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ORIGINAL ARTICLE
Umbelopsis changbaiensis sp. nov. from China
and the typification of Mortierella vinacea
Ya-Ning Wang &Xiao-Yong Liu &Ru-Yong Zheng
Received: 29 April 2013 /Revised: 27 November 2013 / Accepted: 1 December 2013 /Published online: 18 December 2013
#German Mycological Society and Springer-Verlag Berlin Heidelberg 2013
Abstract Umbelopsis changbaiensis is described as a new
species of the genus Umbelopsis , Umbelopsidaceae,
Mucorales. It was isolated from forest soil mix in
Changbai Mountain, Jilin Province, China. The new spe-
cies is distinct from all the previously described species
mainly in colony color, branching type of sporangio-
phores, and shape and size of columellae. Maximum
growth temperatures and phylogenetic analyses of the
internal transcribed spacer (ITS1–5.8S–ITS2 region), par-
tial large subunit (28S D1–D3 region) of rDNA, and
partial actin gene (act -1) support the morphological re-
sult. The closest ally of U. changbaiensis is U. vinacea
whose basionym Mortierella vinacea was published by
Dixon-Stewart in 1932 without any type material indi-
cated. The only figure provided in the protologue is
designated here as the lectotype of M. vinacea and a
dry culture from its authentic strain CBS 212.32 is des-
ignated here as an epitype for this lectotype. Descriptions
and illustrations are provided for both U. changbaiensis
and U. vinacea.
Keywords Mucoromycotina .Phylogeny .Morphology .
Tax on om y .Lectotypification
Introduction
The genus Umbelopsis was described by Amos and Barnett
(1966) based on its type species U. versiformis Amos & H.L.
Barnett. Many species of Umbelopsis were originally de-
scribed as members of the Mortierella section Pusilla
(Linnemann 1941). Turner (1963) clarified the characteristics
of this section and proposed a more reasonable name
“Mortierella isabellina group”, which was also accepted by
Linnemann (1969). Gams (1977) introduced “Mortierella
subgen. Micromucor ”for these fungi. Von Arx (1982)
regarded the fungi of the subgenus Micromucor as belonging
to Mucoraceae rather than to Mortierellaceae,and
reclassified them in two genera, Micromucor and
Umbelopsis, taking account of the different manner of spo-
rangiophores. Yip (1986) discussed that Micromucor and
Umbelopsis should be merged to Umbelopsis because differ-
ent branching manners can be found in U. isabellina,meaning
this criterion to delimit these two genera is somewhat
arbitrary. Based on molecular evidence, Meyer and
Gams (2003) confirmed that these two genera are monophy-
letic and consequently treated all these fungi as members of
Umbelopsis.
Morphological characteristics of the fungi in this genus
mainly include: 1) colonies velvety and often colored; 2) aerial
hyphae low and dense; 3) sporangiophores often arising erect-
ly from a vesicle; 4) columellae small or absent; 5)
sporangiospores variously shaped, similarly colored to the
sporangia (Linnemann 1941;Linnemann1969; Turner 1963;
Gams 1977; Meyer and Gams 2003). In China, five isolates of
Umbelopsis were found to be distinct from all the previously
described species. They constitute a new species which is
closely related to U. vinacea (Dixon-Stew) Arx. No type
Y. <N. Wang :X.<Y. L i u (*):R.<Y. Z he n g (*)
State Key Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101,
People’sRepublicofChina
e-mail: liuxiaoyong@im.ac.cn
e-mail: zhengry@im.ac.cn
Y. <N. Wang
University of Chinese Academy of Sciences, Beijing 100049,
People’sRepublicofChina
Mycol Progress (2014) 13:657–669
DOI 10.1007/s11557-013-0948-9
material was indicated when and after U. vinacea (≡Mortierella
vinacea Dixon-Stew) was published (Dixon-Stewart 1932).
Therefore, the only figure of this species in the protolgue and
a dry culture from its authentic strain are designated here as
lectotype and epitype, respectively.
Materials and methods
Cultures and isolation
Five strains of Umbelopsis changbaiensis were isolated from
forest soil mix in Changbai Mountain, Jilin Province, China.
Other strains of Umbelopsis used in this study were requested
from CBS (Centraalbureau voor Schimmelcultures, Baarn,
Holland), NRRL (USDA Agricultural Research Culture
Collection, Peoria, USA), or isolated by ourselves. Strains
found in China were isolated using the method of Zheng
et al. (2007). Detailed information of these strains is listed in
Tab le 1.
Media and cultivation
CMA (cornmeal agar: 2 % cornmeal and 2 % agar) and MEA
(malt extract agar: 2 % malt extract and 2 % agar) were used
for morphological studies; Czapek’s agar (3 % sucrose, 0.3 %
NaNO
3
, 0.05 % MgSO
4
•7H
2
O, 0.05 % KCl, 0.001 % FeSO
4
,
0.1 % KH
2
PO
4
, and 1.5 % agar) was used to examine growth
capacities; PDA (pH7) was used for determining maximum
temperatures; L-PDA (PDA added with 3 % lecithin) was
used for mating experiments; and ME (malt extract: 2 %,
peptone 0.1 %, and dextrose 2 %) was used for DNA extrac-
tion. Cultivation duration and temperature were 5–10 days at
20 °C for morphological studies, 5–20 days at 20 °C for
testing growth capacities, 7–30 days at 20 °C for mating
experiments, three days at 30–40 °C for determining the
maximum temperature, 4–8 days at room temperature for
DNA extraction. Capitalized color designations in the descrip-
tions referred to the color charts by Ridgway (1912).
Phylogenetic analyses
Total genomic DNA was isolated from mycelia with the
Biospin Fungus Genomic DNA Extraction Kit (Bioer
Technology Company Limited, Beijing, China) according to
the manufacturer’s instructions. Sequences of rDNA includ-
ing the entire ITS1–5.8S–ITS2 and 28S D1–D3 regions, were
amplified with the primers LR5M (5′- GCT ATC CTG AGG
GAA ACT TCG -3′) and NS5M (5′- GGC TTA ATT TGA
CTC AAC ACG G -3′) designed here. Actin genes were
amplified with the primers Act-1 (5′- TGG GAC GAT ATG
GAI AAI ATC TGG CA -3′) and Act-4R (5′-TCITCGTAT
TCT TGC TTI GAI ATC CAC AT -3′)(Voigtand
Wöstemeyer 2000). For rDNA, the PCR reactions began with
an initial denaturation at 94 °C for 5 min, followed by 33 cy-
cles of 94 °C for 30 s, 60 °C for 45 s and 72 °C for 45 s, and
ended up with a final extension of 72 °C for 10 min; While for
actin genes, the PCR program includes an initial denaturation
at 94 °C for 5 min, 38 cycles of 94 °C for 1 min, 55 °C for
1 min and 72 °C for 1 min, and a final extension of 72 °C for
10 min. When actin gene could not be well sequenced directly
in case of strains of Umbelopsis vinacea, PCR products were
ligated into the pUM-T Easy vector (Bioteke Corporation,
Beijing, China) and cloned in E. coli TOP109 competent cells
(Bioteke Corporation, Beijing, China), according to the man-
ufacturer’s instructions. Colony PCRs were performed sepa-
rately with two pairs of primers, Act-1 / Act-4R and M13F (5′-
TGTAAA ACG ACG GCC AGT -3′) / M13R (5′- CAG GAA
ACAGCTATGACC-3′). DNA sequencing was performed
at Majorbio Bio-technology Company Limited, Beijing,
China, with primers ITS4 (5′-TCCTCCGCTTATTGA
TAT GC - 3′) and ITS5 (5′- GGA AGT AAA AGT CGT
AAC AAG G -3′) (White et al. 1990) for ITS1–5.8S–ITS2
regions, primers LROR (5′- ACC CGC TGA ACT TAA GC -
3′) (Vilgalys and Hester 1990) and LR5M for 28S D1–D3
regions, and primers Act-1 and Act-4R for actin genes. For
actin genes three clones of each strain were sequenced. When
these three sequences were obviously different, additional
clones were picked. Generated sequences were assembled
using Sequencher 4.1.4 (Gene Codes Corporation, Ann
Arbor, MI, USA) to obtain consensus sequences.
Multiple alignments were performed using the program
MAFFT 6.952 (Katoh et al. 2005; Katoh and Toh 2008)with
the strategy G-INS-i and were manually optimized using the
program BioEdit 4.7.1 (Hall 1999). Strains of genera
Mortierella,Mucor ,andRhizomucor were selected as
outgroups because they are closely related to Umbelopsis
which was initially divided from Mortierella and included in
the Mucorales as a basal sister-group to other genera
(O’Donnell et al. 2001; Meyer and Gams 2003;Liu2008).
Sequences of these outgroups were downloaded from
GenBank with their accession numbers listed in Table 1.
The pairwise distance matrixes for strains of the two close-
ly related species Umbelopsis changbaiensis and U. vinacea
were calculated following the Kimura two-parameter model
of nucleotide substitution (Kimura 1980) implemented within
the program PAUP* 4.0 b10 (Swofford 2002). Phylogenetic
analysis was done with maximum parsimony and strict clock
Bayesian inferences analysis, respectively. Gaps in sequence
alignment were treated as fifth bases. The maximum parsimo-
ny analysis was performed using PAUP* 4.0 b10. Branch
robustness was estimated by performing 1,000 bootstrap rep-
licates (Felsenstein 1985) with heuristic searches. In order to
integrate branch lengths and bootstrap values into a consensus
tree, the topology generated by bootstrap analyses was loaded
and constrained as the backbone of an additional heuristic
658 Mycol Progress (2014) 13:657–669
Tab l e 1 Sources of isolates and GenBank accession numbers used in this study
Species Strain no.
a
Origin GenBank accession no.
ITS 28S D1–D3 act
Umbelopsis angularis W. Gams & M. Sugiy. CBS 603.68 (HT) Soil, the Netherlands, Maarschalksbos, Baarn, 1968 KC489476 KF727442 KF771842
U. angularis CGMCC 3.6640=Um-1 Soil under Fargesia spathacea, China, Hubei, Shennongjia,
Nanyinzhai, Jul 1984
KC489475 KF727440 KF771831
U. autotrophica (E.H. Evans) W. Gams CBS 310.93 (T of Mortierella
ramanniana var. autotrophica
E.H. Evans)
Soil, UK, Heath, Oxshott KC489480 KF727443 KF771843
U. changbaiensis Y.N. Wang & al. CGMCC 3.16345=Um-18 Forest soil mix, China, Jilin, Changbai Mountain, Jun 2011 KC489481 KF727444 KF771836
U. changbaiensis CGMCC 3.16346=Um-26 ibid. KC489482 KF727445 KF771837
U. changbaiensis CGMCC 3.16347=Um-27 ibid. KC489483 KF727446 KF771838
U. changbaiensis CGMCC 3.16317=Um-28 (HT) ibid. KC489484 KF727447 KF771839
U. changbaiensis CGMCC 3.16348=Um-29 ibid. KC489485 KF727448 KF771840
U. dimorpha Mahoney & W. Gams CBS 110039 (HT) Soil of basaltic parent material, New Zealand, N. part
of South Island, Red Hills area of Mt. Richmond Forest
Park, just N. of Wairau River Valley, alt. 1000 m, Apr 1993
KC489478 KF727471 KF771833
U. dimorpha CGMCC 3.6641=Um-3 Unknown, China, isolated before 1986 KC489477 KF727441 KF771841
U. fusiformis H.Y. Yip CBS 385.85 (T) Soil from forest of Eucalyptus regnans , Australia,
Victoria, near Melbourne
KC489500 KF727463 KF771861
U. gibberispora M. Sugiy. CBS 109328 (HT) Leaf litter, Japan, Kyoto, Miyama city, Ashiu Experimental
Forest of Kyoto University, 18 Nov 1999
KC489479 KF727469 KF771832
U. isabellina (Oudem.) W. Gams CBS 208.32 Sandy loam soil, Australia, Victoria KC489497 KF727460 KF771852
U. isabellina CBS 560.63 Soil from Larix forest, Germany, near Hann.-Münden, 1936 KC489502 KF727466 -
U. isabellina NRRL 1757 Soil, USA, Wisconsin - - AJ287209
b
U. nana (Linnem.) Arx CBS 730.70 Forest soil, Netherlands, Winterswijk KC489506 KF727470 KF771835
U. nana CBS 858.68 (T of M. alba
Manka & Gierczak)
Forest soil, Poland KC489503 KF727467 KF771864
U. nana NRRL 22420 Forest soil, USSR - - AJ287210
b
U. ovata (H.Y.Yip) H.Y.Yip CBS 499.82 (T of M. ovate H.Y. Yip) Rhizoplane, Australia, Victoria, Brisbane Ranges,
90 km SW of Melbourne
KC489501 KF727465 KF771863
U. ramanniana (Möller) W. Gams NRRL 1296 Unknown, USA, Wisconsin KC489493 KF727456 KF771847
U. ramanniana CGMCC 3.16356=Um-78 Soil under Fargesia spathacea, China, Hubei,
Shennongjia, Nanyinzhai, Jul 1984
KC489494 KF727457 KF771848
U. ramanniana NRRL 5844 Associated with Pinus sylvestris, United Kingdom - - AJ287166
b
U. swartii H.Y. Yip CBS 868.85 (T) Soil under Eucalyptus regnans , Australia, Victoria,
Wallaby Creek, 70 km north-north-east
of Melbourne, Jun 1985
KC489504 KF727468 KF771865
U. versiformis Amos &H.L. Barnett CBS 150.81 (T) Root, USA, Virginia, Pendleton Co., 1960 KC489496 KF727459 KF771851
U. versiformis CBS 473.74 (T of M. roseonana
W. Ga ms & Gl ee so n)
Poor quality soil, Australia Victoria , Parkville,
University of Melbourne
KF765510 KF727464 KF771862
U. vinacea (Dixon-Stew.) Arx CBS 212.32 (ET of M. vinacea
Dixon-Stew.)
Sandy loam soil, Australia, Victoria KC489498 KF727461 KF771853 (Type I)
KF771855 (Type I)
Mycol Progress (2014) 13:657–669 659
Tab l e 1 (continued)
Species Strain no.
a
Origin GenBank accession no.
ITS 28S D1–D3 act
KF771856 (Type I)
KF771858 (Type I)
KF771854 (Type II)
KF771857 (Type II)
U. vinacea CBS 236.82
(T of U. multispora Ts. Watan.)
Root of Fragaria , Japan, Shimane Pref. KC489499 KF727462 KF771859 (Type I)
KF771860 (Type III)
U. vinacea CGMCC 3.16349 =Um-53 Soil under herbaceous plant, China, Tibet, Milin,
Nanyi, alt. 3048 m, Aug 2009
KC489486 KF727449 KF771845 (Type I)
KF771844 (Type II)
U. vinacea CGMCC 3.16350 =Um-54 ibid. KC489487 KF727450 -
U. vinacea CGMCC 3.16351 =Um-66 Soil under ferns, China, Tibet, Milin,
Xuekagou, alt. 3000 m, Jul 2004
KC489488 KF727451 -
U. vinacea CGMCC 3.16352 =Um-68 Soil under bush, China, Tibet, Milin,
Nanyi, alt. 3000 m, Jul 2004
KC489489 KF727452 KF771846 (Type I)
U. vinacea CGMCC 3.16353 =Um-69 ibid. KC489490 KF727453 -
U. vinacea CGMCC 3.16354 =Um-70 ibid. KC489491 KF727454 -
U. vinacea CGMCC 3.16355 =Um-71 ibid. KC489492 KF727455 -
U. vinacea CGMCC 3.16357=Um-87 Forest soil mix, China, Jilin,
Changbai Mountain, Jun 2011
KC489495 KF727458 KF771849 (Type I)
KF771850 (Type I)
U. westeae H.Y. Yip CBS 870.85 (T) Soil from acid heathland, Australia,
Victoria, Frankston, 1953
KC489505 KF727472 KF771834
Mucor circinelloides
f. janssenii (Lendn.) Schipper
CBS 205.68 Forest soil, South Africa, Zululand, 1966 HM999952
b
--
M. circinelloides
f. lusitanicus (Bruderlein) Schipper
CBS 277.49 - - - AJ287173
b
Mortierella verticillata Linnem. CBS 220.58 (T of Haplosporangium
fasciculatum Nicot)
Soil under Betula sp ., France,
Fontainebleau, Jun 1952
JN943795
b
JN940873
b
-
M. verticillata NRRL 6337 - - - AJ287170
b
Rhizomucor pusillus (Lindt) Schipper CBS 354.68 (NT) Corn meal, Netherlands, 1966 DQ119005
b
-
R. pusillus NRRL 2543 Bovine mycosis, Great Britain - AJ287192
b
R. variabilis R.Y. Zheng & G.Q. Chen CBS 103.93 (T) Wrist and back of hand of female patient,
China, Jiangsu Province, C. Li, 1989
- KC012656
b
-
a
Abbreviations for status of strains: ET, ex-epitype strain; HT, ex-holotype strain; NT, ex-neotype strain; T, ex-type strain. Abbreviations for culture collections: CBS, Centraalbureau voor
Schimmelcultures, Utrecht, The Netherlands; CGMCC, China General Microbial Culture Collection, Beijing, China; NRRL (ARS), Agricultural Research Service culture collection, Peoria, IL, USA;
Um-, Mucorales Research Group, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
b
Retrieved from GenBank
-No data available
660 Mycol Progress (2014) 13:657–669
search. The Bayesian analysis was performed using MrBayes
3.2.1 (Ronquist et al. 2012). The best-fit models of nucleotide
substitutions were selected by the hierarchical likelihood ratio
tests (Huelsenbeck and Crandall 1997; Posada and Crandall
2001) using the program MrModeltest 2.2 (Nylander 2004).
In order to exactly repeat the analyses, the seed and swapseed
were both set to 200 million. Two independent strict clock
Bayesian analyses ran simultaneously, each with one cold
chain, three heated chains, temperature 0.1, and a random
initial tree. The uniform prior was used for the branch lengths
on clock trees. One out of every 500 trees was sampled for 500
thousand generations. The first 25 % of sampled trees from
each of the two runs were discarded before summarizing the
information in parameter files, and the remaining sampled
trees were used for calculating posterior probabilities. The
convergence of the two runs was diagnosed by the average
standard deviation (usually 0.01–0.05) and the potential scale
reduction factor approaching 1.0 (Gelman and Rubin 1992).
The node reliability was assessed by no less than 70 % of
parsimony bootstrap support values (PBS) and no less than
95 % of Bayesian posterior probability values (BPP; Alfaro
et al. 2003).
Results and discussion
Maximum growth temperatures
The five strains of Umbelopsis changbaiensis were tested
three times for their maximum growth temperatures (MGT),
resulting in a range of 32–33 °C. The authentic strain of
U. vinacea CBS 212.32, however, grows exuberantly at this
temperature and remains active up to 37 °C, which is consis-
tent with the result of Cowley (1962). The MGT of the other
cultures of U. vinacea is 35–36 °C, a little lower than that of
the authentic strain.
The MGT is a quite distinguishable characteristic among
species in the genera of the Mucorales (Zheng and Chen 2001;
Zheng et al. 2007). In Umbelopsis, the growth temperature
was also used by Evans (1971) to distinguish U. ramanniana
(Möller) W. Gams [≡Mortierella ramanniana var.
ramanniana (Möller) Linnem.] and U. autotrophica (E.H.
Evans) W. Gams (≡M. ramanniana var. autotrophica E.H.
Evans). This study finds that the MGTs of U. changbaiensis
(32–33 °C) and U. vinacea (35–37 °C) are significantly
different for separating the former from the latter. This result
confirms the ability of MGT to act as an auxiliary feature in
the classification of Mucorales.
Mating experiments
All strains of Umbelopsis changbaiensis were crossed with
each other in this study, and no response could be detected.
Similarly, Sugiyama et al. (2003) overviewed that no zygo-
spore formation had been observed between any two strains of
Umbelopsis.
Growth on Czapek’sagar
Dixon-Stewart (1932) noticed abundant chlamydospores and
hyphal swellings in Umbelopsis vinacea on Czapek’sagar.
This study confirms his findings. Most cultures of U. vinacea
covered more than half of the Petri dish in 20 days with
numerous chlamydospores and swellings, but almost no spo-
rangium could be detected; while those of U. changbaiensis
grew limitedly, less than 4 cm in diameter, with sparse chla-
mydospores, swellings, and sporangia. Compared to cultures
isolated from soil, one root-associated culture of U. vinacea ,
CBS 236.82, grew exuberantly with abundant sporangia and
few chlamydospores and swellings.
Phylogenetic analyses
All sequences of the ITS1–5.8S–ITS2 region, the 28S D1–D3
region, and actin gene obtained in this study were deposited in
GenBank under the accession numbers given in Table 1.
ITS1–5.8S–ITS2 region varied in length ranging from 531
to 569 bp; the alignment consisted of 655 total characters, of
which 134 characters were constant, 259 variable characters
were parsimony-uninformative, and 340 characters were
parsimony-informative. 28S D1–D3 region varied in length
ranging from 966 to 1097 bp; the alignment consisted of 1146
total characters, of which 625 characters were constant, 305
variable characters were parsimony-uninformative, and 216
characters were parsimony-informative. Partial actin genes
varied in length ranging from 871 to 964 bp; the alignment
consisted of 986 total characters among which 94 (502–595
positions) were intron regions. The intron regions of actin
gene were excluded from phylogentic analysis. The remaining
892 characters were included in analyses, of which 507 char-
acters were constant, 126 variable characters were parsimony-
uninformative, and 259 characters were parsimony-
informative.
Pairwise distance matrixes for strains of Umbelopsis
changbaiensis and U. vinacea based on three genes are
shown in Table 2. ITS rDNA intraspecific variations are 0–
0.2 % and 0–0.7 % in U. changbaiensis and U. vinacea ,
respectively. Intraspecies 28S rDNA evolutionary distances
are 0 and 0–0.5 % in U. changbaiensis and U. vinacea ,
respectively. Intraspecies act evolutionary distances are 0
and 0–1.3 % in U. changbaiensis and U. vinacea,respective-
ly. However, the interspecies dissimilarities between these two
species are significantly larger, that is, 2.3–3.1 %, 0.5–0.6 %
and 12.4–13.1 % for ITS, 28S and act,respectively.Three
types of actin genes were found in strains of U. vinacea .
Among the 17 clones, 13, 3 and 1 belong to type I, II and
Mycol Progress (2014) 13:657–669 661
Tab l e 2 Pairwise distance percentages for strains of Umbelopsis changbaiensis and U. vinacea based on genes ITS, 28S rDNA D1–D3 and act
Gene markers Species Strains U. vinacea U. changbaiensis
12 34 4 567 89 1010 11121314
ITS rDNA U. vinacea 1:CGMCC 3.16350
2:CGMCC 3.16349 0
3:CGMCC 3.16355 00
4:CGMCC 3.16357 0.2 0.2 0
5:CGMCC 3.16351 0.2 0.2 0.2 0.2
6:CGMCC 3.16353 00 00 0
7:CGMCC 3.16352 00 00 00
8:CGMCC 3.16354 00 00 000
9:CBS 236.82 0.7 0.7 0.4 0.5 0.4 0.2 0.2 0.2
10:CBS 212.32 0.4 0.4 0.2 0.2 0.5 0.2 0.2 0.2 0.5
U. changbaiensis 11 :CGMCC 3.16347 3.1 3.1 2.5 2.9 2.5 2.4 2.3 2.3 2.7 2.9
12:CGMCC 3.16345 3.1 3.1 2.5 2.9 2.5 2.4 2.3 2.3 2.7 2.9 0
13:CGMCC 3.16348 3.3 3.3 2.7 3.1 2.7 2.5 2.5 2.5 2.9 3.1 0.2 0.2
14:CGMCC 3.16346 3.1 3.1 2.5 2.9 2.5 2.4 2.3 2.3 2.7 2.9 0.2 0.2 0
15:CGMCC 3.16317 2.9 2.9 2.5 2.9 2.4 2.3 2.3 2.3 2.7 2.9 0.2 0.2 0 0
28S rDNA D1–D3 U. vinacea 1:CGMCC 3.16350
2:CGMCC 3.16349 0
3:CGMCC 3.16355 00
4:CGMCC 3.16357 00 0
5:CGMCC 3.16351 00 00
6:CGMCC 3.16353 00 00 0
7:CGMCC 3.16352 00 00 00
8:CGMCC 3.16354 00 00 000
9:CBS 236.82 0.3 0.3 0.3 0.4 0.3 0.3 0.3 0.3
10:CBS 212.32 0.3 0.3 0.3 0.5 0.3 0.3 0.3 0.3 0.3
U. changbaiensis 11 :CGMCC 3.16347 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6
12:CGMCC 3.16345 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0
13:CGMCC 3.16348 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0 0
14:CGMCC 3.16346 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0 0 0
15:CGMCC 3.16317 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0 0 0 0
act U. vinacea 2:CGMCC 3.16349 c7
4:CGMCC 3.16357 c8 0
4:CGMCC 3.16357 c11 0.3 0.3
7:CGMCC 3.16352 c1 000.3
662 Mycol Progress (2014) 13:657–669
III, respectively (Table 1and Fig. 1c,fourclonesoftypeI
not shown). In Rhizopus americanus, three types of ITS
rDNA were disclosed and the dominant type was selected
as representative for phylogenetic analyses (Abe et al.
2010). Similarly, the dominant actin gene type of
U. vinacea, namely type I was used to test evolutionary
distances with U. changbaiensis. Alastruey-Izquierdo et al.
(2010) also reported multiple copies of actin genes in
Lichtheimia spp.
In most parsimony analyses, a phylogenetic tree resulted in
TL=1421, CI=0.7797, HI=0.2203, RI=0.8790, RC=0.6854
for ITS; TL=763, CI=0.8637, HI =0.1363, RI =0.9132, RC =
0.7887 for 28S D1–D3; and TL=895, CI =0.5486, HI=
0.4514, RI=0.8071, RC=0.4428 for act . The tree topologies
obtained with strict clock Bayesian inferences and maximum
parsimony analyses do not conflict in well-supported groups
for all three genes. The bootstrap values obtained by the latter
are added beside the relative values of the former.
Figure 1a,b,andcshow ITS, 28S D1–D3 and act phylo-
genetic relationships among Umbelopsis species. The new
species U. changbaiensis forms a monophyletic group in all
the three phylograms. In the ITS and 28S D1–D3 phyloge-
netic trees, the clade of U. changbaiensis is a sister group to
the clade (Fig. 1a) or the grade (Fig. 1b)ofU. vinacea.While
in the phylogenetic tree based on actin genes, U. vinacea is
polyphyletic with three lineages (Fig. 1c). According to the
treatment principle of multiple copies in Rhizopus americanus
(Abe et al. 2010), the dominant type of act was applied for
representing the phylogenetic position for U. vinacea .By
considering this treatment, the clade of U. vinacea (type I in
Fig. 1c) was diverged more distantly from U. changbaiensis
in the actin gene than in rDNA. Nevertheless, the minor type
of act (type II in Fig. 1c) suggests a relatively close affinity
between U. changbaiensis and U. vinacea which are sister
groups. Hence, the establishment of the new species
U. changbaiensis and its close affinity with U. vinacea based
on morphological comparisons is phylogenetically confirmed
by ITS, 28S D1–D3 and act . Further study on the phyloge-
netic relationship between these two species will focus on the
phylogenetic nature of multiple copies of actin genes, as well
as more genes.
TAXONO MY
Umbelopsis changbaiensis Y.N. Wang, X.Y. Liu & R.Y.
Zheng, sp. nov. Figs. 2and 4a
MycoBank: MB 803431
Description: Colonies reaching 29–31 mm in diameter on
MEA after five days at 20 °C, less than 1 mm high, flat,
velvety, zonate, Russet (Ridgway, Pl. XV), forming sectors
because of the whitish hyphae upon old sporulation layer; on
CMA reaching 25–27 mm in diameter after five days at 20 °C
, low, slightly russet, sparser than on MEA. Sporangiophores
Tab l e 2 (continued)
Gene markers Species Strains U. vinacea U. changbaiensis
12 34 4 567 89 1010 11121314
9:CBS 236.82 0.6 0.6 0.9 0.6
10:CBS 212.32 c5 1.0 1.0 1.3 1.0 0.8
10:CBS 212.32 3/c7 /cc 0.9 0.9 1.1 0.9 0.5 0.3
U. changbaiensis 11 :CGMCC 3.16347 12.5 12.5 12.8 12.5 12.4 13.1 12.8
12:CGMCC 3.16345 12.5 12.5 12.8 12.5 12.4 13.1 12.8 0
13:CGMCC 3.16348 12.5 12.5 12.8 12.5 12.4 13.1 12.8 0 0
14:CGMCC 3.16346 12.5 12.5 12.8 12.5 12.4 13.1 12.8 0 0 0
15:CGMCC 3.16317 12.5 12.5 12.8 12.5 12.4 13.1 12.8 0 0 0 0
Clone nos.c7 c8 c11 c1 c5 c3 /c7/cc
Strains 1 2 3 4 4 5 6 7 8 9 10 10 11 12 13 14
Mycol Progress (2014) 13:657–669 663
U. autotrophica CBS 310.93
U. vinacea CGMCC 3.16357
U. dimorpha CBS 110039
U. vinacea CGMCC 3.16352
U. vinacea CGMCC 3.16353
U. nana CBS 730.70
U. versiformis CBS 150.81
U. isabellina CBS 208.32
U. vinacea CGMCC 3.16354
U. ovata CBS 499.82
U. changbaiensis CGMCC 3.16347
U. nana CBS 858.68
U. dimorpha CGMCC 3.6641
U. isabellina CBS 560.63
Mortierella verticillata CBS 220.58
U. vinacea CGMCC 3.16350
U. versiformis CBS 473.74
U. changbaiensis CGMCC 3.16348
U. changbaiensis CGMCC 3.16346
U. vinacea CBS 212.32
U. vinacea CGMCC 3.16355
U. changbaiensis CGMCC 3.16317
U. vinacea CBS 236.82
U. vinacea CGMCC 3.16349
U. vinacea CGMCC 3.16351
U. changbaiensis CGMCC 3.16345
U. angularis CBS 603.68
U. angularis CGMCC 3.6640
U. ramanniana CGMCC 3.16356
U. gibberispora CBS 109328
U. ramanniana NRRL 1296
U. swartii CBS 868.85
U. westeae CBS 870.85
U. fusiformis CBS 385.85
Mucor circinelloides f. janssenii
CBS 205.68
Rhizomucor pusillus CBS 354.68
0.1
100/100
100/100
100/100
100
/98
/98
-/72
100/99
100/100
100/100
100/100
100/-
100/95
U. vinacea CBS 236.82
U. changbaiensis CGMCC 3.16346
U. versiformis CBS 473.74
U. versiformis CBS 150.81
U. swartii CBS 868.85
U. vinacea CGMCC 3.16349
U. vinacea CGMCC 3.16351
U. westeae CBS 870.85
Rhizomucor variabilis CBS 103.93
U. vinacea CGMCC 3.16355
U. dimorpha CGMCC 3.6641
U. changbaiensis CGMCC 3.16348
U. ovata CBS 499.82
U. angularis CBS 603.68
U. vinacea CGMCC 3.16350
U. vinacea CGMCC 3.16354
U. changbaiensis CGMCC 3.16347
U. nana CBS 858.68
U. vinacea CGMCC 3.16353
U. gibberispora CBS 109328
U. nana CBS 730.70
U. vinacea CGMCC 3.16352
U. autotrophica CBS 310.93
U. vinacea CGMCC 3.16357
U. changbaiensis CGMCC 3.16345
U. ramanniana CGMCC 3.16356
U. angularis CGMCC 3.6640
U. isabellina CBS 560.63
U. dimorpha CBS 110039
U. vinacea CBS 212.32
U. isabellina CBS 208.32
Mortierella verticillata CBS 220.58
U. changbaiensis CGMCC 3.16317
U. fusiformis CBS 385.85
U. ramanniana NRRL 1296
100/100
100/100
99.8/-
98.5/-
100/98
100/100
99.9/-
100/86
100/-
100/100
98.2/74
100/82
100/100
100/100
0.1
a
b
100
100/-
Fig. 1 Phylogenetic trees of Umbelopsis generated from Bayesian
analyses based on three different genes, ITS (a), 28S D1–D3 rDNA
(b), and act (c) with Mortierella,Mucor and Rhizomucor as
outgroups. Values above the branches represent significant Bayesian
posterior probability values (BPP≥95 %) and parsimony bootstrap
support values (PBS≥70 %). Missing or weakly supported nodes
(BPP<95 % or PBS<70 %) are denoted by a “-”. The bar at the
lower left indicates 0.1 expected changes per site. Hashed line indi-
cates long branches divided by three to improve visibility of branch
lengths. For actin genes in U. vinacea, identifiers of clones (c)are
given after the strain numbers and sequences are discriminated by type
I, II and III
664 Mycol Progress (2014) 13:657–669
arising mainly from distinct vesicles on agar surface, vesicles
subglobose to ampulliform, (6.9–)7.5–13.8×(6.9–)7.5–15.8
(−21.7) μm, umbellately branched, (16–)23–63 (−80) μm
long, (2.4–)2.8–4.6 (−4.9) μm wide near the base, sometimes
tapering about 1 μm in diameter at the middle parts, and 2.4–
4.0 (−4.7) μm near the tip, 1–2 septate, with one septum near
the base. Sporangia globose to subglobose, (9–)11–16 μmin
diameter, reddish-brown, multi-spored; walls deliquescent
leaving, no or small collars. Columellae small but distinct,
depressed globose, 3.2–5.5× 1.6–3.8 μm. Sporangiospores
isodiametrically angular, (2.4–)3.0–4.2 (−4.6)× (2.4–)2.8–
3.6 (−4.3) μm, reddish in mass, without oil droplet.
Chlamydospores absent. Zygospores unknown.
Type: China, Jilin, Changbai Mountain, 42°10′548″–
44°02′274″N, 126°35′399″–128°55′815″E, 360–2654 m
alt., forest soil mix, Xiao-Yong Liu, Jun 2011, HMAS
244432 (holotype) prepared from CGMCC 3.16317 (ex-
holotype).
Etymology: ‘changbaiensis’referring to its collection loca-
tion, Changbai Mountain.
Strains examined: China, Jilin, Changbai Mountain, 42°10′
548″–44°02′274″N, 126°35′399″–128°55′815″E, 360–
2654 m alt., forest soil mix, Xiao-Yong Liu, Jun 2011,
CGMCC 3.16317; ibid, cultures CGMCC 3.16345,
CGMCC 3.16346, CGMCC 3.16347, CGMCC 3.16348.
Note: This fungus possesses typical mycelial habit of
Umbelopsis and is somewhat similar to U. vinacea and
U. angularis. All these three species have reddish, globose
and multi-spored sporangia and angular sporangiospores.
However, several characteristics readily separate
U. changbaiensis from the other two species: 1) colonial
color on MEA is a mixture of orange and yellow, usually
with white sectors when old; 2) sporangiophores are mostly
arising from distinct vesicles, branching short but very
abundant, almost not tapering, and rarely septate below
sporangia; 3) columellae are small but distinct; 4)
sporangiospores are smaller than, and not as sharp as those
in U. vinacea ; 5) unlike U. angularis with abundant
macrochlamydospores, no chlamydospore is found on ei-
ther CMA or MEA.
Umbelopsis vinacea (Dixon-Stew.) Arx, Sydowia 35: 20
(1982) Figs. 3and 4b
≡Mortierella vinacea Dixon-Stew., Trans. Br. Mycol.
Soc. 17: 212 (1932)
0.1
U. changbaiensis CGMCC 3.16317
U. changbaiensis CGMCC 3.16346
U. changbaiensis CGMCC 3.16348
U. changbaiensis CGMCC 3.16347
Type II
Type I
Type III
100/86
U. vinacea CGMCC 3.16349 c7
100/
100
100/91
95.7/-
99.9
/-
100/100
96.5/-
99.9/-
98/-
100/85
99.9/-
100/93
100/82
99.4/-
100/84
99.8/-
100/ 100
99.9/90
96.4/74
96.6/-
100/100
99.7/98
100/ -
-/99
U. changbaiensis CGMCC 3.16345
U. westeae CBS 870.85
U. dimorpha CGMCC 3.6641
U. vinacea CBS 212.32 c6
U. ovata CBS 499.82
U. vinacea CBS 212.32 c3
U. versiformis CBS 473.74
U. vinacea CBS 212.32 cc
U. vinacea CGMCC 3.16349 c10
U. vinacea CGMCC 3.16357 c11
U. vinacea CBS 236.82 cb
U. ramanniana NRRL 5844
U. angularis CBS 603.68
U. vinacea CBS 212.32 c5
Mortierella verticillata NRRL 6337
Rhizomucor pusillus NRRL 2543
U. vinacea CGMCC 3.16352 c1
U. isabellina NRRL 1757
Mucor circinelloides f. lusitanicus
CBS 277.49
U. isabellina CBS 208.32
U. nana CBS 730.70
U. versiformis CBS 150.81 c4
U. ramanniana CGMCC 3.16356
U. autotrophica CBS 310.93
U. vinacea CGMCC 3.16357 c8
U. vinacea CBS 212.32 c4
U. fusiformis CBS 385.85
U. ramanniana NRRL 1296
U. swartii CBS 868.85
U. nana CBS 858.68
U. angularis CGMCC 3.6640
U. vinacea CBS 212.32 c7
U. gibberispora CBS 109328
U. nana NRRL 22420
U. dimorpha CBS 110039
U. vinacea CBS 236.82
c
Fig. 1 (continued)
Mycol Progress (2014) 13:657–669 665
≡Mortierella pusilla var. vinacea (Dixon-Stew.) Zycha,
Krypt.-Fl. Mark Brandenb., 6a: 198, 1935
=Mucor angulisporus Naumov [as 'angulispora'], Opred.
Mukor., Edn 2: 30 (1935)
≡Mortierella ramanniana var. angulispora (Naumov)
Linnem., Mucor.-Gatt. Mortierella Coem. 23: 19 (1941)
≡Micromucor ramannianus var. angulisporus Naumov
ex Váňová, Česká Mykol. 45(1–2): 26 (1991)
=Umbelopsis multispora Ts. Watan., Trans. Mycol. Soc.
Japan 18: 244 (1977)
Description: Colonies reaching35mmindiameteron
MEA after five days at 20 °C, appearing like a cottony felt,
not zonate, light Russet-Vinaceous (Ridgway, Pl. XXXIX)
in the center, lighter gradually towards the edge, sometimes
forming small sectors; on CMA after five days at 20 °C
reaching 26 mm in diameter, low, slightly pinkish in the
center when old, much sparser than on MEA.
Sporangiophores simply branched, (27.7–) 47.4–94.8
(−146.3) μm long, 2.4–2.8 μm wide near the base and
1.8–2.4 μm near the tip, 1–3 septate, often with one septum
near the base and one near the tip. Sporangia globose to
subglobose, (7.9–)9.9–12.3 (−14.2) μmindiameter,
reddish-brown, multi-spored; walls deliquescent leaving
no or small collars. Columellae absent or slightly convex.
Sporangiospores distinctly angular, (4.0–)4.7–5.5
(−6.3)×(3.2–)3.6–4.4 (−4.7) μm, reddish in mass, usually
Fig. 2 Umbelopsis changbaiensis.aSporangiophores showing different branching types; band cDetails of branchings on vesicles and sporangia; d
Sporangiophores with different sizes and shapes of columellae at their tips after the sporangia have been dissolved or detached; eSporangiospores
666 Mycol Progress (2014) 13:657–669
with one oil droplet. Chlamydospores elliptical or glo-
bose, single or bunching, (5.9–)6.9–10.2 (−12.8) ×(3.9–)
5.9–7.9 (−8.9) μm, containing abundant oil droplets.
Zygospores unknown.
Type: Figure 5 in Dixon-Stewart, Species of Mortierella
isolated from soil. Transactions of the British Mycological
Society 17, p.213, 1932 (lectotype designated here for the
basionym Mortierella vinacea). Australia, Victoria, sandy
Fig. 4 Colonies on MEA. a
Umbelopsis changbaiensis;b
U. vinacea
Mycol Progress (2014) 13:657–669 667
Fig. 3 Umbelopsis vinacea .aSporangiophores showing different
branching types; bDetails of sporangia and branching; c
Sporangiophores with no columellae or slightly convex at their tips after
the sporangia have been dissolved or detached, also note the position of
the upmost septa; dSporangiospores with oil droplet; eChlamydospores
loam, HMAS 244433 (epitype designated here for this lecto-
type) prepared from CBS 212.32 (ex-epitype designated
here).
Strains examined: Australia, Victoria, sandy loam, CBS
212.32; Japan, Shimane Pref., root of Fragaria,CBS
236.82; China, Tibet, Milin, Nanyi, alt. 3048 m, soil under
herbaceous plants, Xue-Wei Wang, Aug 2009, CGMCC
3.16349 and CGMCC 3.16350; China, Tibet, Milin,
Xuekagou, alt. 3000 m, soil under ferns, Liang-Dong Guo
and Qing-Ming Gao, Jul 2004, CGMCC 3.16351; China,
Tibet, Milin, Nanyi, alt. 3000 m, soil under bushes, Liang-
Dong Guo and Qing-Ming Gao, Jul 2004, CGMCC 3.16352,
CGMCC 3.16353, CGMCC 3.16354 and CGMCC 3.16355;
China, Jilin, Changbai Mountain, 42°10′548″–44°02′274″N,
126°35′399″–128°55′815″E, 360–2654 m alt., forest soil
mix, Xiao-Yong Liu, Jun 2011, CGMCC 3.16357.
Note: According to the protologue (Dixon-Stewart 1932),
Mortierella vinacea was isolated twice by D. Dixon-Stewart
from a sandy loam in Victoria, i.e., one from cultivated soil
during 1928 and the other from bush soil in 1929. However,
Dixon-Stewart did not designate any number in any herbarium
for those strains and no later typification was found. Hence, it
is necessary to designate a type for this species. The only
figure for M. vinacea in Dixon-Stewart (1932)isdesignated
here as the lectotype of M. vinacea.
CBS 212.32, which was isolated by D. Dixon-Stewart and
deposited by E. McLennan in January 1932, is an authentic
strain of Umbelopsis vinacea in the CBS collections database
(http://www.cbs.knaw.nl/Collections/BioloMICS.aspx?
Table=CBSstraindatabase&Rec=9970&Fields=All accessed
on 23 December 2012). In addition, morphological
characteristics of the strain CBS 212.32, well match the
original description of Mortierella vinacea (≡U. vinacea)
except for the branching pattern of sporangiophores. It is
racemosely branched in the original description but simple
or rarely racemosely branched in CBS 212.32. Due to the
unstability under different culture conditions, a branching
pattern should not be considered as a vital feature for
differentiating Umbelopsis species. No other cultures can fit
the original description better than CBS 212.32, therefore, a
dry culture of this strain is designated here as the epitype of
M. vinacea,basionymofU. vinacea.
Isolates from China are slightly different from CBS 212.32.
Colonies on MEA are denser and lighter in color;
sporangiospores are a little bigger and often without oil drop-
lets. The culture CBS 236.82 from Japan is more vigorously
branched and darker in colony color. These slight variations
might be caused by differences in geographical environment.
Taxonomic status of Mortierella ramanniana var.
incrustacea Chalab. & Zhdanova could not be determined
based on the simple information in the original description and
illustration. Furthermore, there is no access to obtain the
related strains. Therefore, it is considered as a doubtful name
and not mentioned in the synonym list, although it is synon-
ymized with Umbelopsis vinacea in the Index Fungorum
(http://www.indexfungorum.org/ Accessed on 21 Mar 2013).
Morphological variation in Umbelopsis
The studies pertaining to Umbelopsis have mainly concen-
trated on its higher taxonomic position for a long time (Turner
1963;Linnemann1969; Meyer and Gams 2003), while the
criterion for distinguishing species remains very vague. The
U. vinacea and its allies are typical examples. Turner (1963)
mentioned that some strains identified as U. vinacea may be
intermediate between true U. vinacea and Mortierella
ramanniana var. angulispora (Naumov) Linnem. (≡
U. angularis) or even closer to the latter due to their pro-
nounced columellae, which was also reported by Chen (Chen
1986; Chen 1992)inChina.Domschetal.(1980) pointed out
U. vinacea was almost without columella, but there are dis-
tinct columellae in their line drawing. Sugiyama et al. (2003)
found that the ex-type strain of Micromucor ramannianus var.
angulisporus Naumov ex Váňová (≡Mortierella ramanniana
var. angulispora)shouldbeU. vinacea and introduced a new
name U. angularis for other isolates identified under the
former name. Moreover, this research group (Chen 1992;
Wang et al. 2013) isolated strains of U. dimorpha Mahoney
& Gams (CGMCC 3.6641) earlier than its first publication
(Mahoney et al. 2004) but incorrectly preserved under the
name of U. vinacea. All these cases suggest that some old
strains of Umbelopsis might be incorrectly identified or even
belong to cryptic species.
Recently, with the help of phylogenetic analyses, the taxo-
nomic position of this genus has been clarified (Meyerand
Gams 2003). However, the genetic variation in the rDNA
region did not correlate well with the variation of the most
important morphological characteristic, namely the shape of
sporangiospores, in delimiting species of Umbelopsis
(Kendrick et al. 1994; Meyer and Gams 2003; Sugiyama
et al. 2003; Ogawa et al. 2005; Ogawa et al. 2011). It is
implied that some morphological characteristics used for spe-
cies delimitation may not reflect their true affinity.
Umbelopsis species with angular sporangiospores, i.e.,
U. angularis W. Gams & M. Sugiy., U. changbaiensis and
U. vinacea, could be separated by the presence and size of
chlamydospores. On CMA, U. angularis usually has two
types of chlamydospores, i.e., macro- and micro-
chlamydospores (Sugiyama et al. 2003); U. vinacea has
micro-chlamydospores only; and U. changbaiensis has none.
It suggests that the characteristic of chlamydospores, which
was not taken seriously before, might be an important classi-
fication feature for these three angular-sporangiospore-
forming Umbelopsis species. Further studies will focus on
the evaluation of taxonomic characteristics and finding out
interspecific distinguishable criteria in Umbelopsis .
668 Mycol Progress (2014) 13:657–669
Acknowledgments The project was partially supported by the National
Natural Science Foundation of China (Nos. 31070016 and 31070019)
and the Knowledge Innovation Program of the Chinese Academy of
Sciences (No. KSCX2-EW-J-6). CBS and NRRL are acknowledged for
supplying cultures for this study. The straininfo database (http://www.
straininfo.net/) is greatly appreciated for providing information of strains.
Thanks are due to Dr. Gerard Verkley of CBS, and Profs. Yi-Jian Yao and
Wen-Ying Zhuang of this department for their suggestions on nomencla-
ture. We also thank Ms. Hong-Mei Liu of this research group for taking
care of the cultures.
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