 Klondike Loop, Yukon,
Canada
CHITS040-08b
Arctostaphylos uva-ursi DAOM 183586 1982-06-16 Kenora district, Ontario,
Canada
CHITS053-08b
C. cassandrae Picea mariana QFB 25005 2004-09-10 Abitibi, Quebec, Canada CHITS052-08b
Chamaedaphne
calyculata
QFB 25007 2006-08-06 Le ´vis, Quebec, Canada CHITS004-08b
C. chiogenis Gaultheria hispidula QFB 25026 2007-06-22 Charlevoix, Quebec,
Canada
CHITS022-08b
Gaultheria hispidula Only DNA extraction 2007-07-25 Charlevoix, Quebec,
Canada
CHITS031-08b
C. empetri Empetrum nigrum QFB 25033 2007-08-04 Radisson, Quebec,
Canada
CHITS032-08b
Empetrum nigrum QFB 25060 2007-09-05 Charlevoix, Quebec,
Canada
CHITS033-08b
...continued on the nex page
A NEW RUST SPECIES OF DIAPHANOPELLIS Phytotaxa 309 (1) © 2017 Magnolia Press • 57
TABLE 1. (Continued)
Fungal taxon Host plant Specimen no Date of
collection
Geograpphic origin GenBank or
BOLD
accession no.(ITS)
C. ledi Ledum palustre DAOM 138900 1966-09-05 Bialowieza Forest,
Poland
CHITS056-08b
Picea abies DAOM 162213 1975-07-28 Pudasjärvi, Jonku,
Finland
CHITS059-08b
C. ledicola Ledum groenlandicum Only DNA extraction 2005-06-17 Waswanipi River,
Quebec, Canada
CHITS060-08b
Picea glauca QFB 25034 2007-08-04 Chisasibi, Quebec,
Canada
CHITS028-08b
C. monesis Moneses (= Pyrola)
uniflora
DAOM 221985 1957-06-03 Graham Island, British CHITS044-08b
Picea sitchensis DAVFP 10017 1956-09-01 Columbia, Canada CHITS107-09b
C. nagodhii Rhododendron
groenlandicum
Only DNA extraction 2007-06-21 Manicouagan, Quebec,
Canada
CHITS065-08b
Picea mariana QFB 25054 2007-07-25 Charlevoix, Quebec,
Canada
CHITS030-08b
C. neoglandulosi Ledum glandulosum DAOM 229530 1999-08-21 Okanagan, British
Columbia,
Canada
CHITS042-08b
C. piperiana Ledum macrophyllumc DAFVP 14998 1963-06-06 Hope, British Columbia,
Canada
CHITS113-09b
C. pyrolae Picea glauca QFB 25055 2006 Lac St-Jean, Quebec,
Canada
CHITS013-08b
Pyrola sp. QFB 25056 2008-05-31 Bic, Quebec, Canada CHITS066-08b
C. rhododendri Picea abies WM 1183 1999-08-22 Obere Chlusi, Bernese CHITS009-08b
Oberland, Switzerland
Rhododendron
ferrugineum
QFB 19829 1972-07-12 Simplon, Valais,
Switzerland
CHITS036-08b
Ledum lapponicum DAFVP 14606 1962-08-10 Summit Pass, British CHITS105-09b
Columbia, Canada
Ledum lapponicum DAFVP 14607 1962-07-27 Summit Pass, British CHITS106-09b
Columbia, Canada
C. vaccinii Vaccinium parvifolium DAOM 45774 1952-07-08 Graham Island, British CHITS070-08b
Columbia, Canada
Vaccinium parvifolium DAVFP 18160 1968-05-18 Victoria Island, British CHITS115-09b
Columbia, Canada
C. woroninii Ledum groenlandicum QFB 25009 2006-06-26 Charlevoix, Quebec,
Canada
CHITS006-08b
Picea abies DAOM 230441 1996-07-16 Sodankylä, Ruosselkä,
Finland
CHITS072-08b
Coleosporium
campanulae
Campanula sp. HMBF-41501 — China KP017555a
Coleosporium
phellodendri
Phellodendron amurense HMBF-12 — China KP017556a
Coleosporium
phellodendri
Phellodendron chinense BJFC-R00700 2006-07-14 Shaanxi,China KX225406a
Melampsora
epitea
Salix bebbiana SB2002-3 — Minnesota,USA AY471646ab
BJFC: Museum of Beijing Forestry University, Beijing, China; DAFVP: Forest Pathology Herbarium, Canadian Forest Service, Pacific
Forestry Centre,Victoria, British Columbia, Canada; DAOM: Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada; HMAS:
Herbarium Mycologicum Academiae Sinicae, Beijing, China; QFB: Rene Pomerleau Herbarium, Canadian Forest Service, Laurentian
Forestry Centre, Quebec, Canada.
astands for sequences used in the current study in GenBank; bstands for sequences from BOLD; abstands for sequences used as outgroup.
* stands for specimens used in this study.
—stands for no information available.
CAO ET AL.
58 • Phytotaxa 309 (1) © 2017 Magnolia Press
Results
Morphology
The telia of the rust fungus on R. oreodoxa were abundant, pulvinate without stalk cells, and had a waxy or gelatinous
appearance. Teliospores were covered by gelatineous sheaths (Figs 2B, 2C), which is the key characteristics for
identification of the genus Diaphanopellis. The uredinia were surrounded by a conspicuous peridium with square and
ornamented cells, the outter surface of peridium was deeply convave and smooth, while the inner surface was densely
warted.
This taxon clearly differs from D. forrestii (Crane 2005) in its urediniospore surface structure and smaller-size
teliospores. Moreover, the morphology of uredinia peridium is different in these two species. Detailed morphological
comparisons of specimens of the two species, including measurements in the uredinial and telial stages, are presented
in Table 2.
TABLE 2. Comparison of morphological characteristics of Diaphanopellis forrestii and Diaphanopellis purpurea.
Diaphanopellis forrestii Diaphanopellis purpurea
Spermogonial
stage
Unknown Unknown
Aecial stage Unknown Aecia amphigenous, ligulate. Aeciospores ellipsoidal,
oblong,globose,
or subglobose; 16–28× 11–24 m, densely echinulate,
a small numbers of central spines arise frequently;
Peridium outer surface deeply convex, striate or rugulose;
Peridium inner surface flat and smooth
Uredinial stage Urediniosporaes size: 19–35×12–29 m Urediniosporaes size: 22–38×20–30 m
Urediniosporaes surface: densely warted, one side
covered
Urediniosporaes surface: densely warted;
by a shallowly warted, longitudinal cap with a ragged
edge;
one side covered by longitudinal cap,cap with bumps and
broken edge;
Warts crowded, annulate, cylindrical or irregular in shape Warts coronate,fingerlike,cylindrical or irregular in
shape;
with broad tops, interspersed with much smaller,
shallower warts
the heads are acutely and minutely dentate or tubercular.
Peridial cells outer wall surface deeply concave, smooth
or
Peridial cells outer wall surface deeply concave, with
sharply defined edges,
finely warted; smooth to slightly rough surface
Peridial cells inner wall surface densely warted; Peridial cells inner wall surface flat or slightly convex,
warts shallow,
sometimes appearing labyrinthine irregular, and discrete
Telial stage The telial stage occurs on R. selense Franch The telial stage occurs on R. oreodoxa Franch
Teliospores catenulate, covered by a hyaline, thin-walled
sheath
Teliospores catenulate, covered by a hyaline, thin-walled
sheath
Teliospores size:17–33× 6–20 m Teliospores size:10–28× 5–12 m
TABLE 3. Sequence data of related rust genus analyzed in this study or obtained from GenBank and BOLD. (new species
in bold).
Fungal taxon Host plant Specimen no Date of
collection
Geograpphic origin GenBank accession
no. ITS
Diaphanopellis
purpurea
Picea purpurea BJFC-R02299* 2014-07-21 Sichuan,China KX225401
Picea purpurea BJFC-R02300* 2014-07-21 Sichuan,China KX225402
Rhododendron oreodoxa HMAS-55188* 1987-05-01 Sichuan,China KX225403
Rhododendron oreodoxa BJFC-R01698* 2014-05-21 Sichuan,China KX225404
Rhododendron oreodoxa BJFC-R01699* 2014-05-21 Sichuan,China KX225405
Chrysomyxa
arctostaphyli
Picea mariana DAOM 229628 1986-06-30 Klondike Loop,
Yukon,Canada
GU049458b
Arctostaphylos uva-ursi DAOM 183586 1982-06-16 Kenora district,
Ontario,Canada
GU049459b
...continued on the next page
A NEW RUST SPECIES OF DIAPHANOPELLIS Phytotaxa 309 (1) © 2017 Magnolia Press • 59
TABLE 3. (Continued)
Fungal taxon Host plant Specimen no Date of
collection
Geograpphic origin GenBank accession
no. ITS
Chrysomyxa
woroninii
Ledum groenlandicum QFB 25009 2006-06-26 Charlevoix, Quebec,
Canada
GU049495b
Picea abies DAOM 230441 1996-07-16 Sodankylä, Ruosselkä,
Finland
GU049494b
Coleosporium
phellodendri
Phellodendron amurense BJFC-QL12 — — KP017556b
Phellodendron chinense BJFC-R00700 2006-07-14 Shaanxi,China KX225406b
Coleosporium
campanulae
Campanula sp. HMAS-41501 — China KP017555b
— BJFC-ZL001 — China JQ219672b
Melampsora epitea Salix sp. BPI-US0022745 1950 Abisko,Sweden AY471648b
Salix arctica BPI-FC2002-8 — Fort Conger, Ellesmere
Island, Nunavut
AY471634b
Canada
Melampsora
populnea
— — 2006-09-23 France EU808037b
— — 2006-09-23 France EU808036b
Pucciniastrum tiliae Tilia japonica IBA7878 — Aomori,Japan AB221454b
Tilia japonica IBA7670 — Miyazaki,Japan AB221453b
Pucciniastrum
boehmeriae
Boehmeria tricuspis TSH-R21290 — Aomori,Japan AB221449 b
Boehmeria tricuspis TSH-R21289 — Aomori,Japan AB221450b
Melampsoridium
betulinum
Birch sp. — — — EU391657b
— — — — JN581986b
Melampsoridium
hiratsukanum
Alnus incana (alder) — — Trentino region,Italy KC888944b
Alnus rhombifolia PDR1480181 2010-08-08 Santa Cruz County,
California,USA
KC313889b
Thekopsora ostryae Ostrya japonica BJFC -GS-78 2012-08-09 Gansu,China KC415787b
Ostrya japonica BJFC -GS-129 2012-08-11 Gansu,China KC415796b
Thekopsora areolata Picea abies — — Buskerud, Ovre Eiker,
Hokksund,Norway
EF363336b
Picea abies — — Ostfold, Eidsberg,
Ramstad,Norway
DQ087230b
Cronartium ribicola Ribes odoratum — — USA KF387533b
— — 2010-06-01 USA JN587805b
Cronartium
flaccidum
Vincetoxicum nigrum — — France JN802139b
Melampyrum nemorosum — — — AY566270b
Puccinia
melampodii
Parthenium hysterophorus — — — EU659697ab
BJFC: Museum of Beijing Forestry University, Beijing, China; BPI: U.S. National Fungal Herbarium; DAOM: Agriculture and Agri-
Food Canada, Ottawa, Ontario, Canada; HMAS: Herbarium Mycologicum Academiae Sinicae, Beijing, China; QFB: Rene Pomerleau
Herbarium, Canadian Forest Service, Laurentian Forestry Centre, Quebec, Canada; TSH: Mycological Herbarium of the Graduate School
of Life and Environmental Sciences, University of Tsukuba, Japan.
bstands for sequences from GenBank.
abstands for sequences used as outgroup.
* stands for specimens used in this study.
—stands for the information is not found.
Molecular phylogeny
The final alignment consisted of 36 ingroup taxa contained 666 total characters, with 398 constant characters and 93
parsimony-uninformative variable characters. MP analysis yielded a single parsimonious tree (TL = 430, CI = 0.798,
RI = 0.887, and RC = 0.707), resulting in 18 terminal clades (Fig. 3). Bayesian analysis generated a tree of the same
topology. The ITS tree indicated that the rust fungus on P. purpurea and R. oreodoxa formed a monophyletic group in
Coleosporiaceae with strong bootstrap support (100) and Bayesian probabilties (1.00) (Fig. 3). They were also distinct
CAO ET AL.
60 • Phytotaxa 309 (1) © 2017 Magnolia Press
from other known Chrysomyxa species. Moreover, the current rust fungus was phylogenetically distinct from other
Coleosporium species, which also belong to the family of Coleosporiaceae.
The ITS dataset (Fig. 4) of 9 rust genera yielded a highly resolved phylogenetic framework. The data matrix
contained 35 fungal specimens. Of 719 total characters, 332 characters were constant and 37 were parsimony-
uninformative variable characters. MP analysis of sequence data yielded a single parsimonious tree (TL = 845, CI
= 0.611, RI = 0.882, and RC = 0.307). Bayesian analysis resulted in one of identical topology. The topology of the
ITS phylogram indicated that the present rust fungus on P. purpurea and R. oreodoxa formed a sister relationship to
Chrysomyxa species, and they clustered as a distinct clade from Chrysomyxa species and Coleosporium species, all of
which belong to the family of Coleosporiaceae. The present rust fungus represents a new genus because it is different
from Chrysomyxa, Coleosporium and Melampsora.
Life cycle
Sequence data of the rust fungus on P. purpurea and that on R. oreodoxa were identical, and they formed a monophyletic
group with strong bootstrap support and Bayesian probabilities (Fig. 3). This suggested that the fungus on P. purpurea
is the aecial state of the new species Diaphanopellis purpurea. The aecial stage of D. forrestii was not discovered
(Crane 2005). Molecular comparisons and inoculations to obtain its aecial states would facilitate further investigations
of the two species.
Diaphanopellis purpurea produced amphigenous and ligulate, single or confluent aecia on needles of P. purpurea.
Aeciospores were variable in shape from globose to ellipsoidal or oblong, densely echinulate, a small numbers of
central spines arose frequently on a separated flat columnar verruca. Aecial peridium cells were rectangle, larger
than the spores, outer surface was deeply convex, striate or rugulose, while inner surface was flat and smooth. The
aeciospore wall ornamentation of this new species is unique and different from all other described Chrysomyxa species
on Picea, and distinct from other rust fungi with verruose or annulate aeciospores. The connection of the aecial and
telial stages of new species D. purpurea was established by phylogenetic analysis of ITS data.
Taxonomy
Diaphanopellis purpurea C. J. You & J. Cao, sp. nov. (Figs. 1, 2)
MycoBank :—MB819572
Etymology:—Epithet “purpurea” refers to the aecial host of the holotype.
Diagnosis:—Differs from morphologically similar species, Diaphanopellis forrestii in the surface structure of uredinospore and uredium
peridium. In addition, it differs from other known Chrysomyxa species because of teliospore morphology.
Typ e:—CHINA, Sichuan Province: Kangding County, 29°5936N 101°5346E, alt. 3181 m, I on Picea purpurea Mast. (Pinaceae), 21
July 2014, C. J. You (Holotype, BJFC-R02299); CHINA, Sichuan Province: Kangding County, 29°5936N 101°5346E, alt. 3181
m, I on Picea purpurea: 21 July 2014, C. J. You (Isotype: BJFC-R02300).
Other Specimens examined:—CHINA, Sichuan Province: Kangding County, 22°5924N 101°5239E, alt. 3227 m,
II, III on Rhododendron oreodoxa Franch., 21 May 2014, Y. Bai (BJFC-R01698); Yunnan Province: the National
Forest Park of Shangari-La, 29°4535N 99°5941E, alt. 3565 m, I on Picea purpurea: 15 July 2014, C.J. You (BJFC-
R02301; BJFC-R02302): CHINA, Sichuan Province, Kangding County, 29°5921N 101°5240E, alt. 3263 m, 21
May 2014, Y. Bai (BJFC-R01699); Sichuan Province, Kangding County, May 1987, Y. L. Guo (HMAS-55188).
Spermogonia unknown.
Aecia amphigenous, ligulate, 0.3–1.1 mm wide. Aeciospores ellipsoidal, oblong, globose, or subglobose, 16–28 ×
11–24 m, with yellow inclusion, densely echinulate, a small numbers of central spines arise frequently on a separated
flat columnar verruca; wall hyaline, 0.4–0.6 m thick, wall plus spines 1.2–3.4 m thick; Aecial peridium dehiscing
at apex, later shredding, leaving a fringe around sorus; Peridial cells rectangle, larger than the spores, outer surface
deeply convex, striate or rugulose, inner surface flat, wall smooth. Uredinia subepidermal, erumpent, Aecidium-type,
covered by a conspicuous peridium; Peridial cells polygonal, round or square, similar in size or larger than the spores;
Outer peridium surface cells deeply concave, with sharply defined edges, smooth to slightly rough surface; inner
surface flat or slightly convex, warts shallow, irregular, and discrete. Urediniospores catenulate, globose, subglobose
to polygonal or ovoid, occasionally ellipsoidal, 22–38 × 20–30 m, densely warted, warts coronate, fingerlike, flame-
shaped or irregular in shape, the heads are acutely and minutely dentate or tubercular; wall 1.3–1.6 m thick, wall
A NEW RUST SPECIES OF DIAPHANOPELLIS Phytotaxa 309 (1) © 2017 Magnolia Press • 61
plus warts 2.1–3.4 m. Telia in large groups, gelatinous, orange or aurantiaca, erumpent; from round to elongated or
irregular, 130–300 × 100–280 m, often sunken in the center; raised, slightly constricted at the base, without stalk
cells. Teliospores catenulate, 10–28 × 5–12 m, thin-walled, finely tuberculate, enclosed in a loose hyaline sheath with
a thin wall, not laterally adherent.
FIGURE 1. Diaphanopellis purpurea on Picea purpurea (BJFC-R02299). A. Oblong-ellipsoid aeciospores (LM); B. Aecial peridium
with smooth inner surface; C. aeciospores; D. Aecial peridium with deeply convex, striate or rugulose outer surface; E. Densely echinulate
on aeciospores surface. Scale bars: A, C, D, E = 10m; B = 20 m.
Discussion
Diaphanopellis purpurea on R. oreodoxa has all the characteristics of Diaphanopellis, its teliospores are covered
by transparent sheaths (Figs 2B, 2C) and its telia morphology is similar to Diaphanopellis forrestii. It is distinctly
different from Chrysomyxa - the common pathogen infecting Rhododendron, which produces teliospores covered by
hyaline sheath and uredinial anamorph in Aecidium rather than Caeoma (Crane 2005).
Diaphanopellis forrestii on R. selense was described by Crane (2005) and it was characterized by catenulate,
densely warted urediniospores, one side covered by a narrowly warted, longtitudinal cap with a ragged edge. The
peridial cells of uredinia were larger than the urediniospores, outter surface deeply convave, smooth, or slightly warted,
inner surface densely warted, sometimes labyrinthine, side walls striate (Crane 2005). The type specimen of D. forrestii
was unavailable, but detailed morphological examinations of the 2 paratype specimens (HMAS 46927, HMAS 46933)
were provided, and the morphological characteristics fitted well with the original description by Crane (2005). We
were unable to obtain the ITS sequences of the species since these specimens were too old for DNA extraction.
CAO ET AL.
62 • Phytotaxa 309 (1) © 2017 Magnolia Press
FIGURE 2. Diaphanopellis purpurea on Rhododendron oreodoxa (BJFC-R01698). A. Telia in large groups, gelatinous, orange, erumpent;
B. Showing transparent sheaths extending beyond teliospores; C. Cross section of telium, showing transparent sheaths around teliospores;
D. Globose to subglobose urediniospores; E. Urediniospores; F. Urediniospores showing warts coronate, fingerlike, or irregular in shape;
G. Concave outer surface of peridial cells ; H. Warted inner surface of peridial cells. Scale bars: A = 200 m; B = 50 m; C, D, E, G = 10
m; F = 0.5 m; H = 20 m.
The new species D. purpurea is clearly distinct from D. forrestii by its unique urediospore-surface structure,
which is frequently used as important criterion for species identification, and by its smaller teliospores (Figs 2B, 2C).
The urediniospores (Figs 2D, 2E, 2F) are densely verrucose, crowded cylindrical, flame-shaped warts, lacking narrow
and irregular cap on spore surface, and the inner surface of peridial cells (Figs 2G, 2H) are discrete, shallow and
irregular warts, which are different from the densely warted inner surface of D. forrestii. In addition, the teliospore
size (10–28 × 5–12m) of D. purpurea is smaller than that of D. forrestii (17–33 × 6–20m; Crane 2005). The detailed
morphological differences between the two Diaphanopellis species are listed in Table 2. Although the sequence data of
D. forrestii is currently unavailable, the clearly distinct morphological characteristics between the two species suggest
that D. purpurea is a new species. As Crane (2005) mentioned, these unique features of Diaphanopellis including
peridium of uredinia and the gelatinous layer over the telium may be adaptations to avoid desiccation at high altitudes
and climate changes.
Diaphanopellis purpurea differs from other known Chrysomyxa species in its aeciospores surface ornamentation
and aecial peridium. The new species is characterized by its echinulate processes on aeciospores surface, while most
Chrysomyxa species produce aeciospores with annulate warts on spore surface. The densely echinulate aeciospores
with a small numbers of central spines arising frequently on a separated flat columnar verruca (Figs 1C, 1E) closely
resemble the ornamentation on urediniospores of Coleosporium phellodendri Kom. on Phellodendron (Kaneko 1981).
However, in phylogenetic analyses (Fig. 3) the new species on P. purpurea grouped in a distinct clade from two
Coleosporium species and other Chrysomyxa species.
A NEW RUST SPECIES OF DIAPHANOPELLIS Phytotaxa 309 (1) © 2017 Magnolia Press • 63
FIGURE 3. Phylogenetic tree constructed by maximum parsimony and Bayesian analyses based on ITS sequences of rust species of
Coleosporiaceae. Bootstrap values were calculated from 1,000 replications. Parsimony bootstrap (before the slash marks) and Bayesian
posterior probabilities (after the slash marks) greater than 50% are shown. Bars: 10 nucleotide substitutions. New species are shown in
bold.
In the rDNA ITS phylogeny (Fig. 3), the present rust fungus on P. purpurea and R. oreodoxa was phylogenetically
distinct from the other Chrysomyxa species considered in this study. The new species was phylogenetically distinct
from morphologically similar species, i.e., Chrysomyxa cassandrae Tranzschel. and Chrysomyxa rhododendri de Bary.
Moreover, it was nested in all Chrysomyxa group in the rDNA ITS phylogeny, however, at genus rank phylogenetic
work based on molecular data found the present rust fungus to be a new rust genus, different from Chrysomyxa (Fig.
4).
CAO ET AL.
64 • Phytotaxa 309 (1) © 2017 Magnolia Press
FIGURE 4. Phylogenetic tree constructed by maximum parsimony and Bayesian analyses based on ITS sequences of related rust genera.
Bootstrap values were calculated from 1,000 replications. Parsimony bootstrap (before the slash marks) and Bayesian posterior probabilities
(after the slash marks) greater than 50% are shown. Bars: 10 nucleotide substitutions. New species are shown in bold.
The monophyletic status of Chrysomyxa was supported by using 28S sequence data from the two closely related
Chysomyxa species (C. ledi and C. rhododendri) (Maier et al. 2003). However when integrated into a larger phylogenetic
framework based on ITS and 28S phylogenetic signals, Chysomyxa was proved to be polyphyletic. Chysomyxa species
were distributed in three distinct clades, moreover, some close relationships between C. pyrolae, C. monesis and the
Coleosporium genus and between C. weirii and the Melampsora genus were demonstrated (Feau et al. 2010).The
new species D. purpurea is nested in Chrysomyxa group, with a weakly supported sister group relationship with the
genus Chrysomyxa, it indicates that Diaphanopellis is polyphyletic, while the two genus are defined by clearly distinct
morphological characteristics. Further taxonomic investigation and multigene phylogenies are required to identify
evolutionary patterns that drive speciation within the genus Chrysomyxa and Diaphanopellis.
Acknowledgements
This work was supported by National Natural Science Foundation of China (No. 31300540), and The Fundamental
Research Funds for the Central Universities, China (No. BLX2012032) and. We would like to thank the Herbarium
Mycologicum Academiae Sinicae, Beijing, China (HMAS), for providing herbarium materials in this study.
A NEW RUST SPECIES OF DIAPHANOPELLIS Phytotaxa 309 (1) © 2017 Magnolia Press • 65
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