DNA Barcoding in <I>Mucorales</I>: an Inventory Of

Persoonia 30, 2013: 11–47 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158513X665070

DNA barcoding in Mucorales: an inventory of biodiversity

G. Walther1,2,3, J. Pawłowska4, A. Alastruey-Izquierdo5, M. Wrzosek4, J.L. Rodriguez-Tudela5, S. Dolatabadi3,6, A. Chakrabarti7, G.S. de Hoog3,6,8,9

Key words Abstract The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe Backusella immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa biodiversity were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular clinical relevance Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated DNA barcoding for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the intraspecific variability Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and ITS sporangiola, traditionally used in mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an LSU appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability Mucor was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for Mucorales the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 nomenclature possibly undescribed species were detected. Intraspecific variability differed widely among mucorealean species Rhizomucor ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains taxonomy was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly Zygorhynchus for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic Rhizomucor species belong to Mucor. Accordingly, Rhizomucor endophyticus was transferred to Mucor and Rhizomucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa.

Article info Received: 7 September 2012; Accepted: 1 January 2013; Published: 4 March 2013.

INTRODUCTION is increasing in hosts with severe immune or metabolic impair- ment, e.g. due to hemomalignancy, hematopoietic stem cell The order Mucorales represents a phylogenetically ancient transplantation or uncontrolled ketoacidotic diabetes mellitus group of fungi comprising predominantly saprotrophs inhabiting (Skiada et al. 2011). Infections often take a dramatic course and soil, dung and dead plant material, as well as several parasites have a high mortality rate. In risk group patients such as those on plants and on other fungi. Mucoralean strains have been with leukemia or allogenic bone marrow transplant an increase used for centuries in the fermentation of traditional Asian and of 8 % and 2 %, respectively, has been noted (Greenberg et al. African food such as tempeh or furu (fermented tofu) (Nout 2004). In part the clinical strains belong to the same species as & Aidoo 2010), and they also play a role in the production of the ones used in food fermentation. For example, Mucor cir- several kinds of cheese (Hermet et al. 2012). On the other cinelloides is used for starter cultures in Asian food (Hesseltine hand, some members of the Mucorales are responsible for the 1983, Nout & Aidoo 2010), but is also able to infect patients with spoilage of fresh and manufactured food (Pitt & Hocking 2009). an impaired immune system (e.g. Khan et al. 2009). Mucoralean fungi are also known to be involved in human Mucorales are among the best represented groups in fungal cul- infection. Mucormycoses are still very rare, but their incidence ture collections. They easily grow in axenic culture and they 1 Institute of Microbiology, Department of Microbiology and Molecular Biology, have been used as model organisms since the late 19th cen- University of Jena, Jena, Germany; tury. A large share of all species described in the order are corresponding author e-mail: [email protected]. represented today by living cultures publicly available in fungal 2 Leibniz-Institute for Natural Product Research and Infection Biology – Hans- reference collections. For example, the Centraalbureau voor Knöll-Institute, Jena Microbial Resource Collection, Jena, Germany. 3 CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. Schimmelcultures (www.cbs.knaw.nl) possesses 135 ex-type 4 Department of Systematics and Plant Geography, University of Warsaw, or authentic strains out of 227 currently accepted species. This Warsaw, Poland. is a unique situation, compared e.g. with dermatophytes which 5 Instituto de Salud Carlos III Mycology Department, Spanish National Center were described around the same period (Sabouraud 1910). for Microbiology, Madrid, Spain. 6 Institute for Biodiversity and Ecosystem Dynamics, University of Amster- Problematic for the nomenclatural stability of the Mucorales is dam, Amsterdam, The Netherlands. the practice of many early authors to designate a living strain 7 Departments of Medical Microbiology, Postgraduate Institute of Medical as ‘type’ although this was permitted by the International Code Education and Research, Chandigarh, India. of Botanical Nomenclature. Since 2000 Art. 8.4 of ICBN has 8 Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China. allowed deposition of metabolically inactive cultures as types 9 Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. (Greuter et al. 2000). In order to link these original strains to the

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Nothing in this license impairs or restricts the author’s moral rights. 12 Persoonia – Volume 30, 2013 respective names we designated the vial with the lyophilised between members and is not alignable over the entire order. strain that was prepared at time of its accession as lectotypes. ITS sequences of some species differ to such an extent that If the original strain was not lyophilized in the year of its ac- they could not be aligned confidently with their putative sibling cession we lectotypified the name by the original illustration species. To establish the phylogenetic position of species and designated the original strain as epitype. In the case of and to acquire an overview of the entire order that includes Zygorhynchus exponens a neotype was chosen because the all groups recognized on the basis of molecular data, the D1/ original figures were not specific and no other authentic mate- D2 region of the large subunit (LSU) rDNA was sequenced rial is known to exist. from one strain of each Molecular Operational Taxonomic Unit Taxonomy of Mucorales has traditionally been based upon (MOTU). A MOTU is defined by ITS similarities with mutual microscopic morphology and mating experiments. The classi- threshold values of > 99 %. Using this approach a species can cal works of Maria A.A. Schipper (Schipper 1973, 1975, 1976, be represented by a single or by several MOTUs depending 1978a, b, 1979, 1984, 1986, 1990, Schipper & Samson 1994, on intraspecific variability. Schipper & Stalpers 1984, 2003) provided model studies and Analyses of the ITS region as a single locus can not be used have long remained satisfactory for the identification of major to define species boundaries, but, conversely, hypotheses on species. A large number of names were synonymised. However, species limits can be developed by plotting morphospecies on molecular phylogeny has revealed that diversity within and the ITS trees. Therefore only those taxonomic rearrangements between species is much larger than anticipated, and this has were made that did not require exact knowledge on species led to a proliferation of the number of taxa recognised. Since limits. These revisions will be discussed, and nomenclatural the older, morphological synonyms were a priori omitted from status of names analysed. most studies, the respective names remained obscure and ITS barcodes of 668 strains in 203 taxa (178 species, 19 varie- were not included in nomenclatural comparisons. New names ties, 6 forms) were generated for this study covering 78.4 % are being introduced today for species that do not match any of the recognized species in Mucorales. Seventy-six percent of the known taxa deposited in GenBank. Verification of the of the species are represented by ex-type strains or other au- ex-type strains of older, still valid names may prove that some thentic material. LSU sequences were generated for 43.5 % of the new names are later synonyms, and that the historical of the strains. Special attention was paid to the inclusion of names have to be re-installed. a high proportion of clinical strains predominantly provided DNA barcoding was originally aimed to allow faster and more by the Spanish National Center for Microbiology in Madrid precise species identification. However, the accuracy of this (Spain) and the Postgraduate Institute of Medical Education method strongly depends on completeness of taxon sampling and Research in Chandigarh (India). The paper focuses on and on taxonomic elaboration (Meyer & Pauley 2005). Since the genera Actinomucor, Mucor and allies, Rhizomucor and polyphyly was revealed with molecular data in many morphology- Rhizopus because ITS trees have been published for the re- based families and genera (O’Donnell et al. 2001, Voigt & maining medically important genera: Apophysomyces (Álvarez Wöstemeyer 2001), several groups have been revised using et al. 2010b), Cunninghamella (Liu et al. 2001), Lichtheimia molecular phylogenetic analyses, e.g. Actinomucor (Zheng & Liu (Alastruey-Izquierdo et al. 2010), Saksenaea (Álvarez et al. 2005, Khan et al. 2008), Apophysomyces (Álvarez et al. 2010b), 2010a) and Syncephalastrum (Vitale et al. 2012). Cunninghamella (Liu et al. 2001), Lentamyces (Hoffmann & In main traits we follow the nomenclature of the species pro- Voigt 2009), Lichtheimia (Alastruey-Izquierdo et al. 2010), vided by Species Fungorum (http://www.speciesfungorum.org) Pilobolus (Foos et al. 2011), Rhizopus (Abe et al. 2006, 2007, based largely on the 10th edition of the Dictionary of the Fungi. 2010, Liu et al. 2007, Gryganskyi 2010), Saksenaea (Álvarez We adopted the family structure by Hoffmann et al. (2013). et al. 2010a), Siepmannia (Kwaśna & Nirenberg 2008a, b) and Umbelopsis (Meyer & Gams 2003). However, some genera, e.g. Absidia s.str., Circinella and relatives, or Syncephalastrum MATERIALS AND METHODS have not been revised using molecular data. The largest mu- corelean group, Mucor and its relatives, has been investigated Strains only fragmentarily focusing on certain clades (Jacobs & Botha A total number of 668 mucoralean strains belonging to 178 2008, Budziszewska et al. 2010, Álvarez et al. 2011, Madden species, 19 varieties and 6 formae and covering 78.4 % of et al. 2011, Hermet et al. 2012). Only a few publications (Abe the currently accepted species was studied. For 22.2 % of the et al. 2007, Alastruey-Izquierdo et al. 2010, Gryganskyi 2010, taxa it was possible to include five or more strains per lowest Hermet et al. 2012) use at least two unlinked molecular markers taxonomic level (form, variety or species), 60.1 % of the taxa and apply sufficient strain and taxon sampling to adopt concepts were represented by 2 or more isolates. Ex-type strains or other of genealogical concordance phylogenetic species recognition authentic material was available for 75.8 % of the studied spe- (GCPSR, Taylor et al. 2000) satisfactorily. As a consequence, cies. In order to cover intraspecific variability, strains from the the criteria of good taxonomy are insufficiently met, and many most distant localities and from a wide range of substrates were species in Mucorales are poorly delimited. It was, therefore, the selected. Special attention was paid to the inclusion of clinical primary aim of the present study to inventory the genetic diver- isolates. Studied strains originated from the reference collection sity of Mucorales deposited in the CBS culture collection and to of the CBS-KNAW Fungal Biodiversity Centre (CBS; Utrecht, highlight critical groups that need to be studied by a multi-locus The Netherlands), the Instituto de Salud Carlos III National Cen- approach. Our paper provides DNA barcodes for all ex-type tre of Microbiology (CNM-CM; Madrid, Spain), the Departments and authentic strains of Mucorales available in the CBS culture of Medical Microbiology (PGIMER; Chandigarh, India) or the collection, and makes these data available by open access as Belgian Co-ordinated Collections of Micro-organisms (IHEM; reference for subsequent studies on biodiversity and taxonomy. Brussels, Belgium). The studied strains, source information and Recently, the Fungal Barcoding Consortium (Schoch et al. GenBank accession numbers are listed in Table 1. 2012) proposed the rDNA internal transcribed spacer (ITS) as a universal DNA barcode marker for fungi. In our study ITS DNA extraction, PCR amplification, cloning and sequencing was also applied because of its discriminative power in Muco- Genomic DNA was extracted in most cases from 2d-old cultures rales (Meyer & Gams 2003, Kwaśna et al. 2006, Schwarz et grown on malt extract agar (MEA 5 %, Oxoid, Badhoevedorp, al. 2006, Vitale et al. 2012). The ITS region is highly variable (text continues on p. 26) G. Walther et al.: DNA barcoding in Mucorales 13 to that strain, taxa belongs JN206589 JN206594 JN206580 JN206583 LSU JN206595 HM849704 JN206591 JN206587 JN206592 those 103.91 for = ex-holotype CBS only HT strain, JN205814 JN205817 HM849707 JN205822 JN206588 JN205819 JN206579 JN205812 JN205811 HM849703 JN205821 JN206584 JN205818 JN206585 JN205816 JN206582 ITS JN205815 JN206593 JN205847 JN205820 JN206581 JN205813 HM849706 JN205810 HM849705

JN205809 JN206590 JN205827 JN206491 JN205828 JN205826 JN205829 HM849686 JN205823 JN206493 JN205824 JN206281 HM849695 JN206280 JN206536 JN206279 JN205825 JN206492 JN206258 JN206529 JN206257 JN206259 JN206264 (c2) JN206261 JN206263 (c1) JN206524 JN206522 JN206256 JN206270 JN206278 JN206251 JN206265 JN206533 JN206262 (c1) JN206407 JN206523 JN206271 JN206530 JN206268 JN206531 JN206269 JN206255 JN206526 JN206260 literature original Absidia idahoensis * = ex-epitype with ET strain, checked was LSU-sequences: Nomia melanderi ; leaf or = ex-type T Musa sapientum ITS- by: information ; diseased root their Type on soil of pastured hardwood rhizosphere of soil of pine forest myxomycete n.a. sandy soil clay soil forest soil soil soil dung of rabbit dung of rat dung of mouse Source soil brood chamber of manure, in asparagus ﬁeld soil of pine forest virgin soil ambrosia beetle; gland wallpaper n.a. Comandra pallida n.a. n.a. n.a.

n.a. human; wound soil soil of grassy site soil of mango orchard n.a. soil with lichens soil n.a.

Fragaria soil forest soil moist wall agaric soil decaying wood fallen leaf n.a. n.a. n.a. n.a. designated based are material. genera Antilles human; osteomyelitis strains = authentic

different Ex-type to AUT Netherlands USA Honduras Germany n.a. USA USA Denmark n.a. USA USA USA USA Country Cuba USA Germany France former Czechovakia Tanzania soil Canada UK, England n.a. USA n.a. n.a. USA

Egypt Kuwait Nepal Netherlands India India n.a. USA USA Japan

Australia China Macedonia France USA Switzerland Japan and Mucor . belong to Norway strain asterisk belongs an with 428.80 = ex-syntype (designated here) Japan (designated here) USA ST (designated here) Japan (designated here) India CBS

T T T

T Status T

T T T T T IT T IT

T PT

T T of Mucor dispersus T

marked strain,

Strains LT LT

strains. nigra var.

= ex-paratype

PT studied the strain, of A. cylindrospora

Species name at the beginning of the study

A. kuwaitiensis

A. elegans

A. taiwanensis

recurvus M. recurvus var.

Mucor oblongisporus recurvus M. recurvus var. recurvus M. recurvus var. recurvus M. recurvus var. ET Mucor oblongiellipticus

indicus M. recurvus var.

Gongronella , *** Circinella simplex

numbers

= ex-neotype belongs

accession

101757 nigra var. rhizomorpha var. cylindrospora var.

meitauzae var. kuwaitiensis var. strain,

CBS biappendiculata var. spinosa var.

GenBank

and = ex-lectotype LT Absidia fusca Absidia glauca Absidia cylindrospora Absidia cylindrospora Absidia fusca Absidia cylindrospora Absidia cuneospora Absidia cuneospora Absidia coerulea Absidia coerulea Absidia coerulea Absidia coerulea Absidia californica Absidia californica Species Absidia anomala Absidia macrospora Absidia glauca Absidia spinosa Absidia heterospora Absidia idahoensis Absidia pseudocylindrospora Absidia psychrophilia Absidia repens Absidia spinosa Actinomucor elegans Actinomucor elegans Actinomucor elegans Actinomucor elegans Actinomucor elegans Actinomucor elegans Actinomucor elegans Backusella circina Apophysomyces variabilis Apophysomyces elegans Apophysomyces elegans Backusella circina Backusella circina Backusella circina Backusella recurva Backusella circina Backusella oblongielliptica Backusella oblongispora Backusella recurva Backusella recurva Backusella recurva Backusella lamprospora Backusella lamprospora Backusella lamprospora Backusella lamprospora Backusella indica information strain, Circinella lacrymispora ** = ex-isotype are treated in the taxonomy part; remaining data were derived from CBS database. Table 1 Source Table CBS 346.97 CBS 100.48 CBS 127.68 CBS 153.63 CBS 102.35 CBS 100.08 CBS 101.59 CBS 102.59 CBS 628.70B CBS 104.08 CBS 102.28 CBS 101.28 CBS 126.68 CBS 314.78 Strain number CBS 125.68 CBS 697.68 CBS 101.08 CBS 187.64 CBS 101.29 CBS 103.91 CBS 100.62 CBS 128.68 CBS 115583 Circinella ; CBS 106.08 IT CBS 100.09 CBS 100.22 CBS 111556 CBS 154.86 CBS 338.72 CBS 111558 CBS 117697 CBS 128.70 CBS 658.93 CBS 477.78 CBS 476.78 CBS 129.70 CBS 323.69 CBS 382.95 CBS 673.75 CBS 907.73 CBS 568.70 CBS 569.70 CBS 196.71 CBS 317.52 CBS 318.52 CBS 244.67 CBS 195.28 CBS 118.08 CBS 107.09 CBS 786.70 14 Persoonia – Volume 30, 2013 LSU JN206253 (c3) JN206254 (c1) JN206528 HM999964 JN206267 HM849692 ITS JN205881 JN205874 JN206599 JN205871 JN206600 JN206243 JN206410 JN206266 JN205873 JN205872 JN205879 JN205876 JN205877 JN205875 JN206245 JN206408 JN206244 HM849699 JN205859 JN205856 JN205858 HM849722 JN205857 JN206553 JN205850 JN205852 JN205851 JN205853 JN206548 JN205854 JN206550 JN205862 JN205861 JN206552 JN205860 JN206289 JN206608 JN205855 JN206549 JN205849 JN206551 JN205848 JN206294 JN206231 JN206232 JN206586 JN206235 JN206234 JN206233 JN206236 JN206237 JN206514 JN206238 JN206513 JN206021 JN206022 JN206520 JN206019 JN206020 JN206519 JN206230 JN206515 JN206228 JN206229 JN206227 JN206242 JN206409 JN206241 JN206411 JN206023 ; flower

excrements of human Medicago sativa n.a. Source human; sputum n.a. humus-rich soil cultivated soil rotten log forest soil human; tibia human; heart, patient with lymphosarcoma JN205878 HM849701 human; lung (leukemic patient) human human; lung (leukemic patient) dung of rat dung of rabbit dung of mouse soil air dung of dog paramo soil under undisturbed vegetation JN206213 JN206445 n.a. preserved meat soil n.a. gold mine, depth of 600 m n.a. dung of mouse n.a. soil n.a. dung dung of rodent Pisum sativum ; root n.a. n.a. dead insect n.a. n.a. n.a. n.a. n.a. dung of rat litter dung of horse n.a. soil n.a. Hibiscus rosa-sinensis soil dung of lizard dung of rat meadow soil Yugoslavia soil

Egypt Country Spain n.a. India Japan China China USA USA USA USA USA USA USA Germany Kuwait Italy n.a. Colombia USA Netherlands Indonesia n.a. n.a. n.a. Netherlands n.a. Argentina Japan USA USA n.a. n.a. n.a. China n.a. n.a. Japan USA Netherlands Netherlands n.a. n.a. China Sweden Indonesia Panama Spain USA Norway former India

(designated here) India (designated here) India

Status

IT LT LT NT of C. polymorpha NT

T of C. sydowii T

T T NT

PT of B. sinensis PT

T T T

of C. heterospora T.

cucurbitarum cucurbitarum cucurbitarum cucurbitarum cucurbitarum infundibulifera infundibulifera infundibulifera f. f. f. f. f. f. f. f.

Mucor tuberculisporus Mucor tuberculisporus recurvus M. recurvus var. Species name at the beginning of the study

C. polymorpha

Mucor variabilis C. polymorpha C. polymorpha C. polymorpha

C. elegans

C. umbellata

C. infundibulifera C. infundibulifera C. infundibulifera C. infundibulifera C. infundibulifera C. infundibulifera C. infundibulifera C. infundibulifera

B. sinensis

***

sp. Benjaminiella multispora

Backusella tuberculispora Backusella tuberculispora Backusella Species Cunninghamella bertholletiae Cunninghamella bertholletiae Benjaminiella poitrasii Backusella variabilis Cunninghamella bertholletiae Cunninghamella bertholletiae Cunninghamella bertholletiae Cunninghamella bertholletiae Cunninghamella bertholletiae Cunninghamella bertholletiae Cunninghamella bertholletiae Cunninghamella bertholletiae Cokeromyces recurvatus Cokeromyces recurvatus Circinella umbellata Circinella umbellata Circinella umbellata Circinella umbellata Circinella simplex Circinella muscae Circinella muscae Circinella muscae Circinella muscae Circinella muscae Circinella minor Circinella minor Circinella minor Circinella lacrymispora Circinella chinensis Circinella angarensis Choanephora cucurbitarum Choanephora cucurbitarum Choanephora cucurbitarum Choanephora cucurbitarum Choanephora cucurbitarum Choanephora infundibulifera Choanephora infundibulifera Choanephora infundibulifera Circinella angarensis Chaetocladium jonesiii Chaetocladium brefeldii Chaetocladium brefeldii Chaetocladium brefeldii Chaetocladium brefeldii Blakeslea trispora Blakeslea trispora Blakeslea trispora Blakeslea trispora Benjaminiella youngii Chlamydoabsidia padenii

CBS 421.70

CBS 562.66 CBS 570.70 CBS 538.80 Strain number CNM-CM3628 CBS 779.68 CBS 158.60 CBS 564.66 CBS 693.68 CBS 373.95 CBS 372.95 CBS 191.84 CBS 190.84 CBS 186.84 CBS 182.84 CBS 151.80 CBS 168.59 CBS 158.50 CBS 837.97 CBS 195.74 CBS 160.49 CBS 101.16 CBS 428.80 CBS 720.76A CBS 342.79 CBS 159.49 CBS 141.28 CBS 107.13 CBS 143.56 CBS 142.81 CBS 102.16 CBS 101757 CBS 140.28 CBS 173.62 CBS 120.25 CBS 150.51 CBS 178.76 CBS 445.72 CBS 674.93 CBS 153.51 CBS 155.51 CBS 155.58 CBS 172.62 CBS 811.69 CBS 162.82 CBS 156.74 CBS 136.28 CBS 116.24 CBS 564.91 CBS 198.80 CBS 137.49 CBS 130.59 CBS 103.89 CBS 172.67 Table 1 (cont.) Table G. Walther et al.: DNA barcoding in Mucorales 15 JN206610 JN206471 JN206609 JN206539 JN206517 JN206607 JN205889 JN206603 JN205888 JN206602 JN205880 JN205865 JN205868 JN205870 JN205867 HM999958 JN206222 JN206220 HM849691 JN206225 JN205866

JN206055 JN206052 JN206053 JN206193 JN206506

JN206287 JN206288 JN206285 HM849698 JN205846 HM849723 JN206221 JN205844 JN206540 JN205993 JN205992 JN206423 JN205843 JN206557 JN205842 JN205841 JN206556 JN205839 JN206555 JN205840 HM849715 JN205897 HM849693 JN205864 HM849697 JN205892 JN206606 JN205863 JN206605 JN205884 JN205885 JN205887 JN205886 JN205882 HM849700 JN205893 JN206597 JN205894 JN205896 JN206598 JN205895 HM849702 JN205891 JN205890 JN206604 JN205869 JN206601 JN206223 JN206219 JN206284 JN206286 JN206226 JN206218 ; leaf forest ; seed ; fruit ; root soil n.a. human; skin n.a. n.a. Linum issitatissimum n.a. Prunus persica trickling ﬁlter plant Saccharum officinarum wood decaying fruit rice-field soil spoiled beef crackling air-dried raw sausage isopod, on the underside of a disturbed pine stump JN206054 dead isopod (woodlouse) n.a. forest soil with effluent dooryard soil mud from mangrove forest, contaminated JN206290 JN206596 volcanic ash soil soil garden soil dung of shrew poplin n.a. dung of house lizard dung of mouse dung of lizard dung of mouse dung of mouse soil of a cultivated ﬁeld dung of mouse dung of rodent soil of Shorea robusta soil n.a. soil human; lung, biopsy sample n.a. soil under shrub vegetation Linum usitatissimum soil n.a. soil n.a. soil n.a. n.a. Cocos nucifera soil n.a. dung of mouse n.a. n.a. dung of swine n.a.

Brazil n.a. Spain Switzerland n.a. Canada Kuwait USA USA Indonesia Indonesia Croatia Brazil USA Germany UK n.a. India

USA Australia Chile Brazil Japan India USA India USA USA USA USA India USA USA India Indonesia India Japan Spain n.a. former Czechoslovakia n.a. Turkey Canada Chile n.a. Egypt n.a. China n.a. n.a. Indonesia India Pakistan Italy n.a. n.a. India UK China UK, England

T of H. venustellum T

T T

AUT T AUT

T T

T NT T of C. brunnea T T

T of G. urceolifera T of Mucor vesiculosus T indica of G. persicaria var. T

T of G. hainanensis T

persicaria var.

C. elegans C. elegans

persicaria G. persicaria var. persicaria G. persicaria var. persicaria G. persicaria var. Rhizopus stolonifer C. elegans

persicaria G. persicaria var. G. persicaria

C. phaeospora

C. phaeospora C. antarctica

indica G. persicaria var. persicaria G. persicaria var. G. hainanensis Mucor thermophilus

antarctica var.

robustum robustum hesseltinei elegans elegans blakesleeana binariae binariae blakesleeana blakesleeana blakesleeana blakesleeana blakesleeana vesiculosa phaeospora intermedia homothallica elegans elegans elegans elegans elegans echinulata echinulata echinulata echinulata clavata clavata persicaria persicaria persicaria persicaria persicaria persicaria persicaria persicaria persicaria Cunninghamella Cunninghamella Cunninghamella Cunninghamella bertholletiae Cunninghamella Cunninghamella Cunninghamella Cunninghamella Gilbertella Gilbertella Gilbertella Gilbertella Cunninghamella Hesseltinella vesiculosa Helicostylum pulchrum Helicostylum pulchrum Helicostylum pulchrum Helicostylum elegans Helicostylum pulchrum Helicostylum cordense

Gongronella lacrispora Halteromyces radiatus Gongronella butleri Gongronella butleri Gilbertella persicaria Gilbertella Fennellomyces heterothallicus Fennellomyces linderi Fennellomyces heterothallicus Ellisomyces anomalus Ellisomyces anomalus Dichotomocladium Dichotomocladium Dichotomocladium Dichotomocladium Dichotomocladium Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Cunninghamella Gilbertella Gilbertella Gongronella butleri Gongronella butleri Gilbertella Gilbertella Gongronella butleri Gongronella butleri

CBS 133.27 CBS 481.66 CBS 158.28 CNM-CM3650 CBS 177.36 CBS 224.64 CBS 433.84 CBS 720.85 CBS 246.59 CBS 325.71A CBS 325.71D CBS 403.51 CBS 782.68 CBS 197.68 CBS 639.69 CBS 259.68 CBS 258.59 CBS 169.57 CBS 107.23 CBS 223.63

CBS 244.62 CBS 162.75 CBS 969.73 CBS 415.67 CBS 190.32 CBS 421.77 CBS 292.86 CBS 158.54 CBS 290.86 CBS 697.76 CBS 243.57 CBS 440.76 CBS 439.76 CBS 164.61 CBS 714.74 CBS 695.76 CBS 989.96 CBS 692.68 CBS 347.69 CBS 168.53 CNM-CM5114 CBS 781.68 CBS 773.68 CBS 318.78 CBS 167.53 CBS 545.75 CBS 766.68 CBS 656.85 CBS 156.28 CBS 362.95 CBS 100178 CBS 442.64 CBS 532.77 CBS 157.25 CBS 216.58 CBS 565.91 CBS 785.97 CBS 102.44 CBS 179.28 16 Persoonia – Volume 30, 2013 LSU ITS JN206211 JN206439 JN206212 JN206440 GQ342889 GQ342904 JN206293 GQ342885 GQ342886 JN206535 GQ342888 GQ342942 GQ342887 GQ342939 GQ342878 GQ342910 GQ342879 GQ342932 GQ342882 GQ342903 GQ342881 GQ342914 GQ342884 GQ342918 GQ342883 GQ342917 GQ342880 GQ342919 GQ342948 GQ342915 GQ342896 GQ342898; GQ342902 GQ342897 GQ342943 GQ342895 GQ342907 GQ342893 GQ342905 GQ342894 GQ342944 GQ342891 GQ342946 GQ342892 JN206554 GQ342890 GQ342936 GQ342876 GQ342941 GQ342858 GQ342940 GQ342851 GQ342938 GQ342859 GQ342945 GQ342865 GQ342947 GQ342847 GQ342908 GQ342848 GQ342906 GQ342877 GQ342934 GQ342875 GQ342937 GQ342874 GQ342909 GQ342849 GQ342912 GQ342856 GQ342935 GQ342863 GQ342926 GQ342869 GQ342924 GQ342862 GQ342923 GQ342861 GQ342922 GQ342860 GQ342921 GQ342854 GQ342953 GQ342852 GQ342920 GQ342853 GQ342931 GQ342873 GQ342930 GQ342868 GQ342855; GQ342929 GQ342871 GQ342928 GQ342864 GQ342927 ; nut ; stem ; seed ; fruit Source n.a. Burmannia n.a. grassland soil, surface layer n.a. n.a. mouldy shoe indoor air soil aborted cow human; keratomycosis human; ﬁnger tissue human; lung human; bronchia human; bone Bertholletia excelsa n.a. Manihot esculenta fermented food taosi n.a. dung of bird human; wound n.a. n.a. dung of cow n.a. Guinea pig; lung human; cornea Musa sapientum culture contaminant cocoa soil n.a. Theobroma cacao cultivated ﬁeld soil n.a. human; sputum human human; skin human; skin human; skin human; lung human; skin human; bronchoaspirate human; skin

human; skin human; sputum human

Country India Malaysia n.a. n.a. India Germany Afghanistan n.a. France Norway France France France UK n.a. Philippines Spain n.a. n.a. Indonesia n.a. Netherlands Switzerland Netherlands n.a. n.a. Ghana India n.a. Spain Spain Spain Spain Spain Spain Spain Spain Spain

Spain Spain Belgium USA Japan Japan Ghana n.a. India blakesleeana blakesleeana of Absidia of Absidia cristata of Absidia Status T

T of Absidia griseola T NT T of Absidia ornata T

T of Absidia gracilis T

atrospora var. ; T ; T ; var. T ; NT NT

blakesleeana

; Absidia blakesleeana blakesleeana blakesleeana Species name at the beginning of the study

Mycocladus blakesleeanus Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Absidia griseola

Mycocladus blakesleeanus Absidia Absidia Mycocladus blakesleeanus Absidia Mycocladus blakesleeanus Absidia hyalospora atrosporus Mycocladus corymbifer Mycocladus hyalosporus atrospora var.

Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer

Mycocladus corymbifer Mycocladus corymbifer Mycocladus corymbifer

Species Lentamyces parricida Hyphomucor assamensis Hyphomucor assamensis Lichtheimia hyalospora Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia corymbifera Lichtheimia hyalospora Lichtheimia hyalospora

Lichtheimia hyalospora Lichtheimia ornata

Lichtheimia hyalospora Lichtheimia ornata Lichtheimia ornata Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa

Lichtheimia ramosa Strain number CBS 174.67 CBS 415.77 Table 1 (cont.) Table CBS 254.85 CBS 100.28 CBS 100.17 CBS 100.51 CBS 102.48 CBS 115811 CBS 429.75 CBS 100.31 CBS 101040 CBS 109940 CBS 120580 CBS 120581 CBS 120805 CBS 519.71 CBS 102.36 CBS 100.36

CBS 518.71 CBS 291.66

CBS 173.67 CNM-CM4978 CBS 958.68 CBS 100.24 CBS 100.49 CBS 100.55 CBS 101.51 CBS 101.55 CBS 103.35 CBS 124198 CBS 223.78 CBS 271.65 CBS 582.65 CBS 649.78 CBS 713.74 CNM-CM2166 CNM-CM3590 CNM-CM4119 CNM-CM4228 CNM-CM4253 CNM-CM4261 CNM-CM4337 CNM-CM4427 CNM-CM4537 CNM-CM4849 CNM-CM5111

CNM-CM5171 G. Walther et al.: DNA barcoding in Mucorales 17 GQ342867 GQ342955 GQ342850 GQ342913 JN206110 JN206457 GQ342870 GQ342951 GQ342901 JN206111 GQ342916 JN206057 GQ342857 GQ342949 GQ342899 GQ342911 JN205933 JN205969 HM849679 JN205973 JN205956 JN205965 JN206056 JN206461 GQ342866 GQ342900 GQ342954 GQ342933 JN205957 JN205966 JN205958 JN205967 JN205959 JN205972 JN205961 JN205968 JN205942 HM849680 JN205962 JN205955 JN206170 GQ342872 GQ342925 JN205999 JN205954 JN205943 JN206413 JN206014 JN205996 JN205994 JN206430 JN205998 JN206418 JN206015 JN205997 JN206426 JN206428 JN205995 JN205910 JN206424 JN206084 JN206452 JN206083 JN206451 JN206187 JN206497 JN206173 JN206174 JN206499 JN206172 JN206504 HM999957 HM849688 JN206171

sp. Apis mellifera ; fermented fruit juice Dendrobates soil Candida albicans human; wound, double infection with forest soil human; abscess of the flank soil forest soil human; ear n.a. dung of mouse human; face thawing beef meat fermenting rice, component of ‘Chinese yeast’ soil JN205934 garden soil n.a. JN206412 soil human; gastric juice n.a. sufu, chao commercial honey human; thigh necrosis after trauma Artocarpus ; fruit dung of pig n.a. air aestivum Triticum soil air n.a. diseased human; corneal exudate human n.a. sufu starter soil Morinda citrifolia n.a. n.a. soil faeces of diseased forest soil aphid agaric soil dung of lizard Gossypium ; seed dung of lizard garden soil amphibian human Africa

Germany Russia Greece India UK n.a. India Netherlands n.a. Finland India n.a. India Spain India Spain Germany n.a. Netherlands South Netherlands Turkey Germany Ukraine Vietnam Central America Spain Japan Austria China Chile n.a. Turkey Armenia UK, England Hawaii island, USA Netherlands India Japan USA USA India Mozambique Germany Germany China var. China Germany variabilis (designated here) Germany (designated here) India of Rhizomucor thermophila T of Absidia idahoensis T

regularior var.

T NT of M. griseoroseus T

T IT

T ET

var. T janssenii janssenii

racemosus blakesleeanus blakesleeanus blakesleeanus f. f. silvaticus hiemalis hiemalis

variabilis aff. aff. aff. f.

f. f. variisporus

Mycocladus corymbifer

Mycocladus corymbifer M. hiemalis Mycocladus corymbifer

Mycocladus Mycocladus corymbifer regularior M. rouxii

Mycocladus Mycocladus corymbifer M. ramosissimus Rhizomucor

Mycocladus Mycocladus corymbifer M. hiemalis M. racemosus f.

M. circinelloides M. hiemalis M. circinelloides M. hiemalis M. flavus M. plumbeus

M. aff.

circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides

f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f.

Lichtheimia ramosa Lichtheimia ramosa Lichtheimia ramosa

Mucor aligarensis Lichtheimia ramosa Mucor abundans Mucor abundans Mucor aligarensis Lichtheimia ramosa Lichtheimia sphaerocystis Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor amphibiorum Lichtheimia ramosa Lichtheimia sphaerocystis Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor amphibiorum Lichtheimia sphaerocystis (non of the described formae) Mucor circinelloides Mucor circinelloides Mucor circinelloides (non of the described formae) Mucor circinelloides (non of the described formae) Mucor circinelloides (non of the described formae) Mucor circinelloides (non of the described formae) Mucor circinelloides (non of the described formae) Mucor circinelloides (non of the described formae) Mucor circinelloides Mucor brunneogriseus Mucor circinelloides Mucor bainieri Mucor bacilliformis Mucor bacilliformis Mucor ardhlaengiktus Mucor azygosporus Mucor ardhlaengiktus Mucor amphibiorum Mucor ardhlaengiktus AS 3.4808 CBS 112528 CBS 124197

CBS 244.58 CBS 269.65 CBS 388.35 CBS 521.66 CBS 993.70 CNM-CM1638 CBS 648.78 CBS 394.68 CBS 416.77 CBS 479.70 CBS 480.70F CBS 480.70G CBS 185.77 CNM-CM3148 CBS 647.78 CBS 121702 CBS 123973 CBS 172.27 CBS 192.68 CBS 194.68 CBS 195.68 CBS 196.68 CBS 239.35 CBS 247.35 CBS 295.34 CBS 384.95 CBS 763.74 CBS 420.70 CBS 108.16 CBS 111555 CBS 111560 CNM-CM5225 CBS 202.28 CBS 338.71 CBS 526.68 CBS 635.65 CBS 846.73 CBS 129.41 CBS 124110 CBS 293.63 CBS 573.70 CBS 251.53 CBS 650.78 CBS 292.63 CBS 528.73 CNM-CM2934 CBS 210.80 18 Persoonia – Volume 30, 2013 LSU ITS JN205985 JN206427 HM999953 JN206419 JN205982 JN205981 JN205986 JN205984 JN205988 JN205987 JN205979 JN206422 JN205990 (c4) JN205983 (c2) JN205989 (c3) JN205991 (c1) JN206429 JN205980 JN206011 JN206416 JN206009 JN206005 JN206007 JN206008 JN206004 HM999952 JN206425 JN206010 JN206006 JN206012 JN205970 JN205975 JN205952 JN205938 JN205971 JN205964 JN205936 JN205937 JN205960 JN205935 JN205941 JN206003 HM999951 JN206000 JN206421 JN206001 JN206420 JN206002 JN205953 JN205950 JN205939 JN205944 JN205963 JN205945 JN206414 JN205946 JN205947 Source forest soil n.a. n.a. n.a. Zea mays n.a. Zea mays ; grain n.a. Tremella

from sufu factory sufu starter,

from sufu factory sufu starter, n.a. milk powder dung of Dixippus morosus human n.a. n.a. n.a. forest soil iguana; lung grassland soil human; thigh leukemia B cells) human; skin of arm (burned patient) human; exudate (traumatism) human; wound skin human; bronchioaspirate human human; skin of hand, trauma human; wound human human; wound (burned patient) human (burned patient) human soil n.a. n.a. canned strawberries Zea mays esplenomectomy) human; bronchioaspirate (acute lymphoblastic JN205951 amputation) human; skin (surgical wound) human; wound exudate (polytraumatism, JN205974 JN206415 human; nail human; nail human; wound exudate human; peritoneal dialysis human; urine human; reservoir human; catheter (low respiratory tract infection) human; arm wound (traumatism with arm JN205948 JN205949 Africa Africa

Country Russia Mexico n.a. Portugal South Afrika n.a. USA n.a. Germany

China

China n.a. Netherlands n.a. South France n.a. India n.a. USA? Spain Spain Spain Spain Belgium Italy Belgium France Belgium Belgium Belgium Norway n.a. Netherlands Netherlands South Africa

Spain, Canaries Spain human; otic exudate

Spain Spain

Spain Spain Spain Spain Spain Spain Spain Spain Russia (designated here) South Status

T of M. tenellus T

T of M. kurssanovii T

racemosus ramosissimus Species name at the beginning of the study M. fragilis

M. indicus

M. racemosus f.

M. aff.

lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus lusitanicus janssenii janssenii janssenii janssenii janssenii janssenii janssenii janssenii janssenii circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides griseocyanus griseocyanus griseocyanus griseocyanus griseocyanus janssenii circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides circinelloides f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. f. Species Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides

Mucor circinelloides

Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Mucor circinelloides Strain number CBS 969.68 CBS 968.68 CBS 851.71 CBS 847.72 CBS 633.65 CBS 276.49 CBS 253.35 CBS 242.33 CBS 236.35

CBS 111229

CBS 111228 CBS 108.17 CBS 762.74 CBS 365.70 CBS 243.67 CBS 232.29 CBS 227.29 CBS 206.68 CBS 205.68 CBS 204.68 CBS 185.68 CNM-CM4621 CNM-CM4895 CNM-CM4974 CNM-CM5071 CNM-CM5169 IHEM 16415 IHEM 20006 IHEM 21105 IHEM 21158 IHEM 21426 IHEM 22323 CBS 116.08 CBS 198.28 CBS 223.56 CBS 366.70 CBS 698.68 CBS 144.93 CNM-CM4526 CNM-CM4569 CNM-CM4299 CNM-CM4366 CNM-CM2541 CNM-CM2922 CNM-CM3112 CNM-CM3178 CNM-CM3510 CNM-CM3785 CNM-CM3926 CNM-CM2437 Table 1 (cont.) Table G. Walther et al.: DNA barcoding in Mucorales 19 JN206132 JN206449 JN206130 JN206142 JN206141 JN206140 JN206138 JN206127 JN206165 (c4) JN206164 (c3) JN206485 JN206163 JN206486 JN206167 JN206487 JN206168 (c4) JN206169 (c1) JN206488 JN206162 (c4) JN206161 (c1) JN206490 JN206082 JN206475 JN206252 JN206527 JN206247 JN206494 JN206246 JN206047 (c4) JN206044 (c3) JN206046 (c3) JN206436 JN206045 (c1) JN206043 (c2) JN206042 (c1) JN206202 JN206041 JN206435 JN206200 JN206157 JN206447 JN206201 JN206442 JN206204 JN206203 JN206068 (c3) JN206067 (c1) JN206467 JN206063 (c2) JN206062 (c1) JN206465 JN206070 (c3) JN206069 (c2) JN206473 JN206064 JN206051 JN206468 JN206061 JN206464 JN206050 JN206462 JN206049 JN206066 JN206469 JN206470 JN206249 JN206250 JN206509 JN206207 JN206208 JN206112 JN206113 JN206159 JN206456 JN206206 JN206448 JN206441 JN205977 JN205978 JN205976 JN206417 Bryum argenteum ; leaves ; fruit ; brown needle forest soil n.a. human human; nail human; nail agricultural soil n.a.

soil sandy soil in potato ﬁeld n.a.

sediment in drain pipe

cattle feed dung of Periplaneta americana dung of mouse soil soil Malus sylvestris

n.a.

dung of ﬁeld-mouse dung of mouse Picea abies soil cheese cheese n.a. n.a.

coastal gravel flat covered by

forest soil

wheat ﬁeld soil forest litter n.a. dung of roe dung of peacock dung of mouse dung of wood mouse dung of rabbit honeycomb n.a. ploughed ﬁeld soil soil soil aestivum Triticum n.a. dung of pack rat dead plant material desert soil Russia Norway Austria Spain Spain Denmark n.a.

Colombia Netherlands

Sweden

Netherlands Russia India China China Netherlands

USA

Ukraine Finland India Switzerland France France Germany

Antarctica

Russia

Germany Finland Germany Germany Czech Republic floor of room in cave, used for speleotherapy JN206065 JN206472 USA Germany Germany Germany Ukraine China USA Kenya USA India var. France Ukraine

(designated here) Germany (designated here) n.a. (designated here) UK, England of Z. exponens var. UK, England

T T T

echinosporus of M. bedrchanii T T of M. petrinsularis T

NT of M. sciurinus NT

NT T of M. attenuatus T of M. peacockensis T T of M. mephitis T

HT T of M. meridionalis T

HT var var. NT var. LT

heterogamus heterogamus heterogamus heterogamus heterogamus psychrophilus exponens exponens exponens endophyticus rigida rigida

M. hygrophilus

Zygorhynchus Zygorhynchus Zygorhynchus

Zygorhynchus

exponens

smithii (designated here) Zygorhynchus Circinella Rhizomucor Zygorhynchus exponens Zygorhynchus Circinella Backusella ctenidia Backusella ctenidia Backusella ctenidia

corticola corticola

f. f.

Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis

Mucor heterogamus Mucor heterogamus Mucor heterogamus

Mucor heterogamus

Mucor heterogamus Mucor guiliermondii Mucor grandis Mucor gigasporus Mucor genevensis Mucor gigasporus

Mucor genevensis

Mucor fusiformis Mucor genevensis Mucor fuscus Mucor genevensis Mucor fuscus Mucor fuscus Mucor fuscus Mucor fuscus

Mucor flavus

Mucor flavus Mucor flavus Mucor flavus Mucor flavus Mucor flavus Mucor flavus Mucor flavus Mucor falcatus Mucor falcatus Mucor flavus Mucor exponens Mucor durus Mucor endophyticus Mucor exponens Mucor exponens Mucor durus Mucor ctenidius Mucor ctenidius Mucor ctenidius CBS 362.68 CBS 106.09 Fungiscope AS72 CNM-CM5229 CNM-CM2540 CBS 118522 CBS 115.18

CBS 594.83 CBS 580.83 CBS 405.58

CBS 338.74

CBS 252.85 CBS 174.27 CBS 186.87 CBS 566.91 CBS 564.75 CBS 383.95

CBS 535.78

CBS 336.68 CBS 404.71 CBS 530.77 CBS 114.08 CBS 313.78 CBS 254.48 CBS 282.78 CBS 230.29

CBS 992.68

CBS 893.73

CBS 681.73 CBS 664.67 CBS 234.35 CBS 230.35 CBS 182.90 CBS 210.71 CBS 126.70 CBS 252.35 CBS 251.35 CBS 197.71 CBS 508.48 CBS 484.66 CBS 385.95 CBS 141.20 CBS 404.58 CBS 156.51 CBS 696.76 CBS 433.87 CBS 293.66 20 Persoonia – Volume 30, 2013 LSU ITS JN206115 JN206146 JN206145 JN206139 JN206133 JN206137 JN206136 JN206143 JN206128 JN206118 JN206125 HM849683 JN206120 JN206134 JN206121 JN206114 HM849682 JN206119 JN206135 JN206116 JN206131 JN206048 JN206463 JN206126 JN206273 JN206272 JN206521 JN206210 HM999954 JN206148 JN206149 HM849685 JN206147 JN206160 JN206489 JN206209 JN206444 JN206205 JN206443 JN206158 JN206446 JN206156 (c2) JN206151 JN206154 JN206155 (c1) JN206450 JN206183 JN206153 JN206150 JN206152 HM849684 JN206184 JN206186 HM999956 JN206185 HM849690 JN206144 JN206181 JN206180 JN206182 JN206179 JN206501 JN206178 JN206500 JN206177 JN206502 JN206129 Lycopersicon esculentum Lycopersicon ; fruit ; fruit ; immature fruit ; tuber Source human greenhouse soil under soil soil soil n.a. n.a. n.a. human; sputum soil n.a. culture contaminant forest soil soil soil wood mixed with soil lake sediment, submerged n.a. soil n.a. dung of rabbit soil n.a. peat n.a. n.a. n.a. agaric n.a. lake mud cheese forest soil

owl pellet soil n.a. n.a. human; nasolabial infection human; hand garden soil sorghum malt Dioscorea n.a. dung of berber goat agaric human; gastrointestinal infection human; muscle n.a. Diospyros kaki Musa sapientum Spondias mombin Coccinella Africa Country France Netherlands Netherlands Austria Austria Switzerland Germany Austria USA UK, England Netherlands Italy France Norway n.a. Austria Germany Germany Germany Japan Ukraine UK France

India India India China Nigeria Indonesia South Indonesia India Netherlands Germany India n.a. Japan Indonesia Ghana Netherlands Switzerland Germany China India USA France

griseus var. toundrae var. (designated here) USA (designated here) Russia (designated here) France (designated here) Switzerland of M. saximontensis of M. hiemalis Status

T of M. vallesiacus T T of M. hiemalis T of M. adventitius var. T aurantiacus Switzerland

NT IT (designated here)

minuta var.

T of M. griseoochraceus T

T of M. cylindrosporus T T

T T of Rhizomucor variabilis T

ET NT

LT NT

moelleri moelleri moelleri moelleri moelleri moelleri moelleri japonicus macrocarpus luteus luteus luteus luteus luteus hiemalis luteus hiemalis sp. f. f. f. f. f. f. f. f. variosporus Species name at the beginning of the study

Zygorhynchus

Zygorhynchus Zygorhynchus

Zygorhynchus

M. hiemalis M. hiemalis M. hiemalis M. hiemalis

M. fuscus Zygorhynchus M. hiemalis M. hiemalis M. hiemalis Rhizomucor variabilis M. hiemalis M. aff.

Ascidiophora

Zygorhynchus

hiemalis corticola corticola corticola corticola hiemalis hiemalis hiemalis hiemalis hiemalis hiemalis hiemalis hiemalis hiemalis hiemalis

f. f. f. f. f. f. f. f. f. f. f. f. f. f. f.

Species Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor hiemalis Mucor moelleri Mucor hiemalis Mucor moelleri Mucor hiemalis Mucor moelleri Mucor moelleri Mucor moelleri Mucor hiemalis Mucor moelleri Mucor hiemalis Mucor moelleri Mucor hiemalis Mucor minutus Mucor microsporus Mucor luteus Mucor luteus Mucor luteus Mucor megalocarpus Mucor microsporus Mucor luteus Mucor laxorrhizus Mucor laxorrhizus Mucor lanceolatus Mucor japonicus Mucor irregularis Mucor irregularis Mucor irregularis Mucor irregularis Mucor irregularis Mucor irregularis Mucor indicus Mucor indicus Mucor indicus Mucor indicus Mucor hiemalis Mucor indicus Mucor indicus Mucor indicus Mucor indicus Mucor inaequisporus Mucor inaequisporus Mucor inaequisporus

Strain number CBS 107.19 CBS 533.78 CBS 532.78 CBS 366.68 CBS 365.68 CBS 110.19 CBS 117.08 CBS 123972 CBS 201.65 IHEM 21156 CBS 242.35 CBS 501.66 CBS 328.92 CBS 444.65 CBS 460.51 CBS 406.58 CBS 337.71D CBS 380.29 CBS 454.71 CBS 216.27 CBS 528.78 CBS 586.67 CBS 245.35 CBS 243.35 CBS 244.35 CBS 301.74 CBS 567.70A CBS 215.27 CBS 204.28 CBS 237.66 CBS 143.85 CBS 638.74 CBS 154.69 CBS 700.71 CBS 977.68 CBS 100164 CBS 103.93 CBS 609.78 CBS 654.78 CBS 671.79 CBS 535.80 CBS 414.77 CBS 422.71 CBS 980.68 CBS 226.29 CBS 123974 CBS 120585 CBS 120.08 CBS 496.66 CBS 351.50 CBS 255.36 Table 1 (cont.) Table G. Walther et al.: DNA barcoding in Mucorales 21 JN205905 JN205903 JN205904 JN205900 JN205906 JN205898 HM849676 JN205899 JN205909 JN205930 JN205929 JN205908 JN205911 JN205901 JN206188 JN205902 JN206496 JN206190 (c3) JN206189 (c1) JN206498 JN205917 HM999955 HM849677 JN205913 JN205918 JN205915 JN205912 JN205914 JN205916 JN206080 (c4) JN206081 (c1) JN206079 (c4) JN206078 (c2) JN206483 JN206077 (c4) JN206076 (c1) JN206032 JN206034 JN206476 JN206031 JN206029 JN206028 HM849681 JN206030 (c1) JN206109 JN206108 JN206453 JN206194 JN206198 JN206196 JN206197 JN206495 JN206195 JN206176 JN206503 JN206166 JN206484 JN206089 JN206480 JN206092 (c1) JN206091 (c2) JN206093 (c1) JN206085 HM849687 JN206086 JN206087 JN206090 JN206088 JN206017 JN206434 JN206018 JN206016 JN206117 ; decaying fruit ; nut ; lung Agapornis human; skin grassland soil Juglans regia n.a. soya cheese n.a. human human; nail soil halva contaminated cheese n.a. sufu starter

dung of shrew wheat bran human; biopsy material cat; subcutaneous tissue cheese horse meat leaf litter lettuce forest soil

dung of forest-mouse

dung of mouse

dung of rabbit ant hill river water soil and decaying pear fruit n.a. Pyrus communis n.a. soil sandy heath soil soil dung of horse dung of cow laboratory air n.a. n.a. paddy ﬁeld soil dung of rabbit wheat ﬁeld soil

dung of dog cow; nose effluent water dung of ﬁeld-mouse dung of rabbit n.a. dung of mouse dung of Rattus norvegensis dung of mouse soil Tenerife dung n.a. Finland Spain, Netherlands China Spain Spain Austria Ukraine Belgium China India

India Sweden Germany Switzerland France Netherlands Zaire Netherlands Canada

Russia

Netherlands

UK, England Russia Romania USA France n.a. n.a. Austria Australia Japan Netherlands Germany Denmark USA n.a. China Netherlands Germany

n.a. Netherlands Ukraine Armenia Germany Russia India Taiwan India Switzerland chibinensis f. (designated here) of Russia (designated here) Switzerland (designated here) USA of M. dimorphosporus M. racemosus

T of M. sinensis T

T of M. murorum T T

T NT

ET HT circinelloides

chibinensis sphaerosporus chibinensis sphaerosporus f. multiplex californiensis ovalisporus var. ovalisporus var. variisporus M. racemosus f. M. racemosus f. M. sinensis M. racemosus f. M. circinelloides M. racemosus f.

M. aff.

M. laxorrhizus M. laxorrhizus

Zygorhynchus

M. piriformis

Zygorhynchus

racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus racemosus

f. f. f. f. f. f. f. f. f. f. f. f. f. f. californiensis

f. Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor racemosus Mucor prayagensis

Mucor prayagensis Mucor plumbeus Mucor plumbeus Mucor plumbeus Mucor plumbeus Mucor plumbeus Mucor plumbeus Mucor plumbeus Mucor plumbeus

Mucor plasmaticus

Mucor plasmaticus Mucor piriformis Mucor piriformis Mucor piriformis Mucor piriformis Mucor piriformis Mucor piriformis Mucor parviseptatus Mucor parviseptatus Mucor odoratus Mucor odoratus Mucor odoratus Mucor odoratus Mucor odoratus Mucor nederlandicus Mucor multiplex Mucor mucedo Mucor mucedo

Mucor mucedo Mucor mucedo Mucor mucedo Mucor mucedo Mucor mucedo Mucor mucedo Mucor mousanensis Mucor mousanensis Mucor mousanensis Mucor moelleri

CBS 616.63 CBS 636.67 CBS 369.71 CBS 260.68 CBS 271.86 CBS 222.81 CBS 204.74 CNM-CM3862 CNM-CM2569 CBS 661.66 CBS 660.66 CBS 657.68 CBS 111557 CBS 113.08 CBS 816.70

CBS 652.78 CBS 814.96 CBS 634.74 CBS 633.74 CBS 630.74 CBS 312.78 CBS 295.63 CBS 284.78 CBS 226.32

CBS 402.73

CBS 275.49

CBS 177.46 CBS 528.68 CBS 527.68 CBS 256.85 CBS 175.27 CBS 169.25 CBS 111230 CBS 111230 CBS 522.79 CBS 417.77 CBS 572.70 CBS 201.71 CBS 179.76A CBS 130.41 CBS 120.71 CBS 735.70 CBS 110662 CBS 987.68 CBS 836.73

CBS 834.73 CBS 640.67 CBS 542.66 CBS 525.68 CBS 296.35 CBS 228.29 CBS 999.70 CBS 721.76 CBS 531.77 CBS 402.58 22 Persoonia – Volume 30, 2013 LSU JN206547 ITS JN205919 JN206433 JN206099 JN206097 JN206308 JN206307 JN206306 JN206305 HM849662 JN206304 JN206309 JN206026 JN206303 JN206025 (c3) JN206024 (c1) JN206437 JN206027 JN206438 JN205925 JN206214 JN206511 JN206216 JN206217 JN206510 JN205920 JN206215 JN206199 JN206105 JN206107 (c4) JN206106 (c3) JN206104 JN206454 JN206175 JN206508 JN206192 JN206507 JN206191 JN206037 JN206036 JN206035 JN206477 JN206039 (c2) JN206248 JN206013 JN205931 JN206518 JN206033 JN206431 JN206432 JN206482 JN206038 (c1) JN206478 JN206040 JN206479 JN206123 JN205924 JN206122 JN206124 JN206455 JN205922 JN206072 JN206458 JN206075 (c4) JN206073 (c3) JN206074 JN205932 HM849678 JN205928 (c4) JN205927 (c2) JN205926 JN205923 JN205921 Picea abies ; peel, contaminant Pinus wood Source n.a. dung of rabbit n.a. n.a. n.a. n.a. n.a. dung of bat n.a. n.a. n.a. n.a.

n.a. Paeonia storage rot of Cucurbita maxima human; pus of wound washroom in hospital Citrus aurantium wood decaying wood manured soil mould-infected

forest soil under soil n.a. soil of litter layer dung of mouse soil at lake shore soil of dried up bog grassland soil tropical vegetable Zea mays gymnosperm litter Ribes rubrum strawberries n.a. forest soil soil and litter soil of Picea forest forest soil sausage soil

chicken leg fruit body of Hygrophoropsis aurantiaca human; nasal lesion

cheese dung of horse cheese steamed sweet potato

Country Norway UK, Scotland n.a. Germany Germany Netherlands Germany Germany Panama USA Germany USA

n.a. USA n.a. Thailand Germany Netherlands Germany Germany India Sweden

Sweden Germany India Germany Ukraine Austria Norway Ukraine Benin South Africa USA Netherlands

France Germany Germany USA Germany Germany Netherlands

Netherlands Netherlands Uruguay

France Japan France Japan

(designated here) Denmark

Status IT

T T

T NT

T of M. kanivcevii T

griseocyanus griseocyanus racemosus f. f. hiemalis silvaticus silvaticus silvaticus f. f. f. f. Species name at the beginning of the study

piloboloides blakesleeanus var. P.

piloboloides blakesleeanus var. P. piloboloides T blakesleeanus var. P.

P. parasitica P.

P. parasitica P. M. microspora

M. hiemalis M. circinelloides M. circinelloides M. piriformis

M. plasmaticus M. hiemalis M. hiemalis M. hiemalis

M. plasmaticus M. racemosus f.

sphaerosporus sphaerosporus sphaerosporus sphaerosporus sphaerosporus sphaerosporus sphaerosporus sphaerosporus sphaerosporus f. f. f. f. f. f. f. f. f.

sp. sp. Species Mucor racemosus Mucor racemosus Pilaira anomala Phycomyces blakesleeanus Pilaira anomala Phycomyces blakesleeanus Phycomyces blakesleeanus Phycomyces blakesleeanus Phycomyces blakesleeanus Phascolomyces articulosus Phycomyces blakesleeanus Phycomyces blakesleeanus Parasitella

Parasitella Mucor racemosus Parasitella parasitica Mycotypha sp. Mycotypha microspora Mucor racemosus Mycotypha microspora Mycotypha microspora zychae Mucor zychae var. Mucor zonatus

Mucor zonatus Mucor zonatus Mucor variisporus Mucor ucrainicus Mucor ucrainicus Mucor strictus Mucor strictus Mucor sp. Mucor sp. Mucor sp. Mucor strictus Mucor sp.

Mucor sp. Mucor sp. Mucor racemosus Mucor silvaticus Mucor silvaticus Mucor racemosus Mucor silvaticus Mucor saturninus

Mucor saturninus Mucor saturninus Mucor ramosissimus

Mucor racemosus Mucor racemosus Mucor racemosus

Strain number Table 1 (cont.) Table CBS 115.08 CBS 143.70 CBS 396.71C CBS 286.35 CBS 131.23 CBS 284.35 CBS 282.35 CBS 269.32 CBS 270.32 CBS 113.76 CBS 112.20 CBS 188.27 CBS 207.28

CBS 152.69 CBS 238.35 CBS 412.66 CBS 109960 CBS 610.92 CBS 347.87 CBS 230.32 CBS 186.68 CBS 416.67 CBS 529.83

CBS 183.76 CBS 148.69 CBS 837.70 CBS 674.88 CBS 221.71 CBS 576.66 CBS 368.71A CBS 334.71 CBS 541.78 CBS 608.78 CBS 837.73A CBS 100.66

CBS 125018 CBS 122.23 CBS 538.78 CBS 412.71 CBS 509.66 CBS 539.78 CBS 249.35 CBS 974.68

CBS 599.78 CBS 598.78 CBS 135.65

CBS 574.70 CBS 571.70 CBS 634.78 G. Walther et al.: DNA barcoding in Mucorales 23 JN206538 JN206537 JN206558 JN206100 JN206098 JN206460 JN206101 JN206094 JN206459 JN206095 JN206096 JN206275 JN206274 HM849665 JN206299 JN206532 JN206317 HM999967 (c3) HM849674 JN206316 JN206315 JN206314 JN206313 HM999962 JN206312 HM849716 JN206102 JN206512 JN206330 JN206311 JN206310 JN206320 (c4) JN206319 (c1) JN206321 (c2) HM999961 (c4) JN206322 (c1) HM999960 (c2) HM999959 (c1) HM849717 JN206240 JN206239 JN206516 JN206071 JN206481 JN206103 JN206474 JN206327

JN206292 JN206291 HM849663 JN206323 HM849659 JN206326 JN206332 JN206324 JN206328 JN206331 JN206325 JN206333 JN206338 JN206334 JN206340 JN206336 JN206339 JN206337 JN206341 JN206365 JN206404 HM999965 HM849671 JN206335 ; stored grains Parthenium argentatum dung of cow dung dung n.a. n.a. dung of cow n.a. dung of deer dung of mouse human; lung, biopsy sample air human; biopsy sample human; peritoneal fluid n.a. human; sputum soil corn meal soil human; dermal lesions of back pig; kidney forest soil

Hordeum human; kidney and liver (leukemic patient) JN206318

soil

soil beneath Joshua trees dung of mouse n.a. dung of mouse dung of lizard dung of pack rat dung of rodent dung of lizard (?) n.a. human; palate human; sinus (leukemic patient) human; palatum molle rabbit brain Look Pang (sweet fermented glutenous rice) JN206329 n.a. tempeh human n.a. starch-based rice wine starters human; lung air n.a. sorghum malt spaghetti n.a. n.a. human Tobago soil Africa retting Germany Netherlands UK France India Germany Germany Netherlands Armenia USA Spain Spain Spain n.a. Spain USA India Canada n.a. Ukraine

UK, England Australia

USA

USA Sri Lanka and Trinidad USA Armenia Switzerland USA USA USA India USA Netherlands Turkey Greece Netherlands n.a. Japan Singapore India n.a. Vietnam France South n.a. India India India Thailand var. Japan var. n.a. (designated here) Netherlands

T of R. tauricus T

T T of R. maydis T T T T

T of R. oryzae T

T of R. boreas T

NT of Amylomyces rouxii NT

multiplicisporus T of R. delemar T kawasakiensis T of R. javanicus T

americanus var.

Backusella lamprospora

P. caucasica P. R. sexualis

R. oryzae

R. tauricus

P. circinans P.

R. oryzae

R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae R. oryzae

R. oryzae

R. oryzae R. oryzae R. oryzae

arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. arrhizus var. delemar var. delemar var. delemar var. delemar var. delemar var. delemar var. delemar var. delemar var.

caucasica var. pusillus pusillus pusillus pusillus pusillus pusillus pusillus pusillus pusillus americanus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus arrhizus homothallicus caespitosus sp. Pilaira moreaui Pilaira anomala Pilaira anomala Pilaira anomala Pilaira moreaui Pilaira moreaui Pilaira moreaui Pirella circinans Pilobolus umbonatus Pilobolus umbonatus Rhizopus Rhizopus Rhizomucor Rhizomucor Rhizomucor Rhizomucor Rhizomucor Rhizomucor Pirella naumovi Rhizopus Rhizomucor Rhizomucor Rhizomucor

Rhizomucor miehei Rhizomucor miehei

Rhizomucor miehei

Protomycocladus faisalabadensis Poitrasia circinans Poitrasia circinans Pirella Rhizopus Radiomyces embreei Radiomyces spectabilis Rhizomucor miehei Radiomyces embreei Radiomyces embreei Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus

CBS 181.26 CBS 699.71 CBS 695.68 CBS 424.70 CBS 411.67 CBS 496.71 CBS 523.68 CBS 962.68 CBS 425.50 CBS 302.83 CBS 340.62 CBS 109939 CNM-CM5124 CNM-CM4727 CNM-CM2974 CNM-CM2935 CNM-CM2752 CBS 425.78 CBS 524.68 CBS 110.17 CBS 354.68 CBS 219.31 CBS 179.69

CBS 429.70 CBS 360.92 CBS 209.77A

CBS 661.86 CBS 647.70 CBS 153.58 CBS 588.88 CBS 112.07 CBS 254.60 CBS 255.60 CBS 182.67 CBS 206.77 CBS 205.77 CBS 118614 CBS 125017 CBS 146.90 CBS 286.55 CBS 330.53 CBS 515.94 CBS 438.76 Chakrabarti710113 Chakrabarti710113 CBS 111231 CBS 111231 CBS 111760 CBS 120593 CBS 392.95 CBS 402.51 CBS 544.80 CBS 607.68 Chakrabarti710104 CBS 111232 CBS 111232 CBS 427.87 24 Persoonia – Volume 30, 2013 LSU ITS HM999968 HM849670 JN206375 JN206283 (c3) JN206282 (c1) HM849694 JN206366 JN206406 JN206367 JN206368 HM999966 HM849673 JN206350 JN206345 HM849667 HM999971 JN206402 JN206353 JN206349 JN206347 JN206361 JN206346 JN206356 HM999970 JN206358 JN206348 HM849669 JN206362 JN206352 JN206357 JN206364 JN206403 JN206351 JN206360 JN206354 JN206359 JN206355 JN206344 HM849668 JN206373 JN206534 JN206298 HM849664 JN206363 JN206343 HM849666 JN206374 JN206297 JN206295 HM999978 JN206578 HM999972 HM849718 HM999980 (c4) HM999979 (c1) HM999974 HM849719 HM999975 HM849720 HM999976 (c2) HM999977 (c1) JN206575 JN206372 JN206405 JN206296 ; stem leptocephala ; grain ; decaying fruit Source tropical desert soil Hordeum vulgare

rain forest soil Spartocytisus supranubius coffee-ground ragi Fragaria human; bronchial wash of patient with myeloma HM999969 HM849672 koji in brewery sorghum malt n.a. dust in saw mill human; lung herbal tea from Borbonia cordata human; cutaneous lesion human; rhinocerebral infection soil tempeh tempeh probably from tempeh tempeh fermentation human; hand, necrotic tissue maize; ground nuts cow foetus n.a. n.a. n.a. sorghum malt bread n.a. n.a. enterocolitis sufu starter: rice wine tablet tempeh human; liver of premature infant, necrotising JN206342 n.a. fruit bodies of Mycena galericulata fruit body of Mycena cf. air tidal mud-flat soil

human; skin soil soil

n.a. Umbonia fruit body of Mycena pura Yugoslavia stored cereals Africa Africa

Country Guatemala Netherlands

Brazil Spain, Tenerife Netherlands n.a. UK USA China former South Russia Sweden Netherlands USA France n.a. Indonesia Indonesia Indonesia Indonesia Ukraine Australia Mozambique Russia South China France n.a. n.a.

Indonesia Australia Vietnam n.a. Netherlands Germany Kuwait

France China China

Switzerland Ecuador Switzerland Japan India

var. Japan

R. bovinus Status T

T T

NT T of R. pusillus T

NT NT

liquefaciens thermosus var. T of R. pypmaeus T of R. chinensis T of R. pseudochinensis T T

T of

T of S. verruculosum T

T T

Species name at the beginning of the study

reflexus R. stolonifer var.

S. vasiformis R. stolonifer R. stolonifer R. stolonifer

reflexus R. stolonifer var.

S. racemosum

var. var.

tuberosus var. rhizopodiformis var. rhizopodiformis var. rhizopodiformis var. rhizopodiformis var. rhizopodiformis var. rhizopodiformis var. rhizopodiformis var. oligosporus var. oligosporus var. oligosporus var. oligosporus var. rhizopodiformis var. microsporus var. microsporus var. chinensis var. chinensis var. chinensis var. chinensis var. chinensis var. microsporus var. chinensis var. azygosporus var. azygosporus var.

lyococcus homothallicus lyococcus sp. sp. sp. sexualis schipperae microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus microsporus lyococcus microsporus microsporus microsporus microsporus Species Rhizopus Rhizopus Rhizopus Spinellus fusiger

Saksenaea oblongispora Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Rhizopus Spinellus fusiger Rhizopus Rhizopus Rhizopus Rhizopus Spinellus fusiger Spinellus fusiger Syncephalastrum racemosum Syncephalastrum racemosum

monosporum Syncephalastrum racemosum cristatum Syncephalastrum monosporum Syncephalastrum monosporum

Syncephalastrum monosporum Sporodiniella umbellata Strain number Table 1 (cont.) Table CBS 117.43 CBS 336.62 CBS 320.35 CBS 405.63

CBS 133.90 CBS 926.87 CBS 442.74 CBS 126.83 CBS 336.39 CBS 138.95 CBS 113206 CBS 607.73 CBS 536.80 CBS 343.29 CBS 258.79 CBS 220.92 CBS 196.77 CBS 118987 CBS 112587 CBS 228.95 CBS 337.62 CBS 338.62 CBS 102277 CBS 699.68 CBS 308.87 CBS 344.29 CBS 388.34 CBS 394.34 CBS 537.80 CBS 631.82 CBS 112285 CBS 398.95 CBS 515.75 CBS 294.31 CBS 357.93 CBS 359.92 CBS 111563 CBS 633.80 CBS 894.73 CBS 213.78 CBS 199.81

CBS 120811 CBS 567.91 CBS 568.91

CBS 122.12 CBS 195.77 G. Walther et al.: DNA barcoding in Mucorales 25 JN206577 JN206559 JN206565 JN206561 JN206542 JN206570 JN206569

HM999985 HM999982 HM999973 JN206576 HM999983 HM999984 (c4) HM999981 (c2) HM849721 JN206370 (c3) JN206371 (c1)

JN206386 JN206560 JN205837 JN206546 JN206059 JN206058 JN206060 JN206466 JN206369 (c3) JN206401

JN206388 JN206384 JN206564 JN206387 HM849709 JN206380 HM849710 JN206301 JN206302 JN206300 HM849661 JN205833 JN205831 JN205834 JN206543 JN205836 JN206544 JN205832 JN205830 HM849724 JN205835 JN205838 JN206541 JN206396 JN206571 JN206398 JN206397 JN206400 JN206573 JN206389 JN206390 JN206394 JN206392

JN206378 JN206567 JN206393 JN205845 JN206545 JN206277 JN206276 HM849660 JN206379 JN206381 JN206563 JN206385 JN206377 JN206382 JN206376 JN206568 JN206562 JN206383 JN206566 JN206395 JN206572 JN206391 ; rhizoplane ; granulomatous ulcer JN206399 JN206574 ; decaying fruit body Linum usitatissimum ; decaying needle ; leaf litter ; root Thuja occidentalis Eucalyptus regnans ; root environmental dung of coyote soil humose soil under air

soil

Amanita rubescens soil soil of forest Eucalyptus regnans dung of pack rat n.a. dung of bat n.a. n.a. Pinus luchuensis soil of native decidious forest Fagus crenata forest soil soil compost Zea mays dung of mouse municipal compost dung of pig soil under dung of Capromys sp. n.a. dung of rat Bertholletia excelsa dung of rat dung of lizard soil fruit body of Panellus pusillus Varanus comodoensis Varanus soil soil of Larix forest root forest soil forest soil pine stump wood sandy loam Fragaria soil under washed mycorrhizal root dung of mouse dung of rat soil soil of acid heathland soil forest soil Picea abies root soil, wood scraps and other debris Isopogon ceratophyllus forest soil

Spain USA USA Zaire Indonesia

Brazil

Netherlands Australia Australia USA USA Poland USA n.a. Japan Japan USA Sweden New Zealand soil of basaltic parent material Netherlands Germany Nigeria USA India Brazil Canada Cuba France USA USA USA Mexico USA Colombia Netherlands UK Germany USA Belgium Georgia USA Australia Japan Australia Canada Mexico Mexico India Australia Australia Germany China Germany New Zealand litter of ? Carex sp. USA Australia Poland

HT HT

NT T

T of U. versiformis T

AUT of U. multispora T

T T

IT T of Mortierella alba T

U. vinacea U. westeae U. angularis

elegans elegans elegans fusiformis gibberispora fusiformis gibberispora isabellina dimorpha autotrophica dimorpha angularis isabellina isabellina isabellina isabellina nana nana nana nana nana vinacea vinacea westeae nana swartii sp. sp. ramanniana sp. ramanniana ramanniana ovata Syncephalastrum racemosum Syzygites megalocarpus Syncephalastrum racemosum Syncephalastrum racemosum Syncephalastrum racemosum Syncephalastrum racemosum

Syncephalastrum racemosum Syzygites megalocarpus

Umbelopsis Umbelopsis Thamnostylum nigricans Thamnostylum lucknowense Thamnidium Thamnidium Thamnidium Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Thermomucor indicae seudaticae Thermomucor indicae seudaticae Thamnostylum repens Thermomucor indicae seudaticae Thamnostylum piriforme Thamnostylum piriforme Thamnostylum piriforme Thamnostylum piriforme Thamnostylum piriforme Thamnostylum piriforme Thamnostylum piriforme Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Zychaea mexicana Utharomyces epallocaulus Utharomyces epallocaulus Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis Umbelopsis CNM-CM2909 CBS 108947 CBS 441.59 CBS 440.59 CBS 421.63 CBS 370.49

CBS 302.65 CBS 372.39

CBS 919.85 CBS 101745 CBS 690.76 CBS 874.69 CBS 641.69 CBS 411.52 CBS 341.55 CBS 385.85 CBS 109328 CBS 100559 CBS 117350 CBS 212.72 CBS 110039 CBS 603.68 CBS 447.78 CBS 446.78 CBS 692.76 CBS 104.75 CBS 638.69 CBS 480.69 CBS 412.94 CBS 316.66 CBS 233.28 CBS 182.28 CBS 170.57 CBS 167.80 CBS 250.95 CBS 309.93 CBS 560.63 CBS 150.81 CBS 309.52 CBS 373.67 CBS 444.68 CBS 669.83 CBS 212.32 CBS 236.82 CBS 870.85 CBS 858.68 CBS 441.76 CBS 342.73 CBS 329.73 CBS 868.85 CBS 913.85 CBS 559.86 CBS 366.95 CBS 914.85 CBS 112976 CBS 101226 CBS 499.82 26 Persoonia – Volume 30, 2013

The Netherlands) following the protocol given by Möller et al. Morphology (1992) with diverse modifications described in detail by Alas- Strains with a position in the phylogenetic tree that was in truey-Izquierdo et al. (2010). The primer pair V9G (de Hoog & conflict with their original classification were verified microscopi- Gerrits van den Ende 1998) and LR3 (Vilgalys & Hester 1990) cally. These strains were cultivated on MEA at 24 °C. After 2 was used to amplify a DNA segment consisting of the complete and 7 d colonies were observed using a stereomicroscope ITS region and the D1/D2 region of the LSU. The PCR reaction (SMZ 1500; Nikon) and slides for microscopic examination were mixture (25 μl) contained 0.4 μM of each primer, 0.185 mM of made. Fungal material was mounted in lactic acid with cotton each deoxynucleoside triphosphate (GC Biotech, Alphen aan blue (2 mg cotton blue/mL lactic acid) and in lactic acid only den Rijn, The Netherlands), 10× NH4 BioTaq Reaction buffer and examined using a Nikon eclipse 80i microscope (Nikon,

(GC Biotech), 1.5 mM MgCl2, 0.8 U BioTaq DNA polymerase Amstelveen, The Netherlands). Measurements were performed (GC Biotech), and about 20 ng DNA. PCR reactions were con- using the software NIS-Elements D 3.0 (Nikon). For a few ducted on a Thermal cycler 2720 (Applied Biosystems, Nieuwer strains mounts from slide cultures were photographed using a kerk a/d IJssel, The Netherlands) as follows: one initial cycle at Nikon 05SM digital camera. 94 °C for 5 min, followed by 35 cycles of 1 min at 94 °C, 1 min at 53 °C and 2 min at 72 °C, and one final cycle of 7 min at 72 °C. RESULTS AND DISCUSSION PCR products were analysed in 1 % agarose gels. Direct cycle- sequencing was performed for both strands of the PCR products Performance of ITS and LSU sequencing using the Big dye sequencing kit (Applied Biosystems) and the Direct sequencing was possible in 82 % of the strains for primer set ITS1 and ITS4 (White et al. 1990) for the complete the ITS and in all strains for the LSU region. ITS sequences ITS region, and NL1 and LR3 for the D1/D2 region of the LSU of strains that failed direct sequencing could be obtained by (O’Donnell 1993). Cycle-sequencing products were analysed cloning in most taxa, while cloning did not solve the problem on an ABI 3730XL automatic sequencer (Applied Biosystems). in the group of Rhizopus stolonifer; this was probably due to In cases where direct sequencing failed or double bands were extended poly-A- and poly-T-regions. In the majority of cloned detected in the agarose gels PCR products were ligated into strains ITS copies differed only by a single basepair. However, pGEM-T Easy Vector (Promega, Leiden, The Netherlands) and in Syncephalastrum we found ITS sequences of two types cloned in E. coli JM109 competent cells (Promega) following forming two clades in the ITS tree (illustrated in Vitale et al. the manufacturer’s instructions. Colony-PCRs were performed 2012). Divergent copies of ITS have been found in Fusarium using the primer pair M13f (5’-GTAAAACGACGGCCAGT-3’) (O’Donnell & Cigelnik 1997) and Laetiporus (Lindner & Banik and M13r (5’-GGAAACAGCTATGACCATG-3’). 2011). The proportion of strains that had to be cloned was especially high in Absidia, Umbelopsis and Syncephalastrum. Sequence analyses Phylogenetic relationships inferred from LSU data Consensus sequences were constructed by means of the Seq- The family structure of the Mucorales revealed by the LSU Man program v. 7.2.2 (DNASTAR, Lasergene) and deposited in cladogram in main traits corresponds with that given by Hoff- GenBank. A total of 592 ITS sequences (GenBank accession mann et al. (2013), although the Lichtheimiaceae and the numbers JN205809 to JN206400) and 210 LSU sequences Mucoraceae do not form clades (Fig. 1), probably because (GenBank accession numbers JN206401 to JN206610) were our analysis is based on a single locus. Some remaining newly generated for this study. Ninety-two ITS sequences taxonomic inconsistencies between phenotypic and sequence- and 113 LSU sequences of strains included in this study were based classifications were found, particularly in the upper part published in previous papers (Alastruey-Izquierdo et al. 2010, of the LSU cladogram (Fig. 1). The Mycotyphaceae and the Vitale et al. 2012). Choanephoraceae are nested within the Mucoraceae and The highly diverse ITS sequences were divided in subsets of these families together with the Backusellaceae, the Pilobol- alignable sequences for phylogenetic analyses. Additionally, aceae and the Rhizopodaceae compose a well-supported a set of LSU sequences was generated representing every clade (Mucorineae clade; bootstrap 75 %). The Mucorineae MOTU detected in the ITS alignments. The MOTUs were clade is dominated by the polyphyletic genus Mucor (Fig. 2a), defined by a ≥ 99 % similarity threshold. Similarity values based which comprises many groups that are intermingled by other on the uncorrected distances were calculated with BioNumer- sporangia-forming genera such as Circinella p.p. (Fig. 2h), ics (v. 0.4.61) software (Applied Maths, Sint-Martens-Latem, Parasitella, Pilaira, Pirella (Fig. 2f), Rhizomucor p.p. (Fig. 2b), Belgium). and Zygorhynchus (Fig. 2d), sporangia- and sporangiola- ITS and LSU sequences were aligned using the server ver- forming genera such as Backusella (Fig. 8) and Thamnidium sion of the MAFFT program (www.ebi.ac.uk/Tools/mafft) and (Fig. 2i), as well as exclusively sporangiola-forming genera such manually corrected using the program Se-Al v. 2.0a11 (Rambaut as Chaetocladium, Ellisomyces (Fig. 2g), Helicostylum and 2002; http://tree.bio.ed.ac.uk/software/seal/). All alignments Kirkomyces. Based on the LSU (Fig. 1) and ITS trees (Fig. 3 to are lodged in TreeBASE (www.treebase.org/). Mortierella parvi 6), the genera Backusella, Circinella, Mucor, Rhizomucor and spora, a member of the Mortierellales, was selected as outgroup Zygorhynchus appear to be polyphyletic. The lower part of the of the LSU tree because the Mortierellales is the sibling order of LSU tree shows a more resolved taxonomy. The majority of the Mucorales (e.g. O’Donnell et al. 2001, Voigt & Wöstemeyer genera here are clearly defined and appear to be monophyletic 2001). The ITS trees were rooted by the nearest neighbours of based on our sampling. Exceptions were the paraphyletic genus the respective group in the LSU tree. The maximum likelihood Absidia that includes Halteromyces and Chlamydoabsidia, and algorithm with the server version of RAxML-VI-HPC v. 7.0.0 the polyphyletic genera Circinella and Rhizomucor that are (Stamatakis et al. 2008) as implemented on the Cipres portal addressed in more detail below. was used to estimate phylogenetic relationships. The robustness Although the polyphyly of Mucor is indisputable, only few clear of all phylogenetic trees was assessed by bootstrap analyses lineages within Mucor are recognisable. Some of these lineages with 1 000 replicates. ITS trees were only calculated for Actino show a characteristic combination of sporangium size and the mucor, Mucor (4 trees), Rhizomucor and Rhizopus, but all of branching of the tall sporangiophores: in the Mucor mucedo the taxa are included in the LSU tree. group (Fig. 1), consisting, in addition to the name-giving species G. Walther et al.: DNA barcoding in Mucorales 27

0.05 substitutions/site 83 Mucor mucedo CBS987.68 JN206480 Mucor mucedo CBS640.67 HM849687 NT Mucor sp. ‘plasmaticus’ CBS122.23 JN206479 Mucor sp. CBS125018 JN206478 56 Mucor sp. ‘piriformis’ CBS837.73A JN206482 Mucor piriformis CBS169.25 HM849681 NT Mucor mucedo group Mucor piriformis CBS527.68 JN206476 Mucor strictus CBS100.66 JN206477 T of M. kanivcevii Mucor plasmaticus CBS275.49 JN206483 76 Mucor microsporus CBS204.28 JN206521 T of M. cylindrosporus Pirella sp. ‘circinans’ CBS588.88 JN206481 Pirella circinans CBS962.68 JN206512 T Pirella naumovi CBS524.68 JN206474 T Mucor flavus CBS210.71 JN206462 T of M. peacockensis Mucor minutus CBS586.67 JN206463 T of M. griseoochraceus var. minuta Mucor flavus CBS230.35 JN206464 T of M. attenuatus Mucor flavus CBS893.73 JN206465 NT of M. sciurinus Mucor saturninus CBS974.68 JN206458 NT chicken Mucor flavus CBS992.68 JN206467 Mucor flavus CBS681.73 JN206473 Mucor aligarensis CBS993.70 JN206461 T Mucor flavus group Helicostylum pulchrum CBS259.68 EU736315 Mucor flavus CBS182.90 JN206472 Helicostylum elegans CBS169.57 JN206471 Mucor flavus CBS197.71 JN206470 T of M. meridionalis Thamnidium elegans CBS341.55 JN206466 91 Mucor flavus CBS 234.35 JN206468 NT Mucor flavus CBS126.70 JN206469 T of M. mephitis 94 Pilaira moreaui CBS181.26 JN206459 Pilaira anomala CBS695.68 JN206460 100 Zygorhynchus heterogamus CBS405.58 JN206487 ET (designated here) Zygorhynchus heterogamus CBS338.74 JN206488 Zygorhynchus heterogamus CBS580.83 JN206486 92 Zygorhynchus heterogamus CBS594.83 JN206485 Zygorhynchus macrocarpus CBS215.27 JN206489 ET (designated here) Zygorhynchus heterogamus CBS252.85 JN206490 M Zygorhynchus multiplex CBS110662 JN206484 HT 100 Mucor irregularis CBS700.71 JN206450 Mucor irregularis CBS103.93 HM849684 T of Rhizomucor variabilis u 84 Mucor hiemalis f. hiemalis CBS201.65 HM849683 NT Mucor hiemalis f. corticola CBS362.68 JN206449 c 87 Rhizomucor endophyticus CBS385.95 JN206448 HT Mucor hiemalis group Zygorhynchus psychrophilus CBS336.68 JN206447 HT o Zygorhynchus japonicus CBS154.69 JN206446 NT (designated here) 91 Mucor bacilliformis CBS251.53 JN206451 T r Mucor bacilliformis CBS573.70 JN206452 Mucor hiemalis f. silvaticus CBS249.35 JN206455 NT (designated here) Zygorhynchus moelleri CBS444.65 HM849682 LT of Mucor saximontensis (design. here) a Mucor zonatus CBS148.69 JN206454 T Mucor circinelloides ‘f. janssenii’ CBS526.68 JN206426 c Mucor ramosissimus CBS135.65 HM849678 NT Mucor bainieri CBS293.63 JN206424 IT e Ellisomyces anomalus CBS243.57 JN206423 T Mucor circinelloides CBS202.28 JN206418 Mucor circinelloides f. lusitanicus CBS968.68 JN206419 a Mucor circinelloides f. lusitanicus CBS969.68 JN206427 Mucor circinelloides f. lusitanicus ‘fragilis’ CBS236.35 JN206422 e Mucor circinelloides f. griseocyanus CBS198.28 JN206420 Mucor circinelloides f. griseocyanus CBS116.08 JN206421 Mucor circinelloides f. lusitanicus CBS111228 JN206429 Mucor racemosus group Mucor circinelloides f. circinelloides ‘M. rouxii’ CBS416.77 JN206412 Mucor circinelloides f. circinelloides CBS108.16 JN206413 Mucor circinelloides CNM-CM2541 JN206414 94 Mucor circinelloides CBS384.95 HM849679 T of Rhizomucor regularior Mucor circinelloides CNM-CM4366 JN206415 Mucor circinelloides f. circinelloides CBS195.68 HM849680 NT Backusella ctenidia CBS293.66 JN206417 Mucor circinelloides f. janssenii CBS205.68 JN206425 NT (design. here) 88 Mucor circinelloides f. janssenii CBS762.74 JN206416 Mucor circinelloides CBS635.65 JN206428 Mucor circinelloides CBS124110 JN206430 75 Mucor circinelloides ‘f. griseocyanus’ CBS541.78 JN206431 Mucor sp. ‘circinelloides f. griseocyanus’ CBS608.78 JN206432 Mucor racemosus f. sphaerosporus CBS115.08 JN206433 IT Mucor racemosus f. racemosus CBS260.68 HM849676 NT (design. here) 87 Mucor plumbeus CBS634.74 HM849677 Mucor mousanensis CBS999.70 JN206434 T 100 100 Chaetocladium brefeldii CBS116.24 JN206519 87 Chaetocladium brefeldii CBS156.74 JN206520 100 Parasitella sp. ‘parasitica’ CBS152.69 JN206437 Parasitella parasitica CBS412.66 JN206438 T 100 Mucor genevensis CBS114.08 JN206435 T Mucor genevensis CBS535.78 JN206436 Circinella rigida CBS156.51 JN206456 ET (designated here) Mucor abundans CBS521.66 JN206457 Mucor laxorrhizus var. ovalisporus CBS417.77 JN206453 Mucor guiliermondii CBS174.27 JN206475 T * Fig. 1 RAxML phylogram of the Mucorales based on the D1/D2 region of the LSU. Each LSU sequence covers for a MOTU in ITS deﬁned by a similarity threshold of 99 %. Branches with bootstrap values of 75 % or higher are printed in bold. Morphological groups according to sporangiophore branching and diameter of the sporangium: Mucor mucedo group, M. flavus group, M. hiemalis group, M. racemosus group, M. amphibiorum group, M. recurvus group. Black bar highlights the Mucor circinelloides complex. Ex-type strains are designated by: T = ex-type strain, ET = ex-epitype strain, HT = ex-holotype strain, IT = ex-isotype strain, LT = ex-lectotype strain, NT = ex-neotype strain, PT = ex-paratype strain, ST = ex-syntype strain, AUT = authentic material, c = clone, clinical strains are highlighted by red strain and GenBank accession numbers, strains representing MOTUs that comprise clinical strains are marked by red squares. Clinically relevant species are indicated by a red circle if no ITS sequence of a clinical isolate was available for the assignment to a MOTU. Potentially undescribed taxa are indicated by bold blue font. 28 Persoonia – Volume 30, 2013

* Mucor inaequisporus CBS351.50 JN206500 86 Mucor inaequisporus CBS496.66 JN206501 Mucor inaequisporus CBS255.36 JN206502 T Mucor nederlandicus CBS735.70 JN206503

Mucor variosporus CBS837.70 JN206508 T Mucoraceae 98 Mucor indicus CBS226.29 HM849690 ET (designated here) Mucor falcatus CBS251.35 JN206509 HT Mucor amphibiorum CBS763.74 HM849688 T Kirkomyces cordensis CBS223.63 JN206506 IT Mucor ucrainicus CBS674.88 JN206507 Mucor amphibiorum group 87 Mucor azygosporus CBS292.63 JN206497 IT Mucor prayagensis CBS652.78 JN206498 82 Mucor prayagensis CBS816.70 JN206496 Mucor ardhlaengiktus CBS650.78 JN206499 Mucor ardhlaengiktus CBS210.80 JN206504 ET (designated here) Mucor odoratus CBS130.41 JN206495 T 100 Mucor zychae var. zychae CBS416.67 JN206505 T 87 Mycotypha microspora CBS230.32 JN206510 T 78 Mycotypha sp. ‘microspora’ CBS109960 JN206511 Mycotyphaceae Mucor fuscus CBS282.78 JN206442 Mucorac. Mucor lanceolatus CBS638.74 JN206443 Zygorhynchus exponens var. exponens CBS141.20 JN206441 NT (designated here) Mucor laxorrhizus CBS143.85 JN206444 NT 91 Circinella simplex CBS428.80 JN206445 Hyphomucor assamensis CBS415.77 JN206439 T Hyphomucor assamensis CBS254.85 JN206440 100 Choanephora infundibulifera CBS153.51 JN206513 95 Choanephora cucurbitarum CBS674.93 JN206514 100 Blakeslea sinensis CBS564.91 JN206515 PT Poitrasia circinans CBS153.58 JN206517 T Choanephoraceae Gilbertella persicaria CBS 532.77 JN206517 92 Gilbertella persicaria CBS190.32 HM849691 T 100 Benjaminiella youngii CBS103.89 JN206409 T 94 Benjaminiella poitrasii CBS158.60 JN206411 T Mucoraceae Benjaminiella multispora CBS421.70 JN206410 IT Cokeromyces recurvatus CBS158.50 HM849699 T 100 Cokeromyces recurvatus CBS168.59 JN206408 Mucor sp. ‘hiemalis’ CBS334.71 JN206518 Mucor gigasporus CBS566.91 JN206494 T 100 Actinomucor elegans var. elegans CBS100.09 JN206491 Actinomucor elegans ‘var. meitauzae’ CBS111558 JN206492 Actinomucor elegans var. kuwaitiensis CBS117697 JN206493 NT Actinomucor elegans CBS154.86 HM849686 100 Backusella lamprospora CBS195.28 JN206530 100 Backusella lamprospora CBS118.08 JN206531 T Backusellaceae contaminant of CBS523.68 JN206532 Mucor sp. ‘recurvus var. recurvus’ CBS538.80 HM849692 Mucor recurvus var. indicus CBS786.70 JN206526 LT (designated here) Mucor grandis CBS186.87 JN206527 T 100 Mucor variabilis CBS564.66 JN206528 LT (designated here) 100 Backusella circina CBS128.70 JN206529 T Mucor recurvus group Mucor recurvus var. recurvus CBS318.52 JN206522 ET (designated here) 97 Mucor recurvus var. recurvus CBS196.71 JN206523 (Backusella) Mucor recurvus var. recurvus CBS673.75 JN206524 75 87 Mucor tuberculisporus CBS562.66 JN206525 LT (designated here) Mucor oblongiellipticus CBS568.70 JN206533 LT (designated here) Mucor oblongisporus CBS569.70 JN206407 NT (designated here) 95 Utharomyces epallocaulus CBS329.73 HM849660 Pilobolus umbonatus CBS302.83 HM849665 Pilobolaceae Rhizopus microsporus var. microsporus CBS699.68 HM849669 NT 80 Rhizopus microsporus CBS112285 JN206403 78 Rhizopus microsporus var. rhizopodiformis CBS536.80 JN206402 NT Rhizopus caespitosus CBS427.87 HM849671 T 96 Rhizopus schipperae CBS138.95 HM849672 T Rhizopus homothallicus CBS336.62 HM849670 T Rhizopus homothallicus CBS111232 JN206404 Rhizopus arrhizus var. arrhizus CBS112.07 HM849659 T of R. oryzae Rhizopus lyococcus CBS320.35 JN206534 Rhizopodaceae 90 100 Rhizopus sp. ‘stolonifer’ CBS926.87 JN206406 71 Rhizopus stolonifer AFTOL-ID 632 AY997085 99 Sporodiniella umbellata CBS195.77 JN206405 Rhizopus sexualis CBS336.39 HM849673 T 100 Rhizopus americanus CBS340.62 HM849674 T Syzygites megalocarpus CBS372.39 JN206401 ** Fig. 1 (cont.)

M. mucedo, of M. piriformis, M. plasmaticus and M. strictus, racemosus group contains M. circinelloides with the formae all form unbranched tall sporangiophores and large sporangia circinelloides, griseocyanus, lusitanicus and janssenii, Mucor often exceeding 200 µm diam. The morphology of Mucor strictus bainieri, M. plumbeus, M. racemosus, M. ramosissimus and is strongly temperature-dependent. The species develops the Backusella ctenidia. The characteristics of this group are typical group morphology only at low temperatures (Schipper repeatedly sympodially branched sporangiophores and small 1975: 24). The Mucor flavus group, comprising M. aligarensis, sporangia not exceeding 80 µm diam. Species of the Mucor M. minutus and M. saturninus in addition to M. flavus consists amphibiorum group, namely M. amphibiorum, M. ardhlaengik- of species developing sympodially branched tall sporangio- tus, M. azygosporus, M. falcatus, M. inaequisporus, M. indicus, phores and medium-sized sporangia that are over 80 µm but M. nederlandicus, M. odoratus, M. prayagensis, M. ucrainicus, not exceeding 200 µm diam. A clade including M. hiemalis, M. variosporus and M. zychae form − with the exception of M. irregularis, M. luteus, Rhizomucor endophyticus and Zygo M. indicus and M. falcatus − usually unbranched tall sporan- rhynchus psychrophilus, the Mucor hiemalis group, is charac- giophores that bear, unlike species of the M. mucedo group, terised by poorly sympodially branched tall sporangiophores small- to medium-sized sporangia (maximum diameter between and small sporangia not exceeding 80 µm diam. The Mucor 70 and 175 µm). The Mucor recurvus group comprises Mucor G. Walther et al.: DNA barcoding in Mucorales 29

** Circinella umbellata CBS160.49 HM849722 Circinella umbellata CBS101.16 JN206553 Circinella minor CBS142.81 JN206552 Circinella angarensis CBS173.62 JN206551 NT 84 Circinella chinensis CBS140.28 JN206549 T 99 Circinella muscae CBS141.28 JN206548 85 Circinella muscae CBS107.13 JN206550 T of C. sydowii Phascolomyces articulosus CBS113.76 JN206547 T Zychaea mexicana CBS441.76 JN206545 T 100 Thamnostylum lucknowense CBS874.69 JN206546 Lichtheimiaceae 98 75 Fennellomyces heterothallicus CBS292.86 JN206539 AUT 92 Fennellomyces heterothallicus CBS290.86 JN206540 AUT 77 100 Thamnostylum nigricans CBS690.76 JN206541 NT 77 Fennellomyces linderi CBS158.54 HM849723 T Thamnostylum piriforme CBS412.94 JN206543 78 Thamnostylum piriforme CBS316.66 JN206544 T Thamnostylum repens CBS692.76 JN206542 NT 100 Rhizomucor pusillus CBS354.68 HM849716 ET (designated here) Rhizomucor miehei CBS182.67 HM849717 T c1 Syncephalastrum monosporum var. monosporum CBS567.91 HM849719 T Syncephalastrum monosporum var. cristatum CBS568.91 HM849720 T Syncephalastrum monosporum CBS122.12 JN206575 Syncephalastrum racemosum CBS302.65 HM849721 100 Syncephalastrum racemosum CBS213.78 JN206578 Tver Syncephalastraceae Syncephalastrum racemosum CBS199.81 HM849718 Syncephalastrum racemosum CNM-CM2909 JN206577 Syncephalastrum racemosum CBS421.63 JN206576 Lichtheimia hyalospora CBS173.67 GQ342905 NT 93 89 Lichtheimia hyalospora CBS102.36 GQ342907 T of Absidia cristata Lichtheimia hyalospora CBS100.28 GQ342902 T of Absidia blakesleeana 90 Lichtheimia hyalospora CBS100.36 GQ342943 95Lichtheimia sphaerocystis CBS647.78 GQ342911 Lichtheimia sphaerocystis CBS420.70 GQ342933 T 78 93 98Lichtheimia sphaerocystis CBS648.78 GQ342916 Lichtheimia ramosa CNM-CM3148 GQ342925 Lichtheimia ramosa CBS713.74 GQ342935 83 Lichtheimia ramosa CBS124197 GQ342951 99 Lichtheimia ramosa CBS582.65 GQ342909 NT Lichtheimiaceae Lichtheimia ramosa CBS223.78 GQ342934 Lichtheimia corymbifera CBS429.75 GQ342903 NT 100 Lichtheimia corymbifera CBS100.51 GQ342939 Lichtheimia ornata CNM-CM4978 JN206554 77 57 Lichtheimia ornata CBS291.66 GQ342946 T 100 Dichotomocladium elegans CBS714.74 JN206555 T 100 Dichotomocladium elegans CBS695.76 HM849715 Dichotomocladium hesseltinei CBS164.61 JN206556 T 89 Dichotomocladium robustum CBS440.76 JN206557 T ***

Fig. 1 (cont.)

and Backusella species with transitorily recurved sporangio duced taxonomic value of this trait. Circinate sporangiophores phores and is discussed below in more detail. All of these branches (Fig. 2h), considered to be a characteristic of the groups were recognised, to a large extent, by Schipper (1973, genus Circinella, evolved at least three times. 1975, 1976, 1978a). Species identification by DNA barcoding Phenotypic characters with restricted In our study the ITS region turned out to be an appropriate taxonomic relevance DNA barcoding marker in Mucorales because of its power to Genera preferably are defined as monophyletic. This may be discriminate the currently accepted species, including predomi- in conflict with phenotypic definitions when single characters nantly morphologically defined species (morphospecies), and are concerned. For example, sporangiola-forming taxa such as also all species in Mucor (Hermet et al. 2012), Lichtheimia Backusella, Chaetocladium, Ellisomyces, Helicostylum, Kirko- (Alastruey-Izquierdo et al. 2010) and Rhizopus (Abe et al. 2007) myces and Thamnidium, appear sporadically among Mucor recognized by GCPSR studies. The statement of Schwarz et al. species (Fig. 1, 3, 5, 6) and only the clade consisting of the (2006) that ITS is able to discriminate most but not all clinically sporangiola-forming genera Benjaminiella and Cokeromyces relevant members of Mucorales was due to the fact that the (Fig. 1) possess phylogenetic support. Hence we consider the authors treated R. azygosporus as a separate species, while potential to develop sporangiola in addition to or instead of now it is classified as a variety of R. microsporus (Zheng et al. sporangia as a plesiomorphic character in the Mucoraceae that 2007). Indeed the taxonomy of the Mucoraceae is unsatisfactory is genetically fixed but not revealed in all species. Consequently, according to current molecular characteristics and consequently the presence of sporangiola should not be used to define new identification by DNA barcoding is reliable only for species that genera, as was done recently in the newly described genus form clearly delimited clades in the ITS trees. In Rhizomucor Isomucor (de Souza et al. 2012) with sporangiola as the only (Fig. 11) and Rhizopus (Fig. 10), all species can be identified distinctive trait. A classical generic criterion is the presence or by their ITS sequence, and even two varieties of Rp. arrhizus absence of rhizoids. Our finding of rhizoids in Mucor species proved to be distinguishable (see sections on Rhizomucor or (Fig. 2c), the genus originally separated from Rhizomucor by Rhizopus, respectively). ITS data also separate a surprisingly the absence of rhizoids, and the reclassification of Rhizomu- high number of species of Mucor and its allies as monophyle cor species such as R. regularior, R. variabilis (Álvarez et al. tic groups (Fig. 2–5), allowing reliable species identification 2010a), R. chlamydosporus and R. endophyticus (this paper) despite unclear generic boundaries. Problematic for molecular into Mucor suggests that the potential to form rhizoids might be identification are the species complexes of Mucor circinel- plesiomorphic in Mucorales as well. Homo- and heterothallic loides, M. flavus, M. piriformis and Zygorhynchus moelleri. species cluster randomly in our trees demonstrating the re- The complex of Mucor circinelloides constitutes a supported 30 Persoonia – Volume 30, 2013

***

Absidia coerulea CBS102.28 JN206584 98 Absidia coerulea CBS101.28 JN206585 Absidia coerulea CBS104.08 HM849703 100 Absidia macrospora CBS697.68 HM849704 T 72 Absidia californica CBS314.78 JN206582 98 100 Absidia californica CBS126.68 JN206583 T Chlamydoabsidia padenii CBS172.67 JN206586 T Absidia glauca CBS100.48 JN206581 100 Absidia glauca CBS101.08 HM849705 T 100 Absidia pseudocylindrospora CBS100.62 JN206591 T 84 99 Absidia spinosa var. biappendiculata CBS187.64 JN206592 IT 96 Absidia spinosa var. spinosa CBS106.08 JN206590 66 Absidia anomala CBS125.68 JN206593 T 86 Absidia cylindrospora var. nigra CBS127.68 JN206589 T Cunninghamellaceae 86 99 Absidia cylindrospora var. cylindrospora CBS100.08 JN206588 Absidia repens CBS115583 HM849706 IT Absidia psychrophilia CBS128.68 JN206587 T Absidia fusca CBS102.35 HM849707 T 94 98 Absidia cylindrospora var. rhizomorpha CBS153.63 JN206594 T 99 Absidia heterospora CBS101.29 JN206595 T Halteromyces radiatus CBS162.75 JN206596 T 100 Absidia cuneospora CBS102.59 JN206579 Absidia cuneospora CBS101.59 JN206580 T 100 Cunninghamella binariae CBS158.28 JN206602 Cunninghamella binariae CBS481.66 JN206603 Cunninghamella elegans CBS167.53 HM849700 97 Cunninghamella phaeospora CBS692.68 HM849697 NT 98 Cunninghamella blakesleeana CBS782.68 JN206601 Cunninghamella bertholletiae CBS693.68 JN206600 NT of C. polymorpha Cunninghamella bertholletiae CBS190.84 HM849701 Cunninghamella bertholletiae CBS779.68 JN206599 Cunninghamella homothallica CBS168.53 JN206605 T 100 Cunninghamella clavata CBS100178 JN206604 T 100 100 Cunninghamella echinulata CBS156.28 HM849702 99 Cunninghamella echinulata CBS545.75 JN206597 T of C. echinulata var. antarctica Cunninghamella echinulata CBS656.85 JN206598 98 Cunninghamella vesiculosa CBS989.96 HM849693 T Cunninghamella intermedia CBS347.69 JN206606 T of C. brunnea 100 Circinella lacrymispora CBS101757 JN206608 T Gongronella lacrispora CBS244.62 JN206609 T 100 Hesseltinella vesiculosa CBS197.68 JN206609 IT Gongronella butleri CBS157.25 JN206607 T of G. urceolifera Gongronella butleri CBS216.58 HM849698 T of M. vesiculosus Thermomucor indicae-seudaticae CBS104.75 HM849661 T Lichtheimiaceae Lentamyces parricida CBS174.67 JN206535 NT Lentamycetaceae Protomycocladus faisalabadensis CBS661.86 JN206558 T Lichtheimiaceae 100 Apophysomyces variabilis CBS658.93 HM849695 T 98 Apophysomyces elegans CBS477.78 JN206536 Saksenaea oblongispora CBS133.90 HM849694 T Sakseneaceae 100 Radiomyces embreei CBS205.77 100 100 Radiomyces embreei CBS254.60 JN206538 T 77 Radiomyces spectabilis CBS255.60 JN206537 T Radiomycetaceae 100 Spinellus fusiger CBS405.63 HM849664 Phycomyces blakesleeanus CBS270.32 HM849662 Phycomycetaceae 90 Umbelopsis autotrophica CBS212.72 JN206561 Umbelopsis sp. ‘vinacea’ CBS913.85 JN206562 Umbelopsis ramanniana CBS101226 JN206566 89 Umbelopsis swartii CBS868.85 JN206567 T Umbelopsis gibberispora CBS109328 JN206564 T 100 Umbelopsis gibberispora CBS101745 JN206565 Umbelopsis sp. ‘westeae’ CBS559.86 JN206563 Umbelopsis ramanniana CBS112976 JN206568 Umbelopsis fusiformis CBS919.85 JN206559 Umbelopsidaceae 100 Umbelopsis fusiformis CBS385.85 JN206560 T 95 Umbelopsis isabellina CBS560.63 JN206573 80 Umbelopsis isabellina CBS250.95 JN206574 100 Umbelopsis ovata CBS499.82 JN206572 IT of Mortierella ovata 100 86 Umbelopsis isabellina CBS100559 JN206571 Umbelopsis vinacea CBS236.82 JN206569 T of U. multispora 94 Umbelopsis vinacea CBS212.32 JN206570 AUT of Mortierella vinacea Umbelopsis dimorpha CBS110039 HM849709 HT Mortierella parvispora CBS311.52 HM849689 ST Fig. 1 (cont.) clade (bootstrap support 88 %) in the LSU phylogram (Fig. 1) However, in this stage of research we consider ITS and LSU and comprises all formae of M. circinelloides but also other data sufﬁcient to justify taxonomic changes in the following species such as M. bainieri and M. ramosissimus, Ellisomyces cases: 1) synonymy if the ex-type strains have identical ITS anomalus and Backusella ctenidia that cannot be separated by sequences (in case the ITS sequences are identical but large ITS and LSU data from M. circinelloides (Fig. 5). All formae of morphological differences have been reported we maintain va M. circinelloides described by Schipper (1976) can be discrimi- rieties or formae); 2) attribution of the species rank to varieties or nated by their ITS sequences, but there are several strains in the formae if they appear distant to the type variety or forma in ITS complex with a M. circinelloides morphology that do not group and LSU trees; 3) reclassiﬁcations based on polyphyly shown in any of these formae (see sections Mucor and Backusella). by ITS and LSU data (e.g. Zygorhynchus); and 4) extension of a genus based on ITS and LSU analyses and morphological Criteria for taxonomic changes based on synapomorphies (e.g. Backusella). ITS and LSU data In general, multilocus studies are needed to establish boundaries between taxa forming sister clades in single-locus trees. G. Walther et al.: DNA barcoding in Mucorales 31

b c

d e

g h i Fig. 2 Morphological diversity of Mucoraceae. a. CBS 243.35 Mucor luteus, sporangiophore and sporangium with collar, columella and sporangiospores; b. CBS 385.95 Rhizomucor endophyticus (recombined into Mucor endophyticus in this paper), sporangiophore with columella and collar; c. CBS 243.35 Mucor luteus, rhizoids formed on glass slides; d. CBS 110662 Zygorhynchus multiplex (recombined into Mucor multiplex in this paper), zygospore; e. CBS 385.95 Rhizomucor endophyticus (recombined into Mucor endophyticus in this paper), zygospore; f. CBS 588.88 Pirella circinans, lateral sporangium; g. CBS 243.57 Ellisomyces anomalus, multispored sporangiola; h. Circinella rigida (recombined into Mucor durus in this paper), circinate sporangiophore branch with apophysate sporangium; i. CBS 341.55 Thamnidium elegans, multispored sporangiola. — Scale bars = 10 µm, except c = 100 µm. 32 Persoonia – Volume 30, 2013

83 Helicostylum pulchrum CBS259.68 JN206052 0.01 substitutions/site Helicostylum pulchrum CBS639.69 JN206055 Helicostylum pulchrum CBS107.23 JN206053 Helicostylum pulchrum 75 Helicostylum pulchrum CBS258.59 JN206054 T of H. venustellum Mucor flavus CBS664.67 JN206064 92 Mucor flavus CBS182.90 JN206065 Thamnidium elegans CBS411.52 JN206058 Thamnidium elegans CBS641.69 JN206059 Thamnidium elegans CBS341.55 JN206060 Thamnidium elegans 100 Mucor aligarensis CBS993.70 JN206056 T Mucor aligarensis CBS244.58 JN206057 97 Mucor flavus CBS126.70 JN206049 T of M. mephitis Mucor flavus CBS234.35 JN206051 NT Mucor flavus CBS197.71 JN206066 T of M. meridionalis Mucor flavus CBS210.71 JN206050 T of M. peacockensis 99 Mucor flavus CBS992.68 c1 JN206067 Mucor flavus CBS992.68 c3 JN206068 Mucor flavus complex 99 Mucor flavus CBS681.73 c2 JN206069 Mucor flavus CBS681.73 c3 JN206070 83 Mucor flavus CBS230.35 JN206061 T of M. attenuatus 99 Mucor flavus CBS893.73 c1 JN206062 NT of M. sciurinus 79 Mucor flavus CBS893.73 c2 JN206063 NT of M. sciurinus Mucor saturninus CBS974.68 JN206072 NT 100 Mucor saturninus CBS599.78 c3 JN206073 (chicken) Mucor saturninus CBS598.78 JN206074 Mucor saturninus Mucor saturninus CBS599.78 c4 JN206075 (chicken) 81 Mucor minutus CBS586.67 JN206048 T of M. griseoochraceus var. minuta Pilaira anomala CBS131.23 JN206097 Pilaira anomala CBS695.68 JN206098 Pilaira anomala CBS699.71 JN206100 98 Pilaira anomala CBS396.71C JN206099 Pilaira anomala 89 Pilaira anomala CBS424.70 JN206101 100 Pilaira moreaui CBS411.67 JN206095 100 Pilaira moreaui var. caucasica CBS523.68 EF555487 T of P. caucasica Pilaira moreaui CBS496.71 JN206096 Pilaira moreaui Pilaira moreaui CBS181.26 JN206094 90 Pirella sp. ‘circinans’ CBS588.88 JN206071 100 Pirella naumovi CBS524.68 JN206103 T Pirella circinans CBS962.68 JN206102 T Mucor piriformis CBS169.25 JN206028 NT Mucor piriformis CBS175.27 JN206029 99 Mucor piriformis CBS528.68 JN206032 Mucor piriformis CBS527.68 JN206034 Mucor piriformis Mucor piriformis CBS111230 c1 JN206030 Mucor piriformis CBS256.85 JN206031 86 100 Mucor sp. CBS125018 c1 JN206038 Mucor sp. CBS125018 c2 JN206039 97 Mucor sp. ‘piriformis’ CBS837.73A JN206033 100 Mucor strictus CBS100.66 JN206035 T of M. kanivcevii 100 Mucor strictus CBS368.71A JN206036 Mucor strictus 98 Mucor strictus CBS576.66 JN206037 NT Mucor plasmaticus CBS177.46 c4 JN206077 Mucor plasmaticus CBS402.73 c4 JN206080 Mucor plasmaticus CBS275.49 c4 JN206079 100 Mucor plasmaticus CBS275.49 c2 JN206078 Mucor plasmaticus Mucor plasmaticus CBS402.73 c1 JN206081 Mucor plasmaticus CBS177.46 c1 JN206076 100 Mucor mucedo CBS228.29 JN206088 T of M. murorum Mucor mucedo CBS296.35 JN206090 Mucor mucedo CBS987.68 JN206089 85 Mucor mucedo CBS834.73 c2 JN206091 Mucor mucedo CBS836.73 c1 JN206092 Mucor mucedo 96 Mucor mucedo CBS834.73 c1 JN206093 Mucor mucedo CBS542.66 JN206086 98 100 Mucor mucedo CBS525.68 JN206087 Mucor mucedo CBS640.67 JN206085 NT Mucor sp. ‘plasmaticus’ CBS122.23 JN206040 Mucor genevensis CBS404.71 c1 JN206042 98 Mucor genevensis CBS564.75 c3 JN206044 Mucor genevensis CBS404.71 c2 JN206043 100 Mucor genevensis CBS114.08 JN206041 T Mucor genevensis CBS535.78 c3 JN206046 Mucor genevensis 92 Mucor genevensis CBS535.78 c4 JN206047 * Mucor genevensis CBS535.78 c1 JN206045 Fig. 3 RAxML phylogram of the Mucor mucedo group, M. flavus group, M. hiemalis group and related taxa based on the ITS region. Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type strains are designated by: T = ex-type strain, ET = ex-epitype strain, HT = ex-holotype strain, IT = ex- isotype strain, LT = ex-lectotype strain, NT = ex-neotype strain. Ex-type strains of currently accepted taxa are printed in bold. Clones are speciﬁed by a ‘c’ followed by the clone number. Clinical strains are highlighted by red strain and GenBank accession numbers, non-human hosts are given in brackets. Potentially undescribed taxa are indicated by bold blue font. Morphological identiﬁcations are given in quotation marks.

Species concepts: conflicts between grouping (Schell et al. 2011) and Lichtheimia (Alastruey-Izquierdo et al. in the ITS trees and mating results 2010). Consequently, the study of zygospores should not be devaluated by our results but is ideally combined with multi- The broad species concepts in Mucor maintained by Schipper locus studies to recognize species. (1973, 1975, 1976, 1978a), based on positive matings, are partly in conflict with the grouping in our ITS trees. Species in Detection of undescribed species by DNA barcoding the sense of Schipper, e.g. Mucor circinelloides, M. hiemalis Twelve MOTUs are revealed by ITS sequencing that deviate (including M. luteus) and M. flavus are not monophyletic. Their considerably from species included in this study and might clades include other species or even other genera showing that represent undescribed taxa (indicated by bold blue font in all the simple presence of zygospores is not a sufficient marker trees). The species to which they had previously been as- for conspecifity. However, a detailed study of their size, shape, signed, on the basis of morphological features, are given in colour and number may allow to differentiate between inter- quotation marks. In order to establish whether these MOTUs and intraspecific zygospores, as shown for Mucor irregularis deserve species rank we have initiated multilocus-analyses G. Walther et al.: DNA barcoding in Mucorales 33

* Mucor hiemalis f. corticola CBS365.68 JN206133 Mucor hiemalis f. hiemalis CBS110.19 JN206136 T of M. hiemalis var. toundrae Mucor hiemalis f. hiemalis CBS107.19 JN206137 T of M. vallesiacus Mucor hiemalis f. hiemalis CBS242.35 JN206134 IT of M. hiemalis var. griseus Mucor hiemalis f. hiemalis CBS328.92 JN206135 Mucor hiemalis CBS118522 JN206138 Mucor hiemalis Fungiscope AS72 JN206142 Mucor hiemalis f. corticola CBS366.68 JN206139 69 Mucor hiemalis f. hiemalis CBS528.78 JN206144 80 Mucor hiemalis f. corticola CBS532.78 JN206145 Mucor hiemalis f. hiemalis CBS117.08 JN206143 T of M. adventitius var. aurantiacus Mucor hiemalis f. corticola CBS533.78 JN206146 Mucor hiemalis CNM-CM2540 JN206140 Mucor hiemalis 70 Mucor hiemalis f. corticola CBS362.68 JN206132 Mucor hiemalis CNM-CM5229 JN206141 100 Mucor hiemalis f. hiemalis CBS201.65 JN206125 NT Mucor hiemalis f. hiemalis CBS454.71 JN206126 Mucor hiemalis f. hiemalis CBS123972 JN206128 88 Mucor hiemalis CBS115.18 JN206127 Mucor hiemalis f. hiemalis CBS980.68 JN206129 Mucor hiemalis f. hiemalis CBS337.71D JN206131 Mucor hiemalis f. corticola CBS106.09 JN206130 T 79 Zygorhynchus psychrophilus CBS336.68 JN206157 HT Zygorhynchus japonicus CBS154.69 JN206158 NT (designated here) Mucor irregularis CBS654.78 JN206151 Mucor irregularis CBS609.78 JN206152 100 Mucor irregularis CBS103.93 JN206150 T of Rhizomucor variabilis Mucor irregularis CBS100164 JN206153 100 Mucor irregularis PUMC-1 EU616623 98 Mucor irregularis 81 Mucor irregularis CBS700.71 JN206154 Mucor irregularis CBS977.68 c1 JN206155 Mucor irregularis CBS977.68 c2 JN206156 Mucor luteus CBS243.35 HM999954 T 100 Mucor luteus CBS567.70A JN206147 Mucor luteus CBS301.74 JN206149 Mucor luteus Mucor luteus CBS244.35 JN206148 100 85 Rhizomucor endophyticus CBS385.95 JN206159 HT Rhizomucor endophyticus Rm-8 EF583635 100 100 Zygorhynchus heterogamus CBS338.74 c4 JN206168 100 Zygorhynchus heterogamus CBS338.74 c1 JN206169 Zygorhynchus heterogamus CBS405.58 JN206167 ET (designated here) Zygorhynchus multiplex CBS110662 JN206166 HT Zygorhynchus heterogamus CBS594.83 c3 JN206164 Mucor heterogamus 71 Zygorhynchus heterogamus CBS594.83 c4 JN206165 98 Zygorhynchus heterogamus CBS252.85 c1 JN206161 complex 100 100 Zygorhynchus heterogamus CBS252.85 c4 JN206162 Zygorhynchus macrocarpus CBS215.27 JN206160 ET (designated here) Zygorhynchus heterogamus CBS580.83 JN206163 Mucor hiemalis f. silvaticus CBS249.35 JN206122 100 Mucor hiemalis f. silvaticus CBS509.66 JN206123 Mucor hiemalis f. silvaticus CBS412.71 JN206124 NT (designated here) Mucor silvaticus 100 Mucor abundans CBS521.66 JN206110 100 Mucor abundans CBS388.35 JN206111 NT (designated here) 100 Circinella rigida CBS156.51 JN206112 ET (designated here) Circinella rigida CBS484.66 JN206113 99 Mucor bacilliformis CBS251.53 JN206083 T Mucor bacilliformis CBS573.70 JN206084 Mucor guiliermondii CBS174.27 JN206082 T Zygorhynchus moelleri CBS501.66 JN206118 100 Zygorhynchus moelleri CBS380.29 JN206119 Zygorhynchus californiensis CBS402.58 JN206117 ET (designated here) Zygorhynchus moelleri CBS216.27 JN206116 Zygorhynchus moelleri CBS460.51 JN206120 Mucor moelleri 100 Zygorhynchus moelleri CBS406.58 JN206121 NT (designated here) Zygorhynchus moelleri IHEM21156 JN206115 Zygorhynchus moelleri CBS444.65 JN206114 LT of Mucor saximontensis (designated here) Mucor zonatus CBS529.83 JN206105 87 Mucor zonatus CBS183.76 c4 JN206107 98 Mucor zonatus CBS148.69 JN206104 T Mucor zonatus Mucor zonatus CBS183.76 c3 JN206106 100 Mucor laxorrhizus var. ovalisporus CBS417.77 JN206108 T Mucor laxorrhizus var. ovalisporus CBS522.79 JN206109 Mucor amphibiorum CBS763.74 HM999957 T (amphibian)

Fig. 3 (cont.) and detailed morphological studies, which will be published Intraspecific variability in subsequent papers. Regardless of the question if these Intraspeciﬁc variability differs widely among mucorealean spe- MOTUs represent undescribed species, our results suggest cies and genera. The following dissimilarity values were ob- that the used phenotypic criteria for species recognition in the tained for the ITS region of species represented by at least 5 Mucorales underestimated existing diversity. strains: Backusella circina (0 %), Choanephora cucurbitarum The ITS sequences of numerous strains of Absidia and Umbel- (0.5 %), Circinella muscae (1.6 %), Cunninghamella berthol- opsis could not be obtained by direct sequencing and not all of letiae (1.0 %), C. blakesleeana (0.9 %), C. echinulata (13.3 %), them could be cloned. Our ITS dataset is therefore incomplete Gilbertella persicaria (1.2 %), Gongronella butleri (1.1 %), Lich- for these genera. theimia corymbifera (2.0 %), L. hyalospora (6.3 %), L. ramosa ITS barcoding detected cryptic species, but conversely revealed (7.6 %), Mucor circinelloides (5.3 %), M. fuscus (0.2 %), M. hie- growth-reduced mutants of existing taxa that had been main- malis (4.1 %), M. indicus (0.9 %), M. irregularis (2.6 %), M. mucedo tained incorrectly as separate species. Examples include Mucor (3.5 %), M. odoratus (0.3 %), M. piriformis (0.6 %), M. plumbeus sinensis, which is synonymised with M. racemosus f. racemo- (0.2 %), M. racemosus (2.1 %), Phycomyces blakesleeanus sus below, alleged Mucor ramosissimus strains that proved to (0 %), Pilaira anomala (0 %), Rhizomucor pusillus (0.2 %), Rhizo- belong to M. circinelloides f. circinelloides or f. janssenii, and pus arrhizus incl. var. delemar (1.2 %), R. lyococcus (0.7 %), Rhizomucor tauricus which is a synonym of Rm. pusillus. R. microsporus (2.8 %), Thamnostylum piriforme (1.6 %), 34 Persoonia – Volume 30, 2013

Umbelopsis nana (0 %) and Zygorhynchus moelleri (0 %). The clinical strain CNM-CM 5114 (JN205884) isolated in 2008 Complete identity might be the result of undersampling. High from a lung biopsy specimen in Barcelona (Spain) was diag- dissimilarity values of morphologically defined species such as nosed as Cunninghamella elegans by a 99.2 % similarity of Cunninghamella echinulata could be caused by the inclusion the ITS region with the ex-neotype strain of this species CBS of more than one species. However, the taxonomically well- 160.28 (AF254928). This is the first time that the involvement of elaborated genus Lichtheimia also contains species with more C. elegans in human infection has been documented molecu- than 5 % dissimilarity suggesting that intraspecific variability larly. can be comparatively high in Mucorales. The recent detection of species with clinical relevance, such as In agreement with Nilsson et al. (2006) we did not find a unify- Cunninghamella echinulata (Lemmert et al. 2002), Lichtheimia ing threshold for intraspecific variability, a result that should ornata (Alastruey-Izquierdo et al. 2010), Mucor ardhlaengiktus caution against formal ITS-based species delimitation. On the (as M. ellipsoideus, Álvarez et al. 2011) or Rhizopus homothal- other hand, the ≥ 99 % identity threshold given by Balajee et al. licus (Chakrabarti et al. 2010) shows that basically all mucore- (2009) for comparative ITS sequence identification using Gen- alean taxa, including species regarded as strictly environmental, Bank in Mucorales is not covering the intraspecific variability should be included in the database for molecular identification of several clinically important species. Our data suggest that a of clinical strains. Molecular identification by BLAST searches of distinct identity threshold has to be defined for every species GenBank may lead to wrong conclusions because of incomplete for a reliable ITS-based identification. GCPSR studies will be sampling, inconsistent nomenclature and a high percentage required beforehand to define species boundaries in species of misidentified taxa (Bridge et al. 2003, Nilsson et al. 2006, with conflicting morphological and ITS data especially in those Bidartondo et al. 2008, Lian et al. 2011). that are part of a species complex. The following taxa have been reported from proven case studies (de Hoog et al. 2000, Gomes et al. 2011): Actinomucor elegans, Apophysomyces variabilis, A. trapeziformis (Weddle Clinically relevant taxa et al. 2012), Cokeromyces recurvatus, Cunninghamella berthol- Clinical strains are highlighted by red strain numbers and Gen- letiae, C. elegans, Lichtheimia corymbifera, L. ramosa, Mucor Bank accession numbers in all figures. Strains representing a amphibiorum, M. ardhlaengiktus (Sugui et al. 2011), M. circinel- clinically relevant MOTU are marked either by a red square loides (f. circinelloides, f. janssenii, f. lusitanicus), M. hiemalis, (assignment to the respective MOTU by ITS comparison) or M. irregularis (syn. Rhizomucor variabilis), M. luteus (syn. by a red circle (assignment to the respective MOTU by mor- M. hiemalis f. luteus), M. indicus, M. racemosus f. racemosus, phology) in the LSU tree (Fig. 1). The following genera contain M. ramosissimus, Rhizopus microsporus, Rp. arrhizus, Rp. homo- species that are involved in human opportunistic infections: thallicus, Rp. schipperae, Rp. stolonifer, Rhizomucor miehei, Actinomucor, Apophysomyces, Cokeromyces, Cunningha- Rm. pusillus, Saksenaea vasiformis, S. erythrospora (Hospen mella, Lichtheimia, Mucor, Rhizomucor, Rhizopus, Saksenaea, thal et al. 2011) and Syncephalastrum racemosum. Syncephalastrum, and possibly Mycotypha, Thamnostylum The forma circinelloides of Mucor circinelloides contains the (Xess et al. 2012) and Zygorhynchus, the latter genus being highest number of clinical isolates in Mucor and forms a well- synonymised with Mucor below. The recently described clini- supported clade (bootstrap 95 %). Two groups within forma cally relevant species of Apophysomyces, viz. A. ossiformis and A. trapeziformis (Álvarez et al. 2010b) and of Saksenaea, viz. S. erythrospora as well as the pathogenic S. vasiformis (Álvarez et al. 2010a) were not included in our study. No clinical strains of the following species were included in the present study, but clinical relevance of the species, resp. strains originating from clinical samples were reported by other authors: Cokeromyces recurvatus (e.g. Ryan et al. 2011), Cunninghamella blakesleeana (García Rodríguez et al. 2012), C. echinulata (Lemmert et al. 2002), Mucor ardhlaengiktus (as M. ellipsoideus), M. circinelloides f. lusitanicus (as M. lusitanicus) (Álvarez et al. 2011), Rhizopus homothallicus (Chakrabarti et al. 2010), Rhizopus stolonifer (de Hoog et al. 2000) and Thamnostylum lucknowense (Xess et al. 2012). A clinical strain treated as Mucor fragilis by Álvarez et al. (2011) is assigned to M. circinelloides below. a b The involvement of Zygorhynchus moelleri and a possibly un described Mycotypha species in human mucormycoses has not been proven: strain IHEM 21156 of Zygorhynchus moelleri was isolated by J.P. Bouchara at the University Hospital Angers (France) in 2004 but the exact source is unknown, and Myco typha strain CBS 109960 was isolated by N. Poonwan from pus of a wound in the RMSC Pitsunalok (Thailand) in 2002. There is also no case report on Mucor plumbeus but the source of isolation of strain CBS 634.74 (human biopsy material) and CBS 633.74 (subcutaneous tissue of a cat) suggest a pathogenic potential of this species. Mucor aligariensis was isolated in 1958 from human and M. saturninus in 1978 from chicken, but no proven case reports have been published. Based on c d their maximum growth temperatures (< 30 °C, Schipper 1978a) Fig. 4 CBS 388.35 Mucor abundans. a, c. Sporangiophore with columella the involvement of these species in infections is questionable. and collar; b. sporangiospores; d. chlamydospores. — Scale bars = 50 µm. G. Walther et al.: DNA barcoding in Mucorales 35

Mucor circinelloides f. circinelloides CBS195.68 JN205961 NT 0.005 substitutions/site Mucor circinelloides f. circinelloides CBS247.35 JN205962 Mucor circinelloides f. circinelloides CNM-CM2922 JN205963 Mucor circinelloides f. circinelloides IHEM16415 JN205964 Mucor circinelloides f. circinelloides CBS480.70G JN205965 Mucor circinelloides f. circinelloides CNM-CM4621 JN205970 Mucor circinelloides f. circinelloides CNM-CM5169 JN205971 Mucor circinelloides f. circinelloides CBS196.68 JN205968 Mucor circinelloides f. circinelloides CBS394.68 JN205969 Mucor circinelloides f. circinelloides CBS172.27 JN205967 Mucor circinelloides f. circinelloides CBS194.68 JN205972 Mucor circinelloides ‘ramosissimus’ CBS121702 JN205966 78 Mucor circinelloides IHEM21426 JN205935 Mucor circinelloides IHEM20006 JN205936 Mucor circinelloides IHEM21105 JN205937 Mucor circinelloides CBS384.95 JN205933 T of Rhizomucor regularior Mucor circinelloides CNM-CM2437 JN205939 Mucor circinelloides IHEM22323 JN205941 Mucor circinelloides CNM-CM5071 JN205938 Mucor circinelloides Mucor circinelloides f. circinelloides CBS239.35 JN205942 T of M. griseoroseus Mucor circinelloides CBS111555 JN205943 f. circinelloides Mucor circinelloides f. circinelloides ‘M. rouxii’ CBS416.77 JN205934 Mucor circinelloides f. circinelloides CBS479.70 JN205973 Mucor circinelloides f. circinelloides CBS123973 JN205958 Mucor circinelloides CBS111560 JN205957 Mucor circinelloides f. circinelloides CBS480.70F JN205956 Mucor circinelloides f. circinelloides CBS108.16 JN205954 Mucor circinelloides IHEM21158 JN205960 Mucor circinelloides f. circinelloides CBS295.34 JN205955 Mucor circinelloides CNM-CM4569 JN205951 Mucor circinelloides CNM-CM4895 JN205975 Mucor circinelloides CNM-CM4974 JN205952 Mucor circinelloides CNM-CM3926 JN205949 95 Mucor circinelloides CNM-CM2541 JN205944 Mucor circinelloides CNM-CM3178 JN205946 70 Mucor circinelloides CNM-CM3785 JN205948 Mucor circinelloides CNM-CM4299 JN205950 69 Mucor circinelloides CNM-CM3510 JN205947 Mucor circinelloides f. circinelloides CBS192.68 JN205959 Mucor circinelloides CNM-CM4366 JN205974 88 Mucor circinelloides CNM-CM3112 JN205945 Mucor circinelloides CNM-CM4526 JN205953 Backusella ctenidia CBS293.66 JN205976 IT Backusella ctenidia CBS696.76 JN205977 Mucor ctenidius Backusella ctenidia CBS433.87 JN205978 Mucor circinelloides f. griseocyanus CBS223.56 JN206000 Mucor circinelloides f. griseocyanus CBS366.70 JN206001 99 Mucor circinelloides f. griseocyanus CBS698.68 JN206002 96 Mucor circinelloides f. griseocyanus Mucor circinelloides f. griseocyanus CBS198.28 HM999951 100 Mucor circinelloides f. griseocyanus CBS116.08 JN206003 T Mucor circinelloides ‘f. janssenii’ CBS846.73 JN206014 Mucor circinelloides ‘f. janssenii’ CBS526.68 JN206015 Mucor circinelloides f. lusitanicus CBS108.17 JN205980 T Mucor circinelloides f. lusitanicus CBS633.65 JN205986 Mucor circinelloides f. lusitanicus CBS968.68 HM999953 Mucor circinelloides f. lusitanicus CBS847.72 JN205981 Mucor ramosissimus CBS135.65 JN205932 NT Mucor circinelloides f. lusitanicus CBS111229 c2 JN205983 Mucor circinelloides f. lusitanicus CBS851.71 JN205982 Mucor circinelloides f. lusitanicus CBS276.49 JN205984 Mucor circinelloides f. lusitanicus Mucor circinelloides f. lusitanicus CBS969.68 JN205985 Mucor circinelloides f. lusitanicus CBS253.35 JN205988 Mucor circinelloides f. lusitanicus CBS242.33 JN205987 80 Mucor circinelloides f. lusitanicus CBS111229 c4 JN205990 98 Mucor circinelloides f. lusitanicus CBS111228 c3 JN205989 Mucor circinelloides f. lusitanicus CBS111228 c1 JN205991 91 Mucor circinelloides f. lusitanicus ‘fragilis’ CBS236.35 JN205979 Mucor circinelloides CBS338.71 JN205998 100 Mucor circinelloides CNM-CM5225 JN205999 Mucor circinelloides CBS635.65 JN205997 Ellisomyces anomalus CBS243.57 JN205992 T Ellisomyces anomalus CBS697.76 JN205993 Mucor circinelloides CBS202.28 JN205994 Mucor bainieri CBS293.63 JN205995 IT Mucor circinelloides CBS124110 JN205996 Mucor circinelloides f. janssenii CBS232.29 JN206007 T of M. tenellus 94 Mucor circinelloides f. janssenii CBS365.70 JN206009 Mucor circinelloides f. janssenii CBS185.68 JN206006 T of M. kurssanovii Mucor circinelloides f. janssenii CBS206.68 JN206004 80 Mucor circinelloides f. janssenii CBS227.29 JN206008 Mucor circinelloides f. janssenii CBS243.67 JN206005 Mucor circinelloides f. janssenii 96 Mucor circinelloides f. janssenii CBS205.68 HM999952 NT (designated here) Mucor circinelloides f. janssenii CBS762.74 JN206011 Mucor circinelloides f. janssenii ‘aff. ramosissimus’ CBS144.93 JN206012 Mucor circinelloides f. janssenii CBS204.68 JN206010 (iguana, lung) Mucor circinelloides ‘f. griseocyanus’ CBS541.78 JN206013 * Fig. 5 RAxML phylogram of the Mucor racemosus group and related taxa based on the ITS region. Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type strains are designated by: T = ex-type strain, ET = ex-epitype strain, IT = ex-isotype strain, NT = ex-neotype strain. Ex-type strains of currently accepted taxa are printed in bold. Clones are speciﬁed by a ‘c’ followed by the clone number. Clinical strains are highlighted by red strain and Gen- Bank accession numbers, non-human hosts are given in brackets. Potentially undescribed taxa are indicated by bold blue font. Morphological identiﬁcations are given in quotation marks. Black bars indicate groups within Mucor circinelloides f. circinelloides including a large number of clinical strains. 36 Persoonia – Volume 30, 2013

* Mucor racemosus f. chibinensis CBS636.67 JN205904 NT (designated here) Mucor racemosus f. racemosus CBS222.81 JN205906 Mucor racemosus f. racemosus CBS369.71 JN205905 Mucor racemosus f. racemosus CBS616.63 JN205903 (Agapornis, lung) Mucor racemosus f. racemosus CBS661.66 JN205908 Mucor racemosus Mucor racemosus f. chibinensis CBS660.66 JN205911 (halva) Mucor racemosus CNM-CM2569 JN205929 f. racemosus Mucor racemosus CNM-CM3862 JN205930 Mucor racemosus f. racemosus CBS113.08 JN205909 ET of M. dimorphosporus (designate here) Mucor racemosus f. racemosus CBS260.68 JN205898 NT (designated here) Mucor racemosus f. racemosus CBS111557 JN205902 93 Mucor sinensis CBS204.74 JN205899 T 100 Mucor racemosus f. racemosus CBS657.68 JN205901 Mucor racemosus f. ‘sphaerosporus’ CBS271.86 JN205900 Mucor plumbeus CBS226.32 JN205916 82 Mucor plumbeus CBS814.96 JN205917 Mucor plumbeus CBS633.74 JN205913 (cat, subcutaneous tissue) Mucor plumbeus CBS312.78 JN205915 Mucor plumbeus CBS295.63 JN205912 Mucor plumbeus Mucor plumbeus CBS634.74 HM999955 Mucor plumbeus CBS284.78 JN205914 Mucor plumbeus CBS630.74 JN205918 Chaetocladium brefeldii CBS136.28 JN206019 Chaetocladium brefeldii CBS162.82 JN206021 99 Chaetocladium brefeldii CBS116.24 JN206020 Chaetocladium brefeldii 99 Chaetocladium brefeldii CBS156.74 JN206022 Chaetocladium jonesiii CBS811.69 JN206023 Mucor brunneogriseus CBS129.41 JN205910 T Mucor racemosus f. sphaerosporus CBS238.35 JN205920 Mucor racemosus f. sphaerosporus CBS634.78 c2 JN205927 Mucor racemosus f. sphaerosporus CBS115.08 JN205919 IT 83 Mucor racemosus f. sphaerosporus CBS634.78 c4 JN205928 Mucor racemosus f. sphaerosporus CBS574.70 JN205921 Mucor racemosus Mucor racemosus f. sphaerosporus CBS571.70 JN205926 Mucor racemosus f. sphaerosporus CBS539.78 JN205923 f. sphaerosporus Mucor racemosus f. sphaerosporus CBS538.78 JN205922 Mucor racemosus f. sphaerosporus CBS347.87 JN205924 Mucor racemosus f. sphaerosporus CBS143.70 JN205925 Parasitella sp. ‘parasitica’ CBS152.69 c1 JN206024 100 100 Parasitella sp. ‘parasitica’ CBS152.69 c3 JN206025 Parasitella sp. Parasitella sp. ‘parasitica’ CBS207.28 JN206026 Parasitella parasitica CBS412.66 JN206027 NT Mucor sp. ‘circinelloides f. griseocyanus’ CBS608.78 JN205931 Mucor mousanensis CBS531.77 JN206016 100 Mucor mousanensis CBS721.76 JN206018 Mucor mousanensis Mucor mousanensis CBS999.70 JN206017 T Mucor amphibiorum CBS763.74 HM999957 T Fig. 5 (cont.) circinelloides (highlighted by black bars in Fig. 5) contain a pulchrum and Thamnidium elegans. Several ex-type strains large number of clinical strains, which might be explained to of species that were synonymised by Schipper (1975) with high frequency in the human environment, or to an increased M. flavus based on positive matings and morphological simi- virulence. Interestingly, the forma griseocyanus, being the larity are placed in other clades than the neotype strain CBS only forma of Mucor circinelloides that is not able to grow at 234.35. Their correct taxonomic status needs to be assessed 37 °C, does not comprise any clinical isolate. In Mucor race by more detailed studies. mosus we found a similar picture: only the forma racemosus Only two of the four formae established by Schipper (1973) appears to be involved in human infections, while the forma for Mucor hiemalis, viz. f. hiemalis and f. corticola, constitute sphaerosporus contains strains only isolated from food and a clade in the ITS tree (Fig. 3). This clade is divided into two environmental samples. The maximum growth temperature of subclades both composed of strains morphologically assigned forma sphaerosporus is distinctly lower (28 °C > T < 25 °C) max to f. hiemalis and f. corticola. However, the ex-type strains of than that of the clinically relevant forma racemosus (37 °C > both formae are located in the same subclade (Fig. 3). For T < 30 °C) (Schipper 1976). max that reason we consider the small differences in the shape of Many thermotolerant or thermophilic species (Schipper 1973, the spores between the formae as taxonomically insignificant. 1975, 1976, 1978a, b, 1979, 1984, 1986, 1990, Schipper & Strains of Mucor hiemalis f. silvaticus are not part of the well- Stalpers 1984, Schipper & Samson 1994, de Hoog et al. 2000, supported Mucor hiemalis clade, not in the ITS (Fig. 3) nor Zheng & Chen 2001, Hoffmann et al. 2007, Alastruey-Izquierdo in the LSU tree (Fig. 1). Therefore we treat this taxon as a et al. 2010, Álvarez et al. 2010a, b) in the Mucorales are oppor- discrete species. The same applies to Mucor hiemalis f. luteus tunistic pathogens of vertebrates, but there is no direct correla- that recently was reclassified as Mucor luteus (Budziszewska tion between thermotolerance and pathogenicity. Several taxa et al. 2010). including species of Backusella, Cunninghamella and Mucor, No authentic material is known to exist of Mucor abundans. The as well as Protomycocladus faisalabadensis and Thermomucor protologue (Povah 1917) describes M. abundans as a species indicae-seudaticae are thermotolerant or even thermophilic, but with slightly branched sporangiophores, sporangia with a dia have never been reported to cause infections. meter below 100 µm, subglobose to pyriform columellae and small globose to short ellipsoidal sporangiospores. The strains TAXONOMY CBS 388.35 (Fig. 4) and CBS 521.66, the latter deposited as M. abundans in the CBS collection, match these microscopic Mucor characters including the typical size and shape of the sporangiospores. They only differ by features that may have changed Mucor mucedo group, M. flavus group, during prolonged cultivation such as the colour of the colony or M. hiemalis group and related taxa the colour of the young sporangia that are hyaline with a slight In our ITS tree (Fig. 3) Mucor flavus is divided in several small yellow tinge in the studied strains instead of yellowish. Schipper clades that are intermingled with M. aligarensis, Helicostylum (1973) noted the similarity of both strains with M. abundans but G. Walther et al.: DNA barcoding in Mucorales 37 she considered them too similar to M. hiemalis f. corticola to clade of Mucor circinelloides f. lusitanicus. However, the se- recognise a separate species. Our ITS tree (Fig. 3) however, quence differs considerably (19 out of 585 basepairs, 3.2 % supports the separate position of these strains and therefore dissimilarity) from the remaining sequences of M. circinelloides we here designate CBS 388.35 (preserved in a metabolically f. lusitanicus. This is expressed by a long branch in Fig. 5. inactive state by lyophilization, batch nr. 472) as the neotype We therefore retain the species rank for this taxon and await of Mucor abundans. studies on other loci. Other strains that had been identified On MEA at room temperature strain CBS 388.35 shows the morphologically as M. ramosissimus are positioned distantly following features: colonies expanding, cottony, at first white in different groups: CBS 144.93 clusters in the Mucor circinel- later depending on the intensity of sporulation grey beige or loides f. janssenii clade and CBS 121702 in the M. circinelloides pale grey; reverse uncoloured; sporangiophores slightly sym- f. circinelloides clade. These strains differ by slow growth and podially branched, up to 15 µm diam; sporangiophore branches possibly represent growth-reduced mutants of these formae. straight; sporangia dark greyish, young hyaline or with a slight CBS 236.35, the only strain listed as Mucor fragilis in the CBS yellow tinge, small, up to 70 µm diam; columellae subglobose, database, is positioned basally in the Mucor circinelloides f. lu ellipsoidal or slightly pyriform (as illustrated by Povah 1917) sitanicus clade. As far as we are aware no type material exists (Fig. 4a, c), up to 39 by 46 µm in size, often purplish grey; for M. fragilis. Bainier’s (1884) original description assigns the collars distinct; sporangiospores hyaline, smooth, subglobose fungus to the Mucor hiemalis group because the zygospores (3.5–6 µm diam), short ellipsoidal to short cylindrical (4.5–7 by are black and bear characteristically roughened stellate spines. 3.5–5.5 µm) (Fig. 4b); chlamydospores globose, ellipsoidal or Schipper (1976) treated Mucor fragilis as a doubtful species pyriform, intercalary, terminally and laterally formed mainly in because strain CBS 236.35, received as Mucor fragilis from the submerged mycelium (Fig. 4d); zygospores absent. Zycha in 1935, deviated from Bainier’s description. In agreement with Schipper (1976) we are not using this name and re Mucor racemosus group and related taxa identify strain CBS 236.35 as Mucor circinelloides f. lusitanicus. Based on our analyses (Fig. 1, 5) Mucor circinelloides repre Mucor circinelloides f. janssenii splits in two groups in the ITS sents a species complex, which also includes other Mucor tree (Fig. 5): a first group containing the ex-type strain, and species and sporangiola-forming taxa. The backbone of the a second group consisting of CBS 144.93, CBS 204.68 and M. circinelloides part of the ITS tree is poorly resolved, ham- CBS 762.74 at 0.7 % distance. Considering the high degree pering a decision on the rank of the taxa included. Based on positive matings, Schipper (1976) reduced four species related of sequence diversity in Mucorales, expressed for example to Mucor circinelloides to formae, namely f. circinelloides, in an ITS sequence dissimilarity of 2.6 % between Mucor cir- f. griseocyanus, f. janssenii and f. lusitanicus. Recently Álvarez cinelloides f. janssenii and f. lusitanicus, these groups within et al. (2011) proposed species status for f. lusitanicus without jansenii are being treated as taxonomically insignificant. The considering the positive mating results obtained by Schipper ITS sequence of the ex-type strain of the recently described (1976). Strains morphologically assigned to the various formae Mucor velutinosus (FN650646; Álvarez et al. 2011) is identical of M. circinelloides form well-supported clades in the ITS tree to that of CBS 762.74 of f. janssenii (data not shown). According (Fig. 5), but several additional strains that morphologically to their ITS sequences (data not shown) isolates ATCC1209b belong to M. circinelloides are located outside these clades. (HM754254) and UIC-1 (HQ154609) of M. circinelloides form- The strains CBS 338.71 and CBS 635.65, for example, are ing an unknown group in the phylogenetic analyses of Li et al. placed basally to the forma lusitanicus clade in the ITS tree (2011) also belong to f. janssenii but type material of this form (Fig. 5) and develop predominantly globose columellae char- was not included in the study of Li et al. (2011). acteristic of forma lusitanicus, but occasionally they develop The ITS sequence deposited in GenBank (EF203698) for the obovoid columellae typical of forma circinelloides. Schipper’s newly described species Mucor renisporus (Jacobs & Botha (1976) mating results, the presence of intermediate strains and 2008) is identical to that of CBS 480.70F (data not shown) of the absence of compensatory base changes (CBC) between Mucor circinelloides f. circinelloides. However, according to f. circinelloides, f. lusitanicus, and f. janssenii (Pawłowska et al. the description given by Jacobs & Botha (2008) the taxa differ In press) lead us to regard M. circinelloides as a single species significantly in the sizes of sporangia, columellae and sporan- that consists of several still interbreeding lineages which result giospores. A microscopical study of the ex-type strain of Mucor in a high intraspecific ITS variability of 5.3 %. renisporus, and repeated ITS sequencing are necessary to Some confusion exists because of misapplied names for impor- verify conspecifity. tant model strains. Strain CBS 416.77, deposited in the CBS The morphological distinction of f. racemosus and f. sphaero- collection by S. Bartnicki-García as Mucor rouxii, belongs to sporus of M. racemosus is ambiguous while they can be clearly Mucor circinelloides according to its ITS sequence, a fact that differentiated based on ITS data. Some strains of f. racemosus has been noted by several authors (Abe et al. 2006, Schwarz such as CBS 271.86 and CBS 113.08 produce a high proportion et al. 2006, Liu et al. 2007). Ellis et al. (1976) proposed NRRL of spherical sporangiospores. 5866 = CBS 438.76 as the neotype for Amylomyces rouxii (Cal- mette 1892) and found it to be conspecific with that of Rhizopus Mucor racemosus f. chibinensis is grouped with M. racemo- arrhizus. Wehmer (1900) incorrectly believed that the strains sus f. racemosus and represents a synonym of the latter. To he isolated represented Calmette’s Amylomyces rouxii and our knowledge no type material is available for f. chibinensis. proposed the name Mucor rouxii (Calmette) Wehmer for them. Schipper (1976) concluded that CBS 636.67 and CBS 660.66 However, he probably studied strains of Mucor indicus (Schip- strains matched the protologue of Mucor chibinensis (Neo- per 1978a: 10). Consequently Mucor rouxii sensu Wehmer is phytova 1955) and proposed the name Mucor racemosus f. a synonym of Mucor indicus, whereas Mucor rouxii (Calmette) chibinensis. Here we designate CBS 636.67 (preserved in a Wehmer is Rhizopus arrhizus. Strains of M. indicus are very metabolically inactive state by lyophilization, batch nr. 768) as similar morphologically to Mucor circinelloides at some stages neotype of Mucor racemosus f. chibinensis because it matches in the life cycle (Schipper 1978a: 10), but the molecular differ- the description of the basionym M. chibinensis and it is well ences between the two species are unambiguous. described and illustrated (Schipper 1976); in addition the strain Mucor ramosissimus is another member of the Mucor circinel- was isolated in Russia, corresponding which the geographic loides complex. Its ITS sequence clusters in the well-supported origin of the type. 38 Persoonia – Volume 30, 2013

Choanephora cucurbitarum CBS120.25 JN206231 0.05 substitutions/site Choanephora cucurbitarum CBS150.51 JN206232 71 Choanephora cucurbitarum CBS674.93 JN206233 Choanephora cucurbitarum Choanephora cucurbitarum CBS445.72 JN206234 99 Choanephora cucurbitarum CBS178.76 JN206235 Choanephora infundibulifera CBS153.51 JN206236 Choanephora infundibulifera CBS155.51 JN206237 Choanephora infundibulifera Choanephora infundibulifera CBS155.58 JN206238 100 Poitrasia circinans CBS153.58 JN206239 T Poitrasia circinans CBS647.70 JN206240 Blakeslea trispora CBS130.59 JN206227 Blakeslea trispora CBS198.80 JN206228 88 Blakeslea trispora CBS137.49 JN206229 Blakeslea trispora Blakeslea trispora CBS564.91 JN206230 PT of B. sinensis 100 Gilbertella persicaria CBS785.97 JN206218 Gilbertella persicaria CBS403.51 JN206221 Gilbertella persicaria CBS325.71A JN206220 Gilbertella persicaria CBS442.64 JN206219 T of G. persicaria var. indica Gilbertella persicaria CBS565.91 JN206226 T of G. hainanensis 100 Gilbertella persicaria CBS246.59 JN206222 Gilbertella persicaria Gilbertella persicaria CBS421.77 JN206223 Gilbertella persicaria CBS190.32 HM999958 T Gilbertella persicaria CBS532.77 JN206224 Gilbertella persicaria CBS325.71D JN206225 Mycotypha microspora CBS186.68 JN206215 100 Mycotypha microspora CBS610.92 JN206216 Mycotypha microspora 100 Mycotypha microspora CBS230.32 JN206217 T Mycotypha sp. ‘microspora’ CBS109960 JN206214 Mucor fuscus CBS313.78 JN206200 Mucor fuscus CBS282.78 JN206201 100 Mucor fuscus CBS530.77 JN206202 Mucor fuscus 100 Mucor fuscus CBS254.48 JN206203 T of M. bedrchanii Mucor fuscus CBS230.29 JN206204 T of M. petrinsularis var. echinosporus 84 93 Mucor lanceolatus CBS638.74 JN206205 Zygorhynchus exponens var. exponens CBS141.20 JN206206 NT (designated here) 72 Zygorhynchus exponens var. exponens CBS508.48 JN206207 Mucor exponens Zygorhynchus exponens var. smithii CBS 404.58 JN206208 LT (designated here) 94 100 Mucor laxorrhizus CBS143.85 JN206209 NT Mucor laxorrhizus CBS237.66 JN206210 97 99 Hyphomucor assamensis CBS415.77 JN206211 T Hyphomucor assamensis CBS254.85 JN206212 Circinella simplex CBS428.80 JN206213 96 Mucor inaequisporus CBS351.50 JN206178 100 Mucor inaequisporus CBS496.66 JN206179 Mucor inaequisporus 93 Mucor inaequisporus CBS255.36 JN206177 T Mucor nederlandicus CBS735.70 JN206176 100 Mucor falcatus CBS252.35 JN206249 97 Mucor falcatus CBS251.35 JN206250 HT Mucor variosporus CBS837.70 JN206175 T Mucor amphibiorum CBS763.74 HM999957 T 100 Mucor amphibiorum CNM-CM2934 JN206171 Mucor amphibiorum Mucor amphibiorum CBS185.77 JN206170 (Dendrobates sp.) Mucor ardhlaengiktus CBS210.80 JN206172 ET (designated here) Mucor ardhlaengiktus CBS528.73 JN206173 Mucor ardhlangiktus Mucor ardhlaengiktus CBS650.78 JN206174 Mucor indicus CBS123974 JN206181 Mucor indicus CBS120.08 JN206182 Mucor indicus CBS120585 JN206180 Mucor indicus CBS226.29 HM999956 ET (designated here) Mucor indicus 83 100 Mucor indicus CBS671.79 JN206183 Mucor indicus CBS535.80 JN206184 Mucor indicus CBS422.71 JN206186 Mucor indicus CBS414.77 JN206185 98 100 Mucor ucrainicus CBS221.71 JN206191 T 91 Mucor ucrainicus CBS674.88 JN206192 Kirkomyces cordensis CBS223.63 JN206193 IT 96 Mucor prayagensis CBS816.70 JN206188 T 99 Mucor prayagensis CBS652.78 c1 JN206189 Mucor prayagensis 97 Mucor prayagensis CBS652.78 c3 JN206190 76 Mucor azygosporus CBS292.63 JN206187 IT Mucor odoratus CBS572.70 JN206194 Mucor odoratus CBS201.71 JN206198 100 Mucor odoratus CBS130.41 JN206197 T Mucor odoratus 100 Mucor odoratus CBS120.71 JN206195 75 Mucor odoratus CBS179.76A JN206196 Mucor zychae var. zychae CBS416.67 JN206199 T 100 Mucor gigasporus CBS383.95 JN206246 Mucor gigasporus CBS566.91 JN206247 T Benjaminiella youngii CBS103.89 JN206242 T 98 Benjaminiella multispora CBS421.70 JN206243 IT 100 Benjaminiella poitrasii CBS158.60 JN206241 T 100 Cokeromyces recurvatus CBS158.50 JN206244 T Cokeromyces recurvatus CBS168.59 JN206245 Mucor sp. ‘hiemalis’ CBS334.71 JN206248 Mucor oblongisporus CBS569.70 JN206251 NT Fig. 6 RAxML phylogram of the Mucor amphibiorum group and related taxa based on the ITS region. Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type strains are designated by: T = ex-type strain, ET = ex-epitype strain, HT = ex-holotype strain, IT = ex-isotype strain, LT = ex-lectotype strain, NT = ex-neotype strain, PT = ex-paratype strain. Ex-type strains of currently accepted taxa are printed in bold. Clones are speciﬁed by a ‘c’ followed by the clone number. Clinical strains are highlighted by red strain and GenBank accession numbers, non-human hosts are given in brackets. Potentially undescribed taxa are indicated by bold blue font. Morphological identiﬁcations are given in quotation marks. G. Walther et al.: DNA barcoding in Mucorales 39

Mucor sinensis is considered to be conspeciﬁc with M. racemo- = Mucor velutinosus E. Álvarez, Stchigel, Cano, D.A. Sutton & Guarro, in sus f. racemosus because it groups with the ex-neotype CBS Álvarez et al., Med. Mycol. 49: 64. 2011. 260.68 of the latter in the ITS tree. It appears to represent a Neotype. CBS 205.68 (preserved in a metabolically inactive state by morphological variant or a growth-reduced mutant rather than lyophilization, batch nr. 605), designated here. a separate species. Specimens examined. CBS 144.93, CBS 185.68, CBS 204.68, CBS 205.68, CBS 206.68, CBS 227.29, CBS 232.29, CBS 243.67, CBS 365.70, Mucor amphibiorum group and related taxa CBS 762.74. The ITS sequence of the ex-type strain of the recently described Notes — Schipper (1970) described Mucor griseocyanus f. Mucor ellipsoideus (FN650647, Álvarez et al. 2011) is identical janssenii based on strain CBS 205.68 because no authentic to that of CBS 210.80, the ex-type strain of Mucor ardhlaengik- material of M. janssenii existed. However, she did not designate tus, except of an A missing at the 3’ terminus probably due to CBS 205.68 as the neotype though it was considered as such low sequence quality (data not shown). The characteristic azy- in the following years. Here we designate strain CBS 205.68 gospores described in M. ardhlaengiktus (Mehrotra & Mehrotra (in a lyophilized state) as neotype of M. janssenii because it 1978) appear to be a variable feature that was absent from ﬁts the description of this species morphologically and it is well the strain studied by Álvarez et al. (2011) and from strain CBS described (Schipper 1970). 650.78. Mucor ardhlaengiktus is the older name (Mehrotra & Mehrotra 1978) and we therefore consider M. ellipsoideus as a synonym. The varieties of Mucor laxorrhizus, var. laxorrhizus Mucor parviseptatus G. Walther & de Hoog, nom. nov. — and var. ovalisporus, appear distantly positioned in the ITS MycoBank MB800447 (Fig. 6) and LSU (Fig. 1) trees and as a result we recognize ≡ Mucor laxorrhizus Y. Ling var. ovalisporus Schipper, Stud. Mycol. 31: them as separate species. 154. 1989, non Mucor ovalisporus (G. Sm.) Pidopl. & Milko, Atlas Mukor. Grib. (Kiev): 61. 1971. Mucor ardhlaengiktus B.S. Mehrotra & B.M. Mehrotra, Etymology. The epithet parviseptatus refers to the few septa that are Sydowia 31: 94. 1979 [1978]. — MycoBank MB317921 formed unconnected with branching of the sporangiophores.

= Mucor ellipsoideus E. Álvarez, Stchigel, Cano, D.A. Sutton & Guarro, Specimens examined. CBS 417.77 ex-type strain of Mucor laxorrhizus in Álvarez et al., Med. Mycol. 49: 67. 2011. var. ovalisporus, CBS 522.79.

Lectotype. Fig. 1 (Mehrotra & Mehrotra, Sydowia 31: 95. 1979 [1978]), designated here. Mucor racemosus Fresen. f. racemosus Beitr. Mykol. 1: 12. Epitype. CBS 210.80 (preserved in a metabolically inactive state by 1850. — MycoBank MB427116 lyophilization, batch nr. 968), designated here. ≡ Circinomucor racemosus (Fresen.) Arx, Sydowia 35: 18. 1982. Specimens examined. CBS 210.80, CBS 528.73, CBS 650.78. = Mucor racemosus f. brunneus Morini, Malpighia 10: 88. 1896. = Mucor dimorphosporus Lendn., Mat. Fl. Crypt. Suisse 3, 1: 93. 1908. = Mucor christianensis Hagem, Ann. Mycol. 8: 268. 1910. Mucor circinelloides f. janssenii (Lendn.) Schipper, Stud. = Mucor racemosus var. christianensis (Hagem) Naumov, Opred. Mukor. Mycol. 12: 13. 1976. — MycoBank MB348491 (Mucorales): 46. 1935. = Mucor varians Povah, Bull. Torrey Bot. Club 44: 297. 1917. Basionym. Mucor janssenii Lendn. (as ‘janseni’), Bull. Herb. Boissier 2, = Mucor pispekii Naumov, Encycl. Mycol. 9: 47. 1939. Sér. 7: 251. 1908. = Mucor chibinensis Neophyt., Notul. Syst. Inst. Cryptog. Horti Bot. ≡ Mucor griseocyanus Hagem f. janssenii (Lendn.) Schipper, Antonie van Petropol. 10: 160. 1955. Leeuwenhoek 36: 486. 1970. = Mucor racemosus f. chibinensis (Neophyt.) Schipper, Stud. Mycol. 12: ≡ Circinomucor janssenii (Lendn.) Arx, Sydowia 35: 18.1982. 24. 1976. = Mucor tenellus Y. Ling, Rev. Gén. Bot. 42: 736. 1930. = Mucor oudemansii Váňová, Česka Mykol. 45: 25. 1991. ≡ Circinella tenella (Y. Ling) Zycha, Krypt.-Fl. Brandenburg (Leipzig) 6a: = Mucor sinensis Milko & Beliakova, in Pidopl. & Milko, Atlas Mukor. Grib. 99. 1935. (Kiev): 53. 1971. = Mucor stagnalis Novot., Notul. Syst. Inst. Cryptog. Horti Bot. Petropol. 6: 158. 1950. Neotype. CBS 260.68 (preserved in a metabolically inactive state by lyo- = Mucor kurssanovii Milko & Beliakova, Mikrobiologija 36: 118. 1967. philization, batch nr. 87.1018), designated here.

Backusella circina CBS128.70 JN206258 T 0.05 substitutions/site Backusella circina CBS323.69 JN206259 Backusella circina CBS382.95 JN206256 100 Backusella circina CBS129.70 JN206257 PT Backusella circina Backusella circina CBS907.73 JN206260 97 95 Mucor variabilis CBS564.66 c3 JN206253 LT (designated here) 100 Mucor variabilis CBS564.66 c1 JN206254 LT (designated here) 100 Mucor grandis CBS186.87 JN206252 T Mucor recurvus var. indicus CBS786.70 JN206255 LT (designated here) 83 93 Mucor recurvus var. recurvus CBS673.75 c1 JN206263 82 Mucor recurvus var. recurvus CBS673.75 c2 JN206264 Mucor recurvus var. recurvus CBS318.52 JN206261 ET (designated here) 88 Backusella recurva 100 Mucor recurvus var. recurvus CBS317.52 c1 JN206262 100 Mucor recurvus var. recurvus CBS196.71 JN206265 100 Mucor tuberculisporus CBS570.70 JN206266 Mucor tuberculisporus CBS562.66 JN206267 LT (designated here) 100 Mucor ‘recurvus var. recurvus’ CBS538.80 HM999964 Backusella lamprospora CBS107.09 JN206269 T of Mucor dispersus 95 Backusella lamprospora CBS195.28 JN206271 84 Backusella lamprospora CBS244.67 JN206270 Backusella lamprospora Backusella lamprospora CBS118.08 JN206268 T Mucor indicus CBS226.29 HM999956 ET (designated here) Fig. 7 RAxML phylogram of the Mucor recurvus group and Backusella based on the ITS region. Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type strains are designated by: T = ex-type strain, ET = ex-epitype strain, LT = ex-lectotype strain, PT = ex-paratype strain. Ex-type strains of currently accepted taxa are printed in bold. Clones are speciﬁed by a ‘c’ followed by the clone number. Potentially undescribed taxa are indicated by bold blue font. Morphological identiﬁcations are given in quotation marks. 40 Persoonia – Volume 30, 2013

Specimens examined. CBS 113.08 ex-lectotype strain of Mucor dimor- The genus Backusella differs from Mucor only by the formation phosporus (lectotype: Fig. 34 (Lendner, Mat. Fl. Crypt. Suisse 3, 1: 93. of sporangiola in addition to sporangia. However, sporangiola, 1908), designated here; epitype: CBS 113.08, preserved in a metabolically though in low frequency, have also been described in Mucor re- inactive state by lyophilization, batch nr. 479, designated here), CBS 204.74 curvus var. indicus and in M. tuberculisporus (Schipper 1978a). ex-type strain of Mucor sinensis, CBS 222.81, CBS 260.68, CBS 271.86, CBS 369.71, CBS 616.63, CBS 636.67 ex-neotype strain of Mucor racemosus f. In our opinion the clade in the LSU phylogram including Mucor chibinensis, CBS 657.68, CBS 660.66, CBS 661.66, CBS 111557, CNM-CM and Backusella species represents a natural group character- 2569, CNM-CM 3862. ised by transitorily recurved sporangiophores. Consequently, we transfer all Mucor species belonging to that clade to the genus Notes — Strain CBS 260.68 was used by Schipper (1970) Backusella. The two varieties of Mucor recurvus (var. recurvus for the description of Mucor racemosus f. racemosus but not and var. indicus) are located in different supported subclades designated as neotype. Here we designate strain CBS 260.68 of our ITS tree (Fig. 7) and likely represent separate species. (in a lyophilized state) as neotype of this species because it is well characterized (Schipper 1970) and because its morphology Sequence diversity is high in the emended genus Backusella. fully complies with the original description. ITS sequences of M. oblongisporus CBS 569.70, M. oblongi ellipticus CBS 568.70, and a contaminant strain of CBS 523.68 deviate significantly from the remaining members of the group Mucor silvaticus Hagem, Skr. Vidensk.-Selsk. Christiana, and cannot be aligned with confidence. However, their LSU Math.-Naturvidensk. Kl. 7: 31. 1908. — MycoBank MB182519 sequences and the formation of transitorily recurved sporan- ≡ Mucor hiemalis f. silvaticus (Hagem) Schipper, Stud. Mycol. 4: 31. 1973. giophores clearly assign these taxa to the emended genus Backusella. Neotype. CBS 412.71 (preserved in a metabolically inactive state by lyophilization, batch nr. 853), designated here. Backusella ctenidia is positioned inside the Mucor circinelloides complex (Fig. 1, 5) and does not belong in Backusella. For that Specimens examined. CBS 249.35, CBS 412.71, CBS 509.66. reason we propose transferring it to Mucor. Notes — To our knowledge, authentic material of this species We propose the following emendation for Backusella: has been lost. Schipper (1973) studied five strains that matched the description of Hagem (CBS 249.35, CBS 250.35, CBS Backusella Ellis & Hesseltine emend. Walther et al. 508.66, CBS 509.66 and CBS 412.71). Schipper’s description Type species. Backusella circina J.J. Ellis & Hesselt. and drawings are based on CBS 412.71. It was isolated in Den- mark, while the remaining strains originated in Germany. Here Sporophores arising directly from the substrate mycelium, sim- we designate CBS 412.71 as the neotype of Mucor silvaticus, ple or sympodially branched, recurved when young, erect at because it was well described and illustrated as M. hiemalis f. maturity (transitorily recurved), smooth or roughened, produc- silvaticus in Schipper (1973). Compared to the strains available ing terminal sporangia and in some species few to many lat- in international fungal culture collections, it was isolated closest eral, pedicellate sporangiola. Terminal sporangia columellate, to the locality of the type, Norway. multispored, globose to subglobose, size ranging from 90 to 375 µm diam, nonapophysate, wall encrusted, deliquescent; Mucor recurvus group and Backusella columellae subglobose, ellipsoidal, slightly pyriform or conical, smooth. Collars small or consisting of needle-shaped spines. The LSU phylogram (Fig. 1) resolved a clade (Mucor recurvus Sporangiolar pedicels straight, curved, or recurved, simple or group) consisting of Mucor grandis, M. oblongiellipticus, M. branched, smooth or encrusted. Sporangiola columellate, multi- oblongisporus, M. recurvus, M. tuberculisporus, M. variabilis, or unispored; wall verrucose or spinulose or both, persistent. two potentially undescribed species as well as Backusella Sporangiospores of sporangia and multispored sporangia circina and B. lamprospora that exclusively unifies taxa with identical, large, subglobose, ellipsoidal or irregularly polyhedral, transitorily recurved sporangiophores (Fig. 8). In this clade smooth. Columellae, hyphae and sporangiospores in some spe- the sporangiophores are curved during maturation of the spo- cies with yellowish or brownish content. Zygospores globose to rangium and become upright afterwards. Schipper (1978a) subglobose; wall dark, opaque or translucent, ornamented with recognised the Mucor portion of this group on morphological conical or rounded projections; suspensors opposed, smooth grounds. The type species of Backusella, B. circina (Ellis & or roughened, equal or unequal. Hesseltine 1969: 865) (Fig. 8a, b), as well as B. lamprospora (Benny & Benjamin 1975: 320) also have been described to Notes — The emended genus Backusella includes the follow- form transitorily recurved sporangiophores. ing species: Backusella circina, B. grandis, B. indica, B. lam prospora, B. oblongielliptica, B. oblongispora, B. recurva, B. tuberculispora and B. variabilis.

a b c d Fig. 8 Morphology of the genus Backusella. a, b. CBS 128.70 Backusella circina, a. unispored sporangiola; b. transitorily recurved sporangiophore; c. CBS 318.52 Mucor recurvus var. recurvus (Backusella recurva here), transitorily recurved sporangiophore; d. CBS 564.66 Mucor variabilis (Backusella variabilis here), transitorily recurved sporangiophore. — Scale bar: a = 10 µm, b–d = 50 µm. G. Walther et al.: DNA barcoding in Mucorales 41

Backusella grandis (Schipper & Samson) G. Walther & Mucor ctenidius (Durrell & M. Fleming) G. Walther & de Hoog, de Hoog, comb. nov. — MycoBank MB800453 comb. nov. — MycoBank MB800455

Basionym. Mucor grandis Schipper & Samson, Mycotaxon 50: 479. 1994. Basionym. Thamnidium ctenidium Durrell & M. Fleming, Mycologia 58: 797. 1966. Specimen examined. CBS 186.87 ex-type strain of Mucor grandis. ≡ Backusella ctenidia (Durrell & M. Fleming) Pidopl. & Milko, Atlas Mukor. Notes — The ITS sequence of Mucor grandis differs by only Grib. (Kiev): 85. 1971, ex Benny & R.K. Benj., Aliso 8: 325. 1975. 6 basepairs from that of M. variabilis, while different clones of Specimens examined. CBS 293.66 ex-isotype strain of Thamnidium M. variabilis vary at four positions. The small sequence differ- ctenidium, CBS 433.87, CBS 696.76. ences suggest conspecifity but the diameter of the sporangia varies significantly between the species. More detailed taxo- Pilaira nomic studies are needed to clarify species limits. Recently, Zheng & Liu (2009a) studied the genus Pilaira morphologically and reclassified P. caucasica as a variety of P. mo- Backusella indica (Baijal & B.S. Mehrotra) G. Walther & reaui. The variety differed from var. moreaui in the size of de Hoog, comb. nov. — MycoBank MB800449 sporangiophores and sporangiospores (Zheng & Liu 2009a). Basionym. Mucor recurvus var. indicus Baijal & B.S. Mehrotra, Sydowia We found identical ITS sequences for both varieties supporting 19: 207. 1965. conspecifity (Fig. 3). We retain both varieties despite of identical Lectotype. CBS 786.70 (preserved in a metabolically inactive state by ITS sequences because of the clear morphological distinction. lyophilization, batch nr. 344), designated here. Specimen examined. CBS 786.70. Zygorhynchus Phenotypically Zygorhynchus and Mucor differ in the following Backusella oblongielliptica (H. Nagan., Hirahara & Seshita ex features. First, species of Zygorhynchus are exclusively homo- Pidopl. & Milko) G. Walther & de Hoog, comb. nov. — Myco- thallic, while the majority of Mucor species is heterothallic (Wa- Bank MB800451 tanabe 1994). Second, the suspensors of the zygospores are Basionym. Mucor oblongiellipticus H. Nagan., Hirahara & Seshita, Essays unequal in Zygorhynchus and equal in Mucor. Third, the two Stud. Fac. Hiroshima Jogakuin College 18: 167. 1969, nom. inval., Art. 36.1 suspensors originate from the same hypha in Zygorhynchus, the ≡ Mucor oblongiellipticus H. Nagan., Hirahara & Seshita ex Pidopl. & ‘Zygorhynchus pattern’, while they arise from different hyphae Milko, Atlas Mukor. Grib. (Kiev): 81. 1971. in Mucor, the ‘Mucor pattern’ (Hesseltine et al. 1959, Schip- Lectotype. CBS 568.70 (preserved in a metabolically inactive state by per 1986). However, these differences are gradual (Schipper lyophilization, batch nr. 113), designated here. 1986). Zygorhynchus exponens may develop equal but Mucor plumbeus more or less unequal suspensors (Schipper 1986). In Specimen examined. CBS 568.70. Zygorhynchus exponens (Hesseltine et al. 1959), Z. japonicus (Schipper 1986) and Z. moelleri (Green 1927) zygospores are Backusella oblongispora (Naumov) G. Walther & de Hoog, also produced between different hyphae. comb. nov. — MycoBank MB800452 Based on our LSU (Fig. 1) and ITS (Fig. 3, 6) data, Zygorhyn- Basionym. Mucor oblongisporus Naumov, Mater. Mykol. Fitopatol. Rossii chus is polyphyletic. Our analyses indicate that unequal suspen- 1(4): 12. 1915. sors and the Zygorhynchus pattern of zygospore production do Neotype. CBS 569.70 (preserved in a metabolically inactive state by not represent synapomorphies in the genus Zygorhynchus, but lyophilization, batch nr. 55), designated here. appear to be convergent characters within Mucor. Therefore Specimen examined. CBS 569.70. we recombine all Zygorhynchus species in Mucor. The ex-type strains of Zygorhynchus moelleri and Z. califor Backusella recurva (E.E. Butler) G. Walther & de Hoog, comb. niensis have identical ITS sequences suggesting conspecifity. nov. — MycoBank MB800448; Fig. 8c However, Z. californiensis has regularly globose spores, while the spores of Z. moelleri are oblong to ovoidal in shape, 2.0–3.3 Basionym. Mucor recurvus E.E. Butler, Mycologia 44: 561. 1952. × 3.0–6.5 µm (Hesseltine et al. 1959). For that reason we pro- Lectotype. Fig. 1 (Butler, Mycologia 44: 562. 1952). pose reclassifying Z. californiensis as a forma of Z. moelleri. Epitype. CBS 318.52 (preserved in a metabolically inactive state by lyo The two varieties described in Zygorhynchus exponens, var. ex- philization, batch nr. 717), designated here. ponens and var. smithii differ by only a single basepair in their Specimens examined. CBS 196.71, CBS 317.52, CBS 318.52, CBS 673.75. ITS sequences. Also small morphological differences such as the lighter and browner sporangia and columellae in var. Backusella tuberculispora (Schipper) G. Walther & de Hoog, smithii, do not justify the maintenance of a separate variety comb. nov. — MycoBank MB800450 and consequently we consider both varieties as synonymous.

Basionym. Mucor tuberculisporus Schipper, Stud. Mycol. 17: 23. 1978. Mucor exponens (Burgeff) G. Walther & de Hoog, comb. nov. Lectotype. CBS 562.66 (preserved in a metabolically inactive state by — MycoBank MB800461 lyophilization, batch nr. 88.1007), designated here. Basionym. Zygorhynchus exponens Burgeff, Bot. Abh. 4: 34. 1924. Specimens examined. CBS 562.66, CBS 570.70. = Zygorhynchus exponens Burgeff var. smithii Hesselt., C.R. Benj. & B.S. Mehrotra, Mycologia 51: 179. 1959. (A.K. Sarbhoy) G. Walther & de Hoog, Backusella variabilis Neotype. CBS 141.20 (preserved in a metabolically inactive state by lyo- comb. nov. — MycoBank MB800454; Fig. 8d philization, batch nr. 563), designated here.

Basionym. Mucor variabilis A.K. Sarbhoy, Trans. Brit. Mycol. Soc. 48: Specimens examined. CBS 141.20, CBS 404.58 ex-lectotype strain of 559. 1965. Zygorhynchus exponens var. smithii (lectotype: CBS 404.58, preserved in a Lectotype. CBS 564.66 (preserved in a metabolically inactive state by metabolically inactive state by lyophilization, batch nr. 40, designated here), lyophilization, batch nr. 22), designated here. CBS 508.48. Specimen examined. CBS 564.66. 42 Persoonia – Volume 30, 2013

Mucor fusiformis G. Walther & de Hoog, nom. nov. — Myco- Mucor moelleri (Vuill.) Lendn. f. moelleri, Mat. Fl. Crypt. Bank MB800459 Suisse 3, 1: 72. 1908.

≡ Zygorhynchus psychrophilus Schipper & Hintikka, Antonie van Leeu- Basionym. Zygorhynchus moelleri Vuill., Bull. Trimestriel Soc. Mycol. wenhoek 35: 29. 1969, non Mucor psychrophilus Milko, in Pidopl. & Milko, France 19: 117. 1903. Atlas Mukor. Grib. (Kiev): 73. 1971. = Zygorhynchus vuilleminii Namysl., Ann. Mycol. 8: 154. 1910. = Zygorhynchus vuilleminii race agamus Namysl., Bull. Int. Acad. Sci. Etymology. The epithet refers to the shape of the sporangiospores. Cracovie, Cl. Sci. Math., Ser. B, Sci. Nat. 6: 479. 1911. Specimens examined. CBS 336.68 ex-type strain of Zygorhynchus psy = Zygorhynchus dangeardii Moreau, Bull. Soc. Bot. France 59: 717. 1912. chrophilus. = Mucor saximontensis Rall, Mycologia 57: 874. 1965.

Neotype. CBS 406.58 (preserved in a metabolically inactive state by lyo- Mucor heterogamus Vuill., Bull. Séanc. Soc. Sci. Nancy 8: philization, batch nr. 656), designated here. 50. 1887. — MycoBank MB249261 Specimens examined. CBS 216.27, CBS 380.29, CBS 406.58, CBS 444.65 ≡ Zygorhynchus heterogamus (Vuill.) Vuill., Bull. Trimestriel. Soc. Mycol. ex-lectotype strain of Mucor saximontensis (lectotype: CBS 444.65, preserved France 19: 117. 1903. in a metabolically inactive state by lyophilization, batch nr. 803, designated here), CBS 460.51, CBS 501.66, IHEM 21156. Lectotype. Pl. II, f. 27-48 (Vuill., Bull. Séanc. Soc. Sci. Nancy 8. 1887), designated here. Notes — No authentic material of this species is known to Epitype. CBS 405.58 (preserved in a metabolically inactive state by lyo be preserved. Hesseltine et al. (1959) reported NRRL 2660 (= philization, batch nr. 658), designated here. CBS 406.58) as the type of Z. moelleri but the strain studied by Specimens examined. CBS 252.85, CBS 338.74, CBS 405.58, CBS Vuillemin was isolated in Eberswalde (Germany) while NRRL 580.83, CBS 594.83. 2660 originated from soil in Wisconsin (USA). Notes — The original material of this species consists of slides labelled as “Mucor heterogamus P.V. Zygospores Mis de Mucor moelleri f. californiensis (Hesselt., C.R. Benj. & B.S. pain 17-3-86” (Hesseltine et al. 1959). Hesseltine et al. (1959) Mehrotra) G. Walther & de Hoog, comb. nov. — MycoBank studied five strains: NRRL 1489, NRRL 1490, NRRL 1491, MB800460 NRRL 1616 (= CBS 405.58) and a fresh isolate without an Basionym. Zygorhynchus californiensis Hesselt., C.R. Benj. & B.S. NRRL number designated as ‘No. 1957’ and compared these Mehrotra, Mycologia 51: 185. 1959. strains with the original material. The authors found remarkable intraspecific variation in colony appearance, but micromorpho- Lectotype. Fig. 8–10 (Hesseltine, Benjamin & Mehrotra, Mycologia 51: 176. 1959), designated here. logically Vuillemin’s material was almost identical with their living Epitype. CBS 402.58 (preserved in a metabolically inactive state by lyo cultures except for some differences in lengths of the zygospore philization, batch nr. 90.0055), designated here. projections. Therefore they considered the type material and their strains as conspecific. Here we designate CBS 405.58 Specimen examined. CBS 402.58. (NRRL 1616, preserved in a lyophilized state) verified by Hes- seltine et al. (1959) as epitype of Mucor heterogamus. Mucor multiplex (R.Y. Zheng) G. Walther & de Hoog, comb. Isolates of M. heterogamus vary considerably in their ITS se- nov. — MycoBank MB800457 quences (maximum dissimilarity of 10 %) and might represent Basionym. Zygorhynchus multiplex R.Y. Zheng, Mycotaxon 84: 370. 2002. a complex of several species. Isolates that were morphologically assigned to M. heterogamus form a well-supported group Specimen examined. CBS 110662 ex-type strain of Zygorhynchus multi with Z. multiplex and Z. macrocarpus, but at distances to the plex. designated ex-epitype strain of 10.8 % and 7.7 %, respectively. The precise definition of species boundaries awaits detailed Actinomucor multilocus DNA sequence-based analyses. Currently there are three varieties in Actinomucor: A. elegans var. elegans, var. meitauzae (syn. A. taiwanensis, Zheng & Mucor japonicus (Komin.) G. Walther & de Hoog, comb. nov. Liu 2005) and var. kuwaitiensis (Khan et al. 2008). Characters — MycoBank MB800458 distinguishing the varieties are shape, size and ornamentation of the sporangiospores (Zheng & Liu 2005, Khan et al. 2008). Basionym. Zygorhynchus japonicus Komin., Mykol. Zentbl. 5: 3. 1915 (1914). The var. meitauzae and var. kuwaitiensis show reduced growth on Czapek’s agar. In contrast to earlier reports (Jong & Yuan Neotype. CBS 154.69 (preserved in a metabolically inactive state by lyo- 1985), the maximum growth temperature does not discriminate philization, batch nr. 409), designated here. the varieties (Zheng & Liu 2005, Khan et al. 2008). However, Specimen examined. CBS 154.69. the relationships deduced from our ITS data (Fig. 9) contradict current taxonomic concepts. Strains with the characteristics of Notes — The authentic strain of Zygorhynchus japonicus var. meitauzae are scattered over nearly all parts of the tree, and studied by Kominami (1915) has been lost (Schipper 1986). only a part of the strains belonging to var. elegans is included in Strain CBS 154.69 (preserved in a lyophilized state) is selected a well-supported clade around the ex-type strain of var. elegans. as neotype of Z. japonicus because it resembles the original A detailed taxonomic revision is required. strain and it is well described and illustrated (Schipper 1986).

Rhizopus Mucor megalocarpus G. Walther & de Hoog, nom. nov. — MycoBank MB800456 Based on our phylogenetic trees the genus Rhizopus is paraphyletic because Sporodiniella umbellata and Syzygites ≡ Zygorhynchus macrocarpus Y. Ling, Rev. Gén. Bot. 42: 150. 1930, non megalocarpus cluster among Rhizopus species. All currently Mucor macrocarpus Corda, Icon. Fungorum 2: 21. 1838. accepted Rhizopus species are well recognizable in the ITS Lectotype. Fig. 1 (Ling, Rev. Gén. Bot. 42: 152. 1930), designated here. tree. However, three strains of Rhizopus stolonifer, CBS Epitype. CBS 215.27 (preserved in a metabolically inactive state by lyo 126.83, CBS 442.74 and CBS 926.87, exhibit widely deviating philization, batch nr. 748), designated here. ITS sequences, forming a separate group that may represent Specimen examined. CBS 215.27. a new species. In agreement with our results, Vágvölgyi et al. G. Walther et al.: DNA barcoding in Mucorales 43

0.001 substitutions/site 80 Actinomucor elegans CBS111562 AB113009 Actinomucor elegans ATCC46123 AM745430 80 Actinomucor elegans var. meitauzae ATCC52370 AM745432 Actinomucor elegans ATCC22963 AM745429 Actinomucor elegans var. kuwaitiensis CBS117697 JN205823 T Actinomucor elegans CBS154.86 JN205829 Actinomucor elegans var. meitauzae Ac3 AY492087 Actinomucor elegans var. meitauzae ATCC52370 AY492088 T of A. taiwanensis Actinomucor elegans var. elegans Ac35 AY492090 Actinomucor elegans CBS100.09 JN205827 78 Actinomucor elegans CBS100.22 JN205828 Actinomucor elegans var. elegans ATCC22814 AY492092 NT Actinomucor elegans var. elegans Ac16 AY492093 97 86 Actinomucor elegans var. elegans Ac106 AY492091 98 Actinomucor elegans var. meitauzae Ac116 AY492089 NT Actinomucor elegans CBS338.72 JN205824 Fig. 9 RAxML phylogram of the genus Actinomucor based on the ITS region. Actinomucor elegans MUFS089 AJ278368 Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type Actinomucor elegans var. meitauzae ATCC52360 AM745431 strains are designated by: T = ex-type strain, NT = ex-neotype strain. Ex-type Actinomucor elegans var. meitauzae CBS111558 JN205825 strains are printed in bold. Clinical strains are highlighted by red strain and Actinomucor elegans CBS111556 JN205826 GenBank accession numbers.

(2004) found strains morphologically assigned to R. stolonifer The LSU sequences of C. lacrymispora and G. lacrispora dif- with strongly deviating randomly amplified polymorphic DNA fer only in 3 basepairs and conspecifity cannot be excluded. (RAPD) patterns and consequently the authors suspected an We defer recombination until we obtain the ITS sequences of undescribed variety or even species. The varieties arrhizus both taxa and until we perform a detailed morphological study. and delemar of Rhizopus arrhizus are also recognized in the ITS tree, in accordance with Abe et al. (2007) and Gryganskyi Mucor durus G. Walther & de Hoog, nom. nov. — MycoBank (2010) who treated them as separate species. Strains identified MB800462 morphologically as R. arrhizus var. tonkinensis by Zheng et al. (2007) do not form a separate cluster but are distributed in ≡ Circinella rigida G. Sm., Trans. Brit. Mycol. Soc. 34: 19. 1951, non var. arrhizus and var. delemar clades. However, by using short Mucor rigidus Léger, Rech. Struct. Mucor (Thèse, Paris): 71. 1895. tandem repeat motives of IGS rDNA sequences Liu et al. (2008) Lectotype. Pl. 2, Fig. 7–8 (Smith, Trans. Brit. Mycol. Soc. 34: 17–22. were able to characterize all three varieties of R. arrhizus. 1951), designated here. The morphological varieties described in Rhizopus microsporus Epitype. CBS 156.51 (preserved in a metabolically inactive state by lyo are not supported genetically; a single, supported clade includes philization, batch nr. 389), designated here. strains representing the varieties microsporus, chinensis and Etymology. Named after the rigid wall of the sporangium. oligosporus. ITS sequences of the remaining R. microsporus Specimens examined. CBS 156.51, CBS 484.66. strains that had been assigned morphologically to the varieties azygosporus, chinensis, oligosporus, rhizopodiformis and Notes — The species differs markedly from other Mucor tuberosus are all identical. The ITS identities imply in agree- species by the extremely rigid sporangial walls, the often curved ment with Liu et al. (2008), Abe et al. (2010) and Dolatabadi et branches of the sporangiophores, the common formation of al. (In press) that enlarged size and indistinct ornamentation subsporangial septa and the frequent presence of distinct of sporangiospores have no genetic basis in R. microsporus. apophyses (Fig. 2h).

Circinella Rhizomucor The genus Circinella was erected by van Tieghem & le Mon- All non-thermophilic Rhizomucor species, namely Rm. chlamy nier (1873) in order to accommodate strains differing from dosporus, Rm. endophyticus, Rm. regularior and Rm. variabilis Mucor by circinate sporangiophore branches that terminate in belong to Mucor based on our LSU tree (Fig. 1). Rhizomucor globose sporangia with persistent walls (Hesseltine & Fennell regularior and Rm. variabilis have recently been reclassified: 1955). Based on our LSU tree (Fig. 1) Circinella is polyphyletic, Rm. variabilis has been renamed as Mucor irregularis, whereas resolved in a well-supported group around the type species Rm. regularior has been synonymised with M. circinelloides C. umbellata within the Lichtheimiaceae, and two separate (Álvarez et al. 2011). Our ITS data (Fig. 3) indicate that Rhi- species, C. simplex and C. rigida, positioned distantly within zomucor endophyticus represents a discrete species closely different clades of Mucor. As a consequence, we propose as- related to Mucor luteus. The ITS sequence of the ex-type strain signing C. rigida to Mucor. No type material is known to have of Rm. chlamydosporus (GenBank EF583634) is identical to been preserved of C. simplex. We studied five strains of this that of Mucor indicus (data not shown); the morphological species but we only obtained good sequence data for CBS description of the species (Zheng & Liu 2009b) fully matches 428.80. More detailed taxonomic studies on the numerous with that of M. indicus. strains of this species that are available in public collections are necessary to test its monophyly and to select a neotype. Exclud- Mucor hiemalis and M. luteus develop distinct rhizoids when ing the unrelated species C. rigida and C. simplex, the genus they grow over glass slides (Fig. 2c). These findings demon- Circinella is restricted to species that develop sporangiophores strate that not only Rhizomucor, but also Mucor species have either with sterile spines or umbels with circinate branches. the ability to produce rhizoids, at least under certain conditions. The ex-type strain of Circinella lacrymispora clusters with the Consequently, this feature should not be used as sole criterion ex-type strain of Gongronella lacrispora in the Gongronella for the distinction of Mucor and Rhizomucor. clade of the LSU phylogram (Fig. 1). Based on this finding After removal of the above species, Rhizomucor with its type Circinella lacrymispora should be reclassified in Gongronella. species Rhizomucor parasiticus (which is a synonym of Rm. 44 Persoonia – Volume 30, 2013

Rhizopus arrhizus CBS 120593 JN206340 0.05 substitutions/site Rhizopus arrhizus CBS607.68 JN206341 Rhizopus arrhizus CBS402.51 JN20633 T of R. javanicus var. kawasakiensis Rhizopus arrhizus CBS111231 JN206338 Rhizopus arrhizus CBS392.95 JN206336 T of R. delemar var. multiplicisporus Rhizopus arrhizus CBS544.80 JN206337 Rhizopus arrhizus CBS111760 JN206334 81 Rhizopus arrhizus Chakrabarti 710104 JN206335 var. delemar Rhizopus niveus IFO4810 DQ641284 Rhizopus arrhizus var. delemar IFO5442 DQ641289 T of R. javanicus Rhizopus arrhizus var. delemar CBS328.47 DQ641294 T of R. delemar Rhizopus arrhizus var. delemar CBS385.34 DQ641288 T of R. achlamydosporus Rhizopus arrhizus var. delemar CBS393.34 DQ641290 T of R. peka Rhizopus arrhizus var. delemar HUT1233 DQ641286 T of R. chiuniang Rhizopus arrhizus var. tonkinensis IFO5438 DQ641276 T of R. tonkinensis Rhizopus arrhizus CBS330.53 JN206331 T of R. boreas Rhizopus arrhizus Rhizopus arrhizus CBS125017 JN206332 Rhizopus arrhizus CBS109939 JN206330 Rhizopus arrhizus Chakr710113 JN206333 Rhizopus arrhizus CBS120806 DQ119032 Rhizopus arrhizus CBS112.07 JN206323 T of R. oryzae Rhizopus arrhizus CBS146.90 JN206324 Rhizopus arrhizus CBS120809 DQ119028 Rhizopus arrhizus CBS515.94 JN206325 var. arrhizus Rhizopus arrhizus CBS110.17 JN206327 T of R. maydis 100 Rhizopus arrhizus CBS118614 JN206326 Rhizopus arrhizus CBS286.55 JN206328 (rabbit) Rhizopus arrhizus var. arrhizus NRRL1469 DQ641279 NT Rhizopus arrhizus var. tonkinensis HUT1235 DQ641276 Rhizopus arrhizus var. tonkinensis JCM5570 DQ641299 Rhizopus arrhizus ‘var. delemar’ IFO4735 DQ641300 Rhizopus arrhizus CBS438.76 JN206329 NT of Amylomyces rouxii Rhizopus microsporus var. rhizopodiformis CBS607.73 JN206345 Rhizopus microsporus var. rhizopodiformis CBS258.79 JN206349 Rhizopus microsporus var. rhizopodiformis CBS196.77 JN206361 Rhizopus microsporus var. azygosporus CBS357.93 JN206343 T of R. azygosporus Rhizopus microsporus var. chinensis CBS631.82 JN206344 T of R. chinensis Rhizopus microsporus var. rhizopodiformis CBS118987 JN206346 Rhizopus microsporus var. chinensis CBS537.80 JN206355 Rhizopus microsporus var. rhizopodiformis CBS536.80 HM999971 NT Rhizopus microsporus var. rhizopodiformis CBS102277 JN206356 Rhizopus microsporus var. oligosporus CBS338.62 JN206352 Rhizopus microsporus var. chinensis CBS294.31 JN206351 T of R. bovinus (cow) Rhizopus microsporus var. rhizopodiformis CBS343.29 JN206353 T of R. pusillus Rhizopus microsporus var. oligosporus CBS228.95 JN206348 Rhizopus microsporus Rhizopus microsporus var. rhizopodiformis CBS220.92 JN206347 100 Rhizopus microsporus var. tuberosus AS3.1145 DQ641305 T Rhizopus microsporus var. azygosporus CBS359.92 JN206342 Rhizopus microsporus var. chinensis CBS388.34 JN206354 T of R. chinensis var. liquefaciens Rhizopus microsporus var. chinensis CBS394.34 JN206359 T of. R pseudochinensis var. thermosus 91 Rhizopus microsporus var. oligosporus CBS337.62 JN206362 NT Rhizopus microsporus var. microsporus CBS308.87 JN206357 Rhizopus microsporus var. chinensis CBS344.29 JN20636 T of R. pypmaeus Rhizopus microsporus var. oligosporus CBS112587 JN206358 Rhizopus microsporus var. microsporus CBS699.68 HM999970 NT Rhizopus microsporus var. microsporus CBS112285 JN206364 Rhizopus microsporus CBS111563 JN206363 100 100 Rhizopus homothallicus CBS336.62 HM999968 T Rhizopus homothallicus CBS111232 JN206365 Rhizopus caespitosus CBS427.87 HM999965 T Rhizopus caespitosus 33515 AF115730 86 Rhizopus schipperae CBS138.95 HM999969 T 100 Rhizopus schipperae ATCC204270 AY803936 Syzygites megalocarpus CBS372.39 c3 JN206369 100 Syzygites megalocarpus CBS108947 c3 JN206370 Synzygites megalocarpus 100 Syzygites megalocarpus CBS108947 c1 JN206371 100 Sporodiniella umbellata CBS195.77 JN206372 100 Rhizopus stolonifer CBS609.82 AB113023 Rhizopus stolonifer CBS150.83 AB113022 100 Rhizopus stolonifer CBS389.95 DQ641318 NT Rhizopus stolonifer Rhizopus stolonifer TUR5 AM933544 100 Rhizopus stolonifer AFTOL-ID632 AY997085 Rhizopus sp. ‘stolonifer’ CBS926.87 JN206366 Rhizopus sp. ‘stolonifer’ CBS442.74 JN206367 Rhizopus sp. ‘stolonifer’ 100 100 Rhizopus sp. ‘stolonifer’ CBS126.83 JN206368 94 100 Rhizopus sexualis CBS336.39 c2 AB113017 T Rhizopus sexualis CBS336.39 c8 HM99996 T 99 Rhizopus americanus CBS340.62 c3 HM999967 T Rhizopus lyococcus CBS320.35 JN206373 Rhizopus lyococcus CBS319.35 AB100449 Rhizopus lyococcus JCM5589 DQ641319 T of R. reflexus Rhizopus lyococcus Rhizopus lyococcus CBS117.43 JN206375 Rhizopus lyococcus CBS398.95 JN206374 Mucor recurvus var. recurvus CBS538.80 HM999964 T Fig. 10 RAxML phylogram of the genus Rhizopus based on the ITS region. Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type strains are designated by: T = ex-type strain, NT = ex-neotype strain. Ex-type strains of currently accepted taxa are printed in bold. Clones are speciﬁed by a ‘c’ followed by the clone number. Clinical strains are highlighted by red strain and GenBank accession numbers, non-human hosts are given in brackets. Potentially undescribed taxa are indicated by bold blue font. Morphological identiﬁcations are given in quotation marks. G. Walther et al.: DNA barcoding in Mucorales 45

Rhizomucor tauricus CBS179.69 JN206310 LT (designated here) 0.01 substitutions/site Rhizomucor pusillus CBS354.68 JN206312 ET (designated here) Rhizomucor pusillus CBS219.31 JN206311 Rhizomucor pusillus CBS 120586 DQ118999 Rhizomucor pusillus CBS120587 DQ119000 Rhizomucor pusillus CBS425.78 HM999962 Rhizomucor pusillus Rhizomucor pusillus CNM-CM2752 JN206313 100 Rhizomucor pusillus CNM-CM2974 JN206315 Rhizomucor pusillus CNM-CM2935 JN206314 Rhizomucor pusillus CNM-CM4727 JN206316 Rhizomucor pusillus CNM-CM5124 JN206317 Rhizomucor miehei CBS360.92 JN206318 100 Rhizomucor miehei CBS209.77A c1 JN206322 Rhizomucor miehei CBS182.67 c2 HM999960 T Rhizomucor miehei CBS182.67 c4 HM999961 T Rhizomucor miehei CBS429.70 c1 JN206319 Rhizomucor miehei CBS182.67 c1 HM999959 T Rhizomucor miehei Rhizomucor miehei CBS209.77A c2 JN206321 Rhizomucor miehei CBS429.70 c4 JN206320 Syncephalastrum racemosum CBS302.65 c4 HM999984 Fig. 11 RAxML phylogram of the genus Rhizomucor based on the ITS region. Branches with bootstrap values of 75 % or higher are printed in bold. Ex-type strains are designated by: T = ex-type strain, LT = ex-lectotype strain, ET = ex-epitype strain. The identifying ex-type strain of a clade is printed in bold. Clones are speciﬁed by a ‘c’ followed by the clone number. Clinical strains are highlighted by red strain and GenBank accession numbers.

pusillus) is monophyletic. The genus is restricted to thermophilic Specimens examined. CBS 120.08, CBS 226.29, CBS 414.77, CBS species with predominantly subglobose spores, as was recog- 422.71, CBS 535.80, CBS 671.79, CBS 120585, CBS 123974. nized previously by Schipper (1978b) applying only phenotypic characters. Currently four thermophilic Rhizomucor species Rhizomucor pusillus (Lindt) Schipper, Stud. Mycol. 17: 54. are accepted: Rm. miehei, Rm. nainitalensis, Rm. pakistanicus 1978. — MycoBank MB322484 and Rm. pusillus. Rhizomucor tauricus is considered to be Basionym. Mucor pusillus Lindt, Arch. Exp. Path. Pharmacol. 21: 272. conspeciﬁc with Rm. pusillus because their ITS sequences 1886. are identical (Fig. 11). Analysis of its carbon source utilization, isoenzyme patterns and PCR-coupled RFLP of the ITS sug- = Mucor septatus Bezold, Schimmelmyc. Menschl. Ohres: 97. 1889. gested that Rm. tauricus represented a heterothallic mutant ≡ Rhizomucor septatus (Bezold) Lucet & Costantin, Archs Parasitol. 4: 362. 1901. strain of Rm. pusillus (Vágvölgyi et al. 1999). = Mucor parasiticus Lucet & Costatin, Compt. Rend. Hebd. Séances Rhizomucor nainitalensis forms sporangiospores of different Acad. Sci. 129: 1033. 1899. shapes, varying from subglobose to irregularly shaped (Joshi ≡ Rhizomucor parasiticus (Lucet & Costantin) Lucet & Costantin, Rev. 1982). The sporangiospores of Rhizomucor pakistanicus are Gén. Bot. 12: 81. 1900. ≡ Rhizopus parasiticus (Lucet & Costantin) Lendn., Mat. Fl. Crypt. Suisse globose or ovoidal (Mirza et al. 1979). The following description 3: 115. 1908. of Rhizomucor is slightly modiﬁed from Schipper (1978b: 53): = Mucor buntingii Lendn., Bull. Soc. Bot. Genève, Ser. 2, 21: 260. 1930. = Mucor tauricus Milko & Schkur., Novosti Sist. Nizsh. Rast. 7: 139. 1970. ≡ Rhizomucor tauricus (Milko & Schkur.) Schipper, Stud. Mycol. 17: 62. Rhizomucor Lucet & Costantin (1900) 1978. Thermophilic; sporangiophores originating from aerial myce- ≡ Rhizomucor pusillus var. tauricus (Milko & Schkur.) R.Y. Zheng, X.Y. lium, either from short aerial hyphae or from distinct stolons, Liu & R.Y. Li, Sydowia 61: 144. 2009. both with simple or weakly branched rhizoids; sporangio- Lectotype. Pl. II.III, Fig. 1–6 (Lindt, Arch. Exp. Path. Pharmacol. 21: phores branched, each branch bearing a multispored terminal 269–298. 1886), designated here. sporangium; sporangia borne in an upright position, globose, Epitype. CBS 354.68 (preserved in a metabolically inactive state by lyo dark (coloured), distinctly columellate, non-apophysate; spo- philization, batch nr. 85.0901), designated here. rangiospores consistently or partly subglobose; zygospores Specimens examined. CBS 179.69 ex-lectotype strain of Rhizomucor globose, covered with blunt projections, and formed in the tauricus (lectotype: CBS 179.69, preserved in a metabolically inactive state aerial mycelium between non-ornamented, isogamous op- by lyophilization, batch nr. 87.3168, designated here), CBS 219.31, CBS posed suspensors. 354.68, CBS 425.78, CBS 120586, CBS 120587, CNM-CM 2752, CNM-CM 2935, CNM-CM 2974, CNM-CM 4727, CNM-CM 5124.

Mucor endophyticus (R.Y. Zheng & H. Jiang) J. Pawłowska & Umbelopsis G. Walther, comb. nov. — MycoBank MB800463 Our ITS dataset is incomplete for Umbelopsis because of Basionym. Rhizomucor endophyticus R.Y. Zheng & H. Jiang, Mycotaxon the high proportion of strains that needed to be cloned. For 56: 456. 1995. that reason, we refrain from taxonomic changes. The ex-type Specimens examined. CBS 385.95 ex-type strain of Rhizomucor endo- strains of Umbelopsis dimorpha and Umbelopsis nana pos- phyticus. sess identical ITS sequences and it is likely that these species are conspeciﬁc. The ITS sequences of the ex-type strains of U. swartii and U. westeae, as well as those of U. gibberosa and Mucor indicus Lendn., Bull. Soc. Bot. Genève, Ser. 2, 21: 258. U. ramanniana are very similar necessitating a critical revision 1930. — MycoBank MB267842 of Umbelopsis taxonomy. ≡ Zygorhynchus indicus (Lendn.) Arx, Sydowia 35: 16. 1982. = Rhizomucor chlamydosporus R.Y. Zheng, X.Y. Liu & R.Y. Li, Sydowia 61: 142. 2009. Acknowledgement We are grateful to Willem van Boekel for excellent technical assistance. We thank Maria Vehreschild of Fungiscope for providing Lectotype. Fig. 1–3 (Lendner, Bull. Soc. Bot. Genève, Ser. 2, 21: 258– several clinical isolates. We also express our thanks to Kerstin Voigt, Paul 260. 1930), designated here. Kirk and Scott Redhead for critically reading of the manuscript and useful Epitype. CBS 226.29 (preserved in a metabolically inactive state by lyo advice. The reviewers Gerald Benny and Kerry O’Donnell are thanked for philization, batch nr. 679), designated here. their helpful corrections and suggestions. 46 Persoonia – Volume 30, 2013

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DNA Barcoding in &lt;I&gt;Mucorales&lt;/I&gt;: an Inventory Of

DNA Barcoding in <I>Mucorales</I>: an Inventory Of