The family structure of the Mucorales: a synoptic revision based on comprehensive multigene-genealogies

Abstract

The Mucorales (Mucoromycotina) are one of the most ancient groups of fungi comprising ubiquitous, mostly saprotrophic organisms. The first comprehensive molecular studies 11 yr ago revealed the traditional classification scheme, mainly based on morphology, as highly artificial. Since then only single clades have been investigated in detail but a robust classification of the higher levels based on DNA data has not been published yet. Therefore we provide a classification based on a phylogenetic analysis of four molecular markers including the large and the small subunit of the ribosomal DNA, the partial actin gene and the partial gene for the translation elongation factor 1-alpha. The dataset comprises 201 isolates in 103 species and represents about one half of the currently accepted species in this order. Previous family concepts are reviewed and the family structure inferred from the multilocus phylogeny is introduced and discussed. Main differences between the current classification and preceding concepts affects the existing families Lichtheimiaceae and Cunninghamellaceae, as well as the genera Backusella and Lentamyces which recently obtained the status of families along with the Rhizopodaceae comprising Rhizopus, Sporodiniella and Syzygites. Compensatory base change analyses in the Lichtheimiaceae confirmed the lower level classification of Lichtheimia and Rhizomucor while genera such as Circinella or Syncephalastrum completely lacked compensatory base changes.

INTRODUCTION

The fungal order Mucorales – evolutionary position and characterisation

As a member of the Mucoromycotina, the Mucorales belong to the early diverging, ancient fungi along with the Kickxellomycotina, Zoopagomycotina, Entomophthoromycotina, Mortiellomycotina, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, and Cryptomycota and Microsporidia with the latter two still highly discussed (Schüßler et al. 2001, James et al. 2006, Hibbett et al. 2007, Hoffmann et al. 2011, Jones et al. 2011a, b, Benny 2012). The Entomophthoromycotina were later elevated to the phylum Entomophthoromycota (Humber 2012).

Mucorales are characterised by a usually abundant, rapidly growing mycelium as well as anamorph structures usually formed in large quantities. The mycelium is typically unseptate or irregularly septate. Anamorphic sporangiospores are produced in multi-spored sporangia, few-spored sporangiola or merosporangia. Chlamydospores, arthrospores and yeast cells are, in most species, rarely formed. Sporangia are characterised by the inclusion of a variously shaped columella. This well-developed columella counts as a synapomorphic character for the Mucorales. Conjugation in homothallic species or between compatible mating types of heterothallic species results in the formation of zygospores. Zygospores often display a specific exospore ornamentation (smooth, rough, warty) and protecting appendages (finger-like, antler-like) born on the supporting cells (suspensors) (Zycha et al. 1969). Some species of the Mucorales exhibit dimorphism, possessing the ability to switch between a filamentous, multi-cellular state to a yeast-like state (Bartnicki-Garcia & Nickerson 1962).

Life styles and applications — Fig. 1

Fig. 1.

General life cycle, important fields of scientific research and main applications of Mucorales. Exemplary and prominent species are given.

Mucoralean fungi are ubiquitous, predominantly saprobic soil organisms on decaying organic material but parasites of plants, fungi and animals also are known. As one of the largest orders in the basal Fungi, the Mucorales is also one of the most studied groups in the early diverging fungi. These studies on mucoralean fungi encompass physiology and biochemistry, as well as taxonomy and systematics, and potential applications in industry. In general, mucoralean fungi reproduce anamorphically via non-motile sporangiospores released from variously shaped sporangia. If not homothallic, a compatible mating partner is needed for the formation of the zygospore, where meiosis occurs. The different sexual modality of either homo- or heterothally in the Mucorales was discovered more than 100 yr ago, with most species found to be heterothallic (Blakeslee 1904). Volatiles are responsible for the formation of sexual reproductive structures (Burgeff 1924). These volatiles were identified as trisporoids, derivatives of beta-carotene (van den Ende 1967, Gooday 1968). The trisporic acid precursors are mutually processed by the compatible mating partners, resulting in the formation of a mature zygospore (Werkman 1976). Although the composition of the compounds is species specific to allow only intra-species matings (Sutter et al. 1989), inter-species zygospores are also described with some impact on systematics (Blakeslee & Cartledge 1927, Stalpers & Schipper 1980). Combining an order-wide trisporoid profiling with the current knowledge on phylogenetic relationships would most likely reveal the ‘languages’ of the different clades and their potentials for interspecific mutual recognition. But currently, only profiles for few species are known: e.g. Phycomyces blakesleeanus (Miller & Sutter 1984) and Blakeslea trispora (Caglioti et al. 1966).

Although a general trisporic acid biosynthesis pathway (Schachtschabel et al. 2005) is widely accepted, the genetic background is resolved only in parts. The synthesis and degradation of beta-carotene is well studied and understood (Almeida & Cerdá-Olmedo 2008, Polaino et al. 2010, Tagua et al. 2012) but most enzymes responsible for trisporic acid production remain undiscovered. So far, only 4-dihydro-methyltrisporate dehydrogenase and 4-dihydrotrisporin dehydrogenase are verified (Czempinski et al. 1996, Wetzel et al. 2009).

Since an interaction of compatible mating types is essential for matured zygospores to be produced, the information for the mating type is probably genetically coded. The appropriate regions were identified first in Phycomyces blakesleeanus (Idnurm et al. 2008) and subsequently discovered in Rhizopus delemar, R. oryzae (Gryganskyi et al. 2010), Mucor circinelloides (Lee et al. 2010) and even in a homothallic species, Syzygites megalocarpus (Idnurm 2011). Although heterothallic strains possess only one gene coding for either plus or minus mating type, the phenomenon of rare switches between mating types (Schipper & Stalpers 1980) is not yet explained.

The importance of zygospores for reproduction and distribution compared to the asexual sporangiospores is still unknown, since germination in the natural habitat could not be observed and germination under laboratory conditions has only been described and illustrated for few species (Michailides & Spotts 1988, Yu & Ko 1997). Nevertheless, zygomycetes are reported from the fossil records. The earliest zygomycotan fossil known, exclusive of the Glomeromycota, may be Jimwhitea circumtecta, possible Endogonaceae, from the middle Triassic (Krings et al. 2012). Many fossil zygomycetes have been found in the Carboniferous and later, including Protoascon missouriensis and others (Taylor et al. 2005, Kar et al. 2010). Calculations of the diverging time of zygomycetes using molecular data suggest an origin of around 600 mya (Berbee & Taylor 2001).

The zygomycetes are known to be useful for a variety of different applications, including food and food additive production and food preservation. Zygomycetes are used as starter cultures for the fermentation of soybean- or rice-based products in Asia, Africa and South America, e.g. beverages, or the well-known tempeh (Henkel 2004, 2005, Hesseltine 1983, 1991, Nout & Kiers 2005, Tamang & Thapa 2006).

Mucorales also are used for diverse biological transformations (Gładkowski et al. 2004, 2011) as well as the production of additives for food, feed, pharmaceuticals (like lycopene) or various applications of chitosan (reviewed by Shahidi et al. 1999), a cell wall component only known to be produced by Mucorales. Yet, Mucorales also are reported as spoilage agents in stored cereals and other food, especially fruits and vegetables (Martin 1964, Wade & Morris 1982, Ray & Ravi 2005). In addition, some organisms also infect living plants, especially the fruits (e.g., strawberry, yellow summer squash or green beans; Fig. 2c, d) (Dennis 1983). Thus, these fungi play an important role as plant pathogens as well (Shtienberg 1997). Furthermore, some species of the Mucorales are facultative parasites of other fungi. They can be biotrophic or necrotrophic parasites with a few species (Syzygites megalocarpus, Dicranophora fulva, Spinellus fusiger) able to infect the fruit body of agarics (Fig. 2a; Zycha et al. 1969), a feature that is thus far not well studied. However, well studied is the biotrophic fusion parasitism (Fig. 2b) between Absidia glauca and Parasitella parasitica, a model system for studies of horizontal gene transfer and the link between sexual and parasitic interactions (Burgeff 1924, Kellner et al. 1993, Schultze et al. 2005). Trisporic acid and its precursors are also believed to be responsible for recognition of potential hosts for Chaetocladium (another parasite) and Parasitella, which was assumed from an observed mating-type dependent infection (Burgeff 1924, Schultze et al. 2005). Yet, a strict mating-type dependency was rejected as early as 1926 by a mere tendency which, in addition, seems to be restricted to only few species (Satina & Blakeslee 1926). Currently, an order-wide comprehensive survey of host-ranges for all known biotrophic fusion parasites is lacking. A recent investigation revealed an unstudied mycoparasite, Lentamyces parricida, as the most basal with the highest mycoparasitic potential to infect other mucoralean hosts (Hoffmann & Voigt 2009).

Fig. 2.

a. Syzygites megalocarpus on Pleurotus ostreatus (artificially infected); b. Parasitella parasitica on Mucor circinelloides. Galls (g) and sikyotic cells (s) are marked; c. Choanephora cucurbitarum on yellow summer squash. d. Rhizopus stolonifer on strawberries. — Scale bar = 20 μm.

Mucoralean fungi are also known as human and animal pathogens. Mucor corymbifer (currently Lichtheimia corymbifera) was first reported as causative agent of mycosis in a rabbit (Platauf 1885). In the last decades, the reported number of infections caused by members of the Mucorales (mucormycoses) has constantly increased. This is probably due to a rising awarness, an improved identification by the use of molecular methods, as well as a permanent worldwide increase of risk factors such as immunosupression, malignancies and diabetes (Roden et al. 2005, Skiada et al. 2011).

The symptoms of infections by Mucorales remain unspecific for a long time, making a diagnosis extremely difficult. A fast, proper and effective therapy is required, since these infections can result in death within hours to a few days. Survival rates for mucormycosis are highly dependent on the location of the infection, but they are very low overall at 53 % (Skiada et al. 2011). The large and still increasing numbers of studies pertaining to the susceptibility of Mucorales to known and new fungicides indicate a pressing need for an effective therapy. And with the discovery of species-specific susceptibility profiles, it became obvious, that the causative agents should be identified correctly to species level (e.g. Vitale et al. 2012). To investigate and to understand mucormycoses, their susceptibility and their evolutionary relationships need to be comparatively investigated. Understanding evolutionary relationships will elucidate approaches to improve existing or to invent new applications in industry, agriculture or medicine.

Morphology-based families

Traditionally, Mucorales were classified using their observable characters, for example physiology, biochemistry and, especially, morphology (Table 1). Unfortunately, Mucorales display only a small number of distinguishable morphological characters and only a few of them have proven to be useful for distinction between species, genera and families.

Table 1.

Morphological featured observable in mucoralean fungi.

Feature/character	Criteria
Mycelium	Height, colour, rhizoids, arthrospores, chlamydospores, giant cells
Sporangia incl. sporangiophore	Height, origin, branching pattern, size, shape, colour, number of spores, septation, dissolving of the wall, release of spores, response to light
Sporangiospores	Shape, size, ornamentation, colour, appendages
Zygospores	Homo-/heterothallism, air-borne or submerged, relative placement and size of suspensors, shape, size, colour, ornamentation, appendages

Nevertheless, in early mucoralean systematics, clustering of morphologically similar species resulted in well-defined genera and families accepted before the implementation of molecular data in phylogenetic reconstruction (Table 2).

Table 2.

Morphology based family structure of the Mucorales adopted from Zycha et al. 1969, Hesseltine & Ellis 1973, Benjamin 1979, Benny 1982, von Arx 1982.

Family	Main characteristics
Chaetocladiaceae	Unispored sporangiola formed on fertile vesicles, discoid columella, dichotomous branched fertile hyphae, sterile spines, chlamydospores absent, zygospores rough-walled, suspensors opposed
Choanephoraceae	Sporangia and sporangiola, on different sporangiophores, zygospores striate, suspensors apposed or tongs-like
Cunninghamellaceae	(Fig. 3a), unispored sporangiola, sporangia absent, zygospores warty, suspensors opposed
Gilbertellaceae	Sporangiospores appendaged; zygospores rough-walled, suspensors opposed
Mucoraceae	Sporangia present, specialized sporangiola absent, zygospores smooth to warty, variously shaped suspensors: opposed, naked, appendaged, polyphyletic
Mycotyphaceae	Sporangiola on pedicels (Fig. 4k)
Phycomycetaceae	Sporangiophores large and unbranched, zygospores with coiled tongs-like suspensors and branched appendages
Pilobolaceae	Spores are actively liberated, zygospores smooth, suspensors tongs-like or apposed (Fig. 5)
Radiomycetaceae	Sporangia absent, sporophores with a primary vesicle bearing secondary ampullae, sporangiola originating from ampullae, zygospores smooth, suspensors apposed, appendaged
Saksenaeaceae	Provisionally classified together with Lobosporangium (currently Mortierellomycotina) because of the unusual-shaped sporangia
Syncephalastraceae	Merosporangia, zygospores warty, suspensors opposed (Fig. 4g)
Thamnidiaceae	Sporangiola present, sporangia absent or apically on the sporangiophores, zygospores warty, suspenors opposed (Fig. 4n, o)

Delimitations of morphology

Traditional approaches used to classify fungi – fossil records, biochemistry and, especially, morphology (e.g., Paterson & Bridge 1994, Benny 1995, Hawksworth et al. 1995) became less important following the emergence of molecular systematics (White et al. 1990). Applying molecular data to phylogenetic analyses has led to the breakdown of the former phylum Zygomycota, combined by the morphological feature ‘zygospore’ into the subphyla Mucoromycotina, Kickxellomycotina, Zoopagomycotina and Entomophthoromycotina (James et al. 2006, Hibbett et al. 2007).

The family structure of the Mucorales is still rather unstable, but with the discovery of new, potentially phylogenetic informative characters (molecular data) and with the availability of higher resolution microscopy (e.g., fluorescence, SEM, TEM) it becomes feasible to reveal smaller, presumably monophyletic clades.

The most significant changes have affected the Thamnidiaceae, Mucoraceae, Chaetocladiceae and Absidiaceae. The first molecular studies addressing the entire order (O’Donnell et al. 2001, Voigt & Wöstemeyer 2001) showed that species traditionally assigned to Thamnidiaceae and Mucoraceae were scattered over the entire order. A widely accepted classification predominantly based on morphological traits was published by Benny et al. (2001) and is summarised with the molecular studies in Table 3.

Table 3.

Summary of the family structure of the Mucorales based predominantly on morphology (Benny et al. 2001) as well as on combination with molecular data (O’Donnell et al. 2001, Voigt & Wöstemeyer 2001).

Family	Genera
Chaetocladiaceae	Chaetocladium, Dichotomocladium
Choanephoraceae	Blakeslea, Choanephora, Poitrasia
Cunninghamellaceae	Cunninghamella
Gilbertellaceae	Gilbertella
Mortierellaceae	Aquamortierella, Dissophora, Echinosporangium, Modicella, Mortierella, Umbelopsis
Mucoraceae	Absidia, Actinomucor, Apophysomyces, Chlamydoabsidia, Circinella, Circinomucor, Dicranophora, Gongronella, Halteromyces, Hyphomucor, Micromucor, Mucor, Mycocladus, Parasitella, Protomycocladus, Rhizomucor, Rhizopodopsis, Rhizopus, Spinellus, Sporodiniella, Syzygites, Thermomucor, Zygorhynchus
Mycotyphaceae	Benjaminiella, Mycotypha
Phycomycetaceae	Phycomyces
Pilobolaceae	Pilaira, Pilobolus, Utharomyces
Radiomycetaceae	Hesseltinella, Radiomyces
Saksenaeaceae	Saksenaea
Syncephalstraceae	Syncephalastrum
Thamnidiaceae	Backusella, Cokeromyces, Ellisomyces, Fennellomyces, Helicostylum, Kirkomyces, Phascolomyces, Pirella, Thamnidium, Thamnostylum, Zychaea

Over the following years, several species and genera were studied in more detail, re-evaluated and revised (for a complete list see Walther et al. 2013 in this issue of Persoonia). In the following only studies that influenced family concepts by the dissection of the genus, the exclusion of a genus from a family or the fusion of families are addressed.

Absidia, Lichtheimia and Lentamyces

The genus Absidia was originally defined by its pyriform, apophysate sporangia (Fig. 3b, c, 4h–j). The first phylogenetic analyses (O’Donnell et al. 2001, Voigt & Wöstemeyer 2001) revealed a paraphyletic origin of this genus, a separation was accomplished later. Mesophilic species were retained in the genus Absidia (Cunninghamellaceae), whereas thermotolerant species form a separate phylogenetic clade as genus Lichtheimia (Hoffmann et al. 2007, 2009). In addition to the thermotolerant species separated from Absidia, potential mycoparasitic species were also distinguished in a new genus, Lentamyces (Hoffmann & Voigt 2009). This genus harbours two species, L. parricida and L. zychae. At the same time, two new species were isolated from nature and described as Siepmannia lariceti and S. pineti (Kwaśna & Nirenberg 2008a, b). This genus also was supposed to include both species of Lentamyces. Since molecular data for Siepmannia includes only ITS sequences, with no living material accessible, the relationship between the two genera remains unclear.

Fig. 3.

a. Cunninghamella sp. Sporangiophore with apical vesicle and sporangiola on stalks; b. apophysate sporangia of Absidia sp.; c. columella of Absidia sp. with typical apical projection and subsporangial septae; d, e. sporangium of Gilbertella persicaria: d. ruptured sporangial wall and released spores; f. branching sporangiophore of Blakeslea trispora with apical vesicles bearing few spored sporangiola. — Scale bars: a = 5 μm; b, c = 20 μm; d–f = 50 μm.

Fig. 4.

a. Umbelopsidaceae. Sporangium and sporangiophore with the highly reduced columella (arrow). — b, c. Lentamycetaceae. b. Warty zygospore, species are homothallic; c. sporangium. — d–f. Dichotomocladium. d. Zygospores; e, f. dichotomous branched sporangiophores. — g. Syncephalastrum racemosum, merosporangia. — h–j. Lichtheimia. h. Columella; i, j. apophysate sporangia. — k. Mycotypha sp., cylindrical vesicle covered with sporangiola. — l, m. Chaetocladium sp., branched fertile head with sporangiola. Branches often terminate in sterile spines. — n, o. Thamnidium elegans. n. Dichotomous branched sporangiophores with sporangiola; o. main multi-spored sporangia. — p, q. Columella and sporangia borne on circinate sporangiophores of Circinella sp. — Scale bars: all = 20 μm.

Choanephora and Gilbertella

Although there are morphological differences in zygosporogenesis in the Gilbertellaceae and the Choanephoraceae, a molecular study combined with ultrastructure supported merging these two families, under the older name, Choanephoraceae (Voigt & Olsson 2008).

Pilaira

Due to morphological similarities, this genus was placed traditionally within the Pilobolaceae together with Pilobolus and Utharomyces. But molecular data (O’Donnell et al. 2001, Voigt & Wöstemeyer 2001) revealed a non-relationship of Pilaira to both other genera, followed by an assignment to the Mucoraceae as published in Index Fungorum. This classification was also suggested on the base of a comprehensive molecular study of the Pilobolaceae (Foos et al. 2011).

Molecular systematics and implications on Mucorales

Molecular systematics is rapidly developing. Taxon samplings, possibilities to combine data and the number of applicable analytical tools are constantly increasing. In addition, with the ability to sequence whole genomes at relatively moderately cost combined with appropriate annotation software, computing capability and open access, genome-wide phylogeny comes within reach (Fitzpatrick et al. 2006, Kuramae et al. 2006, Huerta-Cepas et al. 2008). However, as only a few mucoralean fungi are fully sequenced, elucidating the phylogenetic relationships within this order is usually based on single genes or the combination of a few genes. Currently (April 2012), 24 genome/transcriptome projects for Mucorales are listed in the JGI Genome Online Database (GOLD; Fig. 6), but this includes only four different taxa (Mucor circinelloides, Rhizopus oryzae, Rhizopus stolonifer (each one project), and Phycomyces blakesleeanus (21 projects).

Fig. 6.

Schematic fungal tree and important data about the fungal groups. The topology resembles the current understanding of the relationships of the fungal groups according to Hibbett et al. (2007), James et al. (2006) and Schoch et al. (2012) (data retrieved April 2012).

There are currently more than 6 000 sequences of zygomycota deposited in GenBank, approximately one-third of these are protein coding sequences. This is the third largest fraction for basal fungi, but still far behind the derived fungi, the Dikarya (Ascomycota and Basidiomycota; Fig. 6). Molecular data for the Mucorales have been submitted to GenBank since 1993, with a constantly increasing number, reaching more than 1 000 sequences in 2010 and more then 1 400 last year (Fig. 7). Nevertheless, the submitted sequences are restricted to only a few genera and species, with half of the sequences from the two genera Mucor and Rhizopus (Table 4). Around 50 species for Mucor and nine species for Rhizopus are listed in the 10th edition of the Dictionary of the Fungi (Kirk et al. 2008) which is 24 % and 4 %, respectively, of all species accepted in the Mucorales.

Fig. 7.

Chronology of sequences submitted to GenBank since 1993 for the Mucorales (data retrieved April 2012).

Table 4.

Sequences available at GenBank (April 2012) for mucoralean genera.

Genus	No. of seq.	Genus	No. of seq.	Genus	No. of seq.
Absidia	184	Gongronella	31	Rhizomucor	200
Actinomucor	68	Halteromyces	5	Rhizopus	1928
Ambomucor	3	Helicostylum	20	Saksenaea	50
Amylomyces	163	Hesseltinella	4	Siepmannia	2
Apophysomyces	69	Hyphomucor	4	Spinellus	7
Backusella	6	Kirkomyces	4	Sporodiniella	4
Benjaminiella	12	Lentamyces	23	Syncephalastrum	64
Blakeslea	93	Lichtheimia	679	Syzygites	26
Chaetocladium	25	Mucor	1501	Thamnidium	11
Chlamydoabsidia	6	Mycotypha	11	Thamnostylum	14
Choanephora	27	Parasitella	17	Thermomucor	6
Circinella	6	Phascolomyces	6	Umbelopsis	243
Cokeromyces	16	Phycomyces	110	Utharomyces	20
Cunninghamella	141	Pilaira	59	Zychaea	4
Dichotomocladium	29	Pilobolus	149
Dicranophora	4	Pirella	5
Ellisomyces	8	Poitrasia	11	environmental/
Fennellomyces	10	Protomycocladus	4	uncultured/
Gilbertella	16	Radiomyces	8	unclassified	108

Studies predominately concerned with molecular phylogenetic as-pects of zygomycetes, especially Mucorales, are still relatively rare. Searching NCBI and the ISI Web of Science with ‘zygomycetes or Mucorales AND phylogeny’ resulted only in between 40 and 50 analyses including 15 studies where at least 2 loci were applied (April 2012, Fig. 8).

Fig. 8.

Number of publications predominantly focused on mucoralean phylogeny retrieved from NCBI and ISI Web of Science by searching ‘Zygomycota/ Mucorales AND phylogeny’. Publications are separated by the molecular marker applied for phylogeny. Nearly half of all published studies included more than one molecular marker. Published combinations of molecular markers are indicated by different colours (data retrieved April 2012).

Commonly applied markers for phylogeny are sequences coding for rDNA (especially 18S rDNA for relationship levels of families, orders and above-order as well as ITS1 & 2 for relationships of species and genera). Therefore, the majority of studies are using rDNA sequences for phylogenetic approaches although ITS sequences represent the largest fraction of sequences in GenBank (Fig. 9). Protein coding genes predominantely applied so far are actin and translation elongation factor 1-alpha. Establishment of alternative protein coding markers for the whole order remains difficult. Whereas largest and second-largest subunit of RNA polymerase II (RPB1 & 2), ATPase subunit 6 (ATP6), a DNA replication licensing factor (MCM7), a gene required for rRNA accumulation (TSR1) or cytochrome c oxidase I (COX1) proved to be suitable for other fungal groups mostly belonging to the Basidiomycota and Ascomycota (Matheny et al. 2002, Reeb et al. 2004, Seifert et al. 2007, Schmitt et al. 2009), these genes have not be successfully amplified for a broad range of Mucorales and are still under represented in GenBank (Schoch et al. 2012).

Fig. 9.

Distribution of available sequences in GenBank for the Mucoromycotina. Also the total number of available sequences for all basal fungal linages are given (data retrieved April 2012).

The present study focuses on the family structure of the Mucorales. Family boundaries are inferred from a molecular phylogeny based on four markers and including 201 isolates and all currently accepted genera. Historical approaches and changes in recent years are revised, the support of the families by the current data is discussed and the families are characterised morphologically and ecologically. The resulting changes on the higher level nomenclature of the Mucorales were already briefly introduced by Voigt (2012). In order to ensure that these changes were based on a stable lower level taxonomy the internal transcribed spacer 2 region (ITS2) was analysed for of compensatory base changes (CBCs) as indicators for species boundaries (Müller et al. 2007).

MATERIAL AND METHODS

Strains, DNA isolation, PCR

Strains used for the generation of additional sequences (bold accession numbers in Table 5) were cultivated on 3 % malt extract medium at room temperature. Genomic DNA was extracted as described in Hoffmann et al. (2007). For phylogenetic analyses, sequences of large (LSU) and small (SSU) subunit of ribosomal DNA, ITS (internal transcribed spacer 1 & 2, incl. 5.8 SrDNA), actin (act) and translation elongation factor 1-alpha (tef) were either generated in this study or retrieved from GenBank (www.ncbi.nlm.nih.gov/; Table 5). Primers used for the amplification of LSU were NL1 and NL4 (O’Donnell 1993), NS1 and NS4 for SSU (White et al. 1990), ITS1 and ITS4 for ITS (White et al. 1990), Act1/Act1b and Act4R/Act4Ra for actin (Voigt & Wöstemeyer 2000) and MEF1 and MEF4/UEF4 for Tef (O’Donnell et al. 2001). PCR fragments were purified using the protocol of Vogelstein & Gillespie (1979) and sequenced on an Applied Biosystems 3730xL DNA Analyzer (ABI, Carlsbad) according to the manufacturer’s instructions.

Table 5.

Taxa and sequences used for the phylogenetic analyses. GenBank accession numbers in bold are generated within this study.

Internal no.	Species	Isolate	18S rDNA	28S rDNA	Act	Tef
	Ascomycota
KV5	Archaeorhizomyces finlayi	Ny10	JF836020	JF836022	na	JF836025
P248	Saccharomyces bayanus	CBS380	X97777	AF113892	na	na
	Basidiomycota
P249	Agaricus bisporus	AFTOL448	AY787216	AY635775	na	na
	Blastocladiomycota
P251	Blastocladiella emersonii	AFTOL302	AY635842	DQ273808	na	na
	Chytridiomycota
P250	Batrachochytrium dendrobatidis	AFTOL21	AH009052	NG_027619	na	na
	Eccrinales
KV1	Enterobryus sp.		AY336711	AY336693	na	na
KV2	Enteromyces callianassae	CA12c8	AY336702	AY336696	na	na
KV4	Palavascia patagonica	ARGD1c15	AY682845	AY336695	na	na
KV3	Taeniellopsis sp.	MA5C17	AY336704	AY336697	na	na
	Endogonales
P011	Endogone pisiformis	AFTOL539	DQ322628	DQ273811	AB609182	DQ282618
	Entomophthoromycotina
P006	Basidiobolus ranarum	AFTOL301	AY635841	DQ273807	na	DQ282610
P021	Batkoa major	ARSEF2936	EF392559	EF392401	na	na
P017	Conidiobolus coronatus	NRRL28638	NG_017182	NG_027617	HM117709	na
P024	Entomophaga maimaiga	ARSEF1400	EF392556	EF392395	na	na
P025	Entomophthora muscae	ARSEF3074	NG_017183	NG_027647	na	na
P026	Erynia radicans	ATCC60281	JQ014018	JN939182	na	na
P027	Eryniopsis caroliniana	ARSEF640	EF392552	EF392387	na	na
P030	Massospora cicadina	ARSEF374	EF392548	EF392377	na	na
P033	Pandora neoaphidis	ARSEF3240/ARSEF835	EF392560	EF392405	na	na
P007	Schizangiella serpentis	ARSEF203	AF368523	EF392428	na	na
P037	Zoophthora radicans	ARSEF4784/ARSEF6003	EF392561	EF392406	na	na
	Glomeromycota
P253	Glomus intraradices	AFTOL845	DQ322630	DQ273828	na	na
	Kickxellomycotina
P053	Austrosmittium biforme	32-1-9/ 32-1-8	DQ367462	DQ367494	na	na
P056	Bojamyces repens	ME-JL-2	DQ367447	DQ367478	na	na
P057	Capniomyces stellatus	mis-21-127	EF396191	EF396194	na	na
P088	Coemansia reversa	NRRL1564	NG_017186	NG_027615	AB609183	DQ282615
P091	Dipsacomyces acuminosporus	NRRL2925	AF007534	AF031065	na	na
P062	Furculomyces boomerangus	AFTOL303	AF007535	DQ273809	na	na
P065	Genistelloides hibernus	2-16-2	DQ367448	DQ367479	na	na
P066	Genistellospora homothallica	VT-3-W14	DQ367454	DQ367495	na	na
P048	Harpella melusinae	NF-15-4b	DQ367514	DQ367518	na	na
P049	Harpellomyces sp.	PA-3-1d	EF396192	EF396195	na	na
P092	Kickxella alabastrina	NRRL2693	AF007537	AF031064	na	na
P093	Linderina pennispora	NRRL3781	AF007538	AF031063	na	na
P095	Martensiomyces pterosporus	NRRL2642	AF007539	AF031066	na	na
P097	Myconymphaea yatsukahoi	NBRC100467	AB287984	AB287998	na	na
P075	Pennella simulii	NY-5-3/ NF-19-8	DQ367515	DQ367502	na	na
P089	Pinnaticoemansia coronantispora	NBRC100470	AB287986	AB288000	na	na
P076	Plecopteromyces sp.	37-1-2	DQ367445	DQ367476	na	na
P080	Smittium culisetae	AFTOL29/IAM14394/BL023	AF007540	DQ273773	HM117719	AB077104
P100	Spirodactylon aureum	NRRL2810	AF007541	AF031068	na	na
P087	Zygopolaris ephemeridarum	CA-4-W9	DQ367463	DQ367508	na	na
	Mortierellomycotina
P106	Dissophora decumbens	NRRL22416	AF157133	AF157187	AJ287155	AF157247
P107	Echinosporangium transversale	NRRL3116	AF113424	AF113462	AJ287156	AF157248
P108	Gamsiella multidivaricata	NRRL6456	AF157144	AF157198	AJ287168	AF157260
P111	Mortierella longicollis	CBS209.32	JQ040249	JN940876	na	na
P110	Mortierella verticillata	CBS374.95	HQ667482	JN940872	na	na
KH001		NRRL6337	AB016017	DQ273794	AJ287170	AF157262
	Mucoromycotina
P121a	Absidia caerulea	NRRL1315	AF113405	AF113443	AJ287133	AF157226
P121f	Absidia californica	CBS126.68	EU736273	EU736300	AY944758	EU736246
P121b	Absidia glauca	CBS101.48	AF157118	AF157172	AJ287135	X54730
P121e	Absidia macrospora	CBS696.68	EU736276	EU736303	AY944760	EU736249
P121d	Absidia psychrophilia	CBS128.68	EU736279	EU736306	AY944762	EU736252
P121	Absidia repens	NRRL1336	AF113410	AF113448	AJ287136	AF157228
P121c	Absidia spinosa	ATCC22755	EU736280	EU736307	EU736227	EU736253
P137	Actinomucor elegans	NRRL3104/CBS111559	AF157119	AF157173	AJ287137	AF157229
P190	Apophysomyces elegans	NRRL22325	AF113411	FN554250	na	na
P190a		NRRL28632	AF113412	AF113450	AJ287139	AF157231
P140	Backusella circina	NRRL2446	AF157121	AF157175	AJ287140	AF157232
kH1		FSU2455	JX644458	JX644491	na	na
kH9		FSU10121	JX644459	JX644492	na	na
kH10		FSU10122	JX644460	JX644493	na	na
kH11		FSU10123	JX644461	JX644494	na	na
kH12		FSU10124	JX644462	JX644495	na	na
P169g	Backusella recurva	NRRL3247	AF157146	AF157200	AJ287179	AF157270
kH5		FSU10115	JX644463	JX644496	na	na
kH6		FSU10116	JX644464	JX644497	na	na
kH7		FSU10117	JX644465	JX644498	na	na
kH8		FSU10118	JX644466	JX644499	na	na
P143	Benjaminiella poitrasii	NRRL2845	AF157123	AF157177	AJ287142	AF157234
P114	Blakeslea trispora	CBS130.59	AF157124	AF157178	AJ287143	AF157235
P146	Chaetocladium brefeldii	CBS136.28	EU736284	EU736311	EU736230	EU736257
P146a		NRRL1349	AF157125	AF157179	AJ287144	AF157236
P146b	Chaetocladium jonesii	NRRL2343	AF157126	AF157180	AJ287145	AF157237
P123	Chlamydoabsidia padenii	NRRL2977	AF113415	AF113453	AJ287146	AF157238
P115	Choanephora infundibulifera	CBS150.51/NRRL2744	AF157127	AF157181	AJ287147	AF157239
P151a	Circinella sp.	NRRL13768	JX644467	JX644500	JX644524	JX644574
P151b		NRRL13768	JX644468	JX644501	JX644525	JX644575
P151	Circinella umbellata	NRRL1351	AF157128	AF157182	AJ287148	AF157240
P154	Cokeromyces recurvatus	AFTOL627	AY635843	DQ273812	na	na
P154a		NRRL2243	AF113416	AF113454	AJ287150	AF157242
P124a	Cunninghamella bainierii	FSU319/NRRL1375	EU736286	EU736313	EU736232	EU736259
P124b	Cunninghamella bertholletiae	NRRL6436	AF113421	AF113459	AJ287151	AF157243
P124	Cunninghamella echinulata	NRRL1382/CBS156.28	AF157130	AF157184	AJ287152	AF157244
P194	Dichotomocladium elegans	NRRL6236	AF157131	AF157185	AJ287153	AF157245
P194a		NRRL2664	JQ775463	JQ775492	EU826394	EU826399
P194e	Dichotomocladium floridanum	FSU8694	JQ775462	JQ775491	JX644526	JX644576
P194b	Dichotomocladium hesseltinei	NRRL5912	JQ775464	JQ775493	JX644527	JX644577
P194c	Dichotomocladium robustum	NRRL6234	JQ775465	JQ775494	JX644528	JX644578
P194d		NRRL6235	JQ775466	JQ775495	JX644529	na
P194f	Dichotomocladium sphaerosporum	FSU8696	JQ775469	JQ775498	na	JX644579
P194g		FSU8697	JQ775467	JQ775496	JX644530	JX644580
P194h	Dichotomocladium sphaerosporum 2	FSU8698	JQ775468	JQ775497	JX644531	JX644581
P194i	Dichotomocladium sphaerosporum 3	FSU8698	JX644469	JX644502	JX644532	JX644582
P156	Dicranophora fulva	NRRL22204	AF157132	AF157186	AJ287154	AF157246
P157	Ellisomyces anomalus	NRRL2749	AF157134	AF157188	AJ287157	AF157249
P195	Fennellomyces linderi	NRRL2342	AF157135	AF157189	AJ287158	AF157250
P119	Gilbertella persicaria	NRRL2357/CBS442.64	AF157136	AF157190	AJ287159	AF157251
P125	Gongronella butleri	NRRL1340/ATCC8989	AF157137	AF157191	AJ287160	AF157252
P126	Halteromyces radiatus	NRRL6197	AF157138	AF157192	AJ287161	AF157253
P160	Helicostylum elegans	NRRL2568/CBS258.59	AF157139	AF157193	AJ287162	AF157254
P160c	Helicostylum pulchrum	CBS639.69	EU736289	EU736316	EU736235	EU736262
P160b		CBS259.68	EU736288	EU736315	EU736234	EU736261
P127	Hesseltinella vesiculosa	CBS197.68	AF157140	AF157194	AJ287163	AF157255
P162	Hyphomucor assamensis	NRRL22324	AF157141	AF157195	AJ287164	AF157256
P164	Kirkomyces cordensis	NRRL22618	AF157142	AF157196	AJ287165	AF157257
P160a		CBS223.63	EU736287	EU736314	EU736233	EU736260
P216a	Lentamyces zychae	CBS104.35	EU736282	EU736309	EU736228	EU736255
P134	Lichtheimia corymbifera	CBS429.75	JQ014052	GQ342903	GQ342831	FJ719483
P134a		NRRL2982	AF113407	FJ719429	AJ287134	AF157227
P134b	Lichtheimia hyalospora	NRRL1304	AF157117	AF157171	AJ287132	AF157225
P134d		NRRL2916	EU826360	EU826368	EF030531	JX644583
P134c	Lichtheimia ramosa	FSU6197	JX644470	JX644503	JX644533	JX644584
P169a	Mucor amphibiorum	NRRL28633	AF113426	AF113466	AJ287172	AF157263
P152	Mucor circinelloides	NRRL22652	AF157129	AF157183	AJ287149	AF157241
P169	Mucor circinelloides f. circinelloides	CBS195.68/FSU6169	EU484248	FN650667	na	na
P169h		CBS416.77	EU736294	EU736321	EU736240	EU736267
P169b	Mucor circinelloides f. lusitanicus	NRRL3631	AF113427	AF113467	AJ287173	AF157264
P141	Mucor ctenidius	NRRL6238	AF157122	AF157176	AJ287141	AF157233
kH2		FSU10112	JX644471	JX644504	na	na
kH3		FSU10113	JX644472	JX644505	na	na
kH4		FSU10114	JX644473	JX644506	na	na
P169c	Mucor hiemalis	NRRL3624	AF113428	AF113468	AJ287174	AF157265
P169d	Mucor indicus	NRRL28634	AF113429	AF113469	AJ287175	AF157266
P135c	Mucor irregularis	NRRL28773	AF113435	AF113476	AJ287193	AF157284
P180	Mucor moelleri	FSU779/FSU514	EU736298	EU736325	EU736244	EU736271
P180b		FSU531	EU736297	EU736324	EU736243	EU736270
P168	Mucor mucedo	CBS144.24	X89434	AF113470	AJ287176	AF157267
P169i	Mucor plumbeus	FSU283	EU736295	EU736322	EU736241	EU736268
P169j		FSU289	EU736296	EU736323	EU736242	EU736269
P169e	Mucor racemosus	NRRL3640	AF113430	AF113471	AJ287177	AF157268
P169f	Mucor ramosissimus	NRRL3042	AF113431	AF113472	AJ287178	AF157269
P182a	Mycotypha africana	NRRL2978	AF157147	AF157201	AJ287180	AF157271
P182	Mycotypha microspora	NRRL1572/F169	AF157148	AF157202	AJ287181	AF157272
P170	Parasitella parasitica	NRRL1461/CBS412.66/NRRL2501	HQ845295	HQ845307	AJ287182	HQ845318
P197	Phascolomyces articulosus	NRRL2880	AF157150	AF157204	AJ287183	AF157274
P197a		CBS113.76	JX644474	JX644507	JX644534	na
P183	Phycomyces blakesleeanus	NRRL1555	NG_017190	NG_027559	genome1	DQ282620
kH20	Pilaira sp.	FSU2463	JX644475	JX644508	na	na
P171	Pilaira anomala	NRRL2526	AF157152	AF157206	AJ287185	AF157276
kH19		FSU774	JX644476	JX644509	JX644535	JX644585
kH22		NRRL2526	AF157152	na	AJ287185	AF157276
P171a	Pilaira caucasica	NRRL6282	JX644477	JX644510	JX644536	JX644586
kH14		FSU10081	JX644478	JX644511	JX644537	na
kH16		FSU10083	JX644479	JX644512	JX644538	na
kH17		FSU10084	JX644480	JX644513	JX644539	na
kH18		FSU10085	JX644481	JX644514	JX644540	na
KH21		FSU6229	EU826363	EU826369	EU826376	EU826385
kH13	Pilaira sp.	FSU10080	JX644482	JX644515	JX644541	na
kH15	.	FSU10082	JX644483	JX644516	JX644542	na
kH25	Pilobolus crystallinus	FSU6210	JX644484	JX644517	na	na
kH28	Pilobolus longipes	IUE563	EU595654	na	na	na
KH29		IUE409	DQ211054	na	na	na
KH30		IUE340	DQ211053	na	na	na
KH31	Pilobolus roridus	IUE415	EU595649	na	na	na
kH23	Pilobolus sp.	DSM1343	JX644485	JX644518	na	JX644587
P186	Pilobolus umbonatus	NRRL6349	AF157153	AF157207	AJ287186	AF157277
kH24		CBS302.83	JX644486	JX644519	na	na
kH26		UAMH7297	DQ211050	na	na	na
kH27		NRRL6349	AF157153	na	na	na
KH32		UAMH7298	DQ211051	na	na	na
P172	Pirella circinans	NRRL2402/Kh-BI-O	AF157154	AF157208	AJ287187	AF157278
P120	Poitrasia cicinans	CBS153.58	AF157155	AF157209	AJ287188	AF157279
P198	Protomycocladus faisalabadensis	NRRL22826	AF157156	AF157210	AJ287189	AF157280
P198a		CBS661.86	JX644487	JX644520	na	na
P191	Radiomyces spectabilis	NRRL2753	AF157157	AF157211	AJ287190	AF157281
P135a	Rhizomucor miehei	NRRL28774	AF113432	AF113473	AJ287191	AF157282
P135d		CBS182.67	JX644488	JX644521	JX644543	na
P135	Rhizomucor pusillus	NRRL3695	HQ845298	HQ845310	na	HQ845321
P135b		NRRL2543	AF113433	AF113474	AJ287192	AF157283
P135e		CBS354.68	JX644489	JX644522	na	HQ845320
P175	Rhizopus arrhizus	CBS112.07	AB250164	AB250187	AB281499	AB281528
P205		CBS438.76	AB250171	AB250194	na	na
P205a		NRRL3139	AF157120	AF157174	AJ287138	AF157230
P176a	Rhizopus microsporus var. azygosporus	NRRL28627	AF113436	AF113477	AJ287194	AF157285
P176	Rhizopus microsporus var. microsporus	CBS699.68	AB250155	JN939137	AB512247	AB512270
P176b		NRRL28775	AF113438	AF113479	AJ287195	AF157286
P176c	Rhizopus microsporus var. oligosporus	NRRL2710	AF157158	AF157212	AJ287197	AF157288
P176d	Rhizopus microsporus var. rhizopodiformis	NRRL28630	AF113439	AF113480	AJ287196	AF157287
P176e	Rhizopus stolonifer	NRRL1477	AF113441	AF113482	AJ287199	AF157290
P193	Saksenaea vasiformis	NRRL2443	AF113442	AF113483	AJ287200	AF157291
P184	Spinellus fusiger	NRRL22323	AF157159	AF157213	AJ287201	AF157292
P213	Sporodiniella umbellata	NRRL20824	AF157160	AF157214	AJ287202	AF157293
P199	Syncephalastrum monosporum	NRRL54019/NRRL22812	AF157161	AF157215	AJ287203	AF157294
P199b	S. monosporum var. pluriproliferum	CBS569.91	JX644490	JX644523	na	JX644588
P199a	Syncephalastrum racemosum	NRRL2496	X89437	AF113484	AJ287204	AF157295
P215	Syzygites megalocarpus	NRRL6288/xsd08121	AF157162	AF157216	AJ287205	AF157296
P178	Thamnidium elegans	NRRL2467/CBS341.55	AF157163	AF157217	AJ287206	AF157297
P200	Thamnostylum piriforme	NRRL6240	AF157164	AF157218	AJ287207	AF157298
P136	Thermomucor indicae-seudaticae	NRRL6429	AF157165	AF157219	AJ287208	AF157299
P202a	Umbelopsis isabellina	NRRL1757	AF157166	AF157220	AJ287209	AF157300
P202c	Umbelopsis nana	NRRL22420	AF157167	AF157221	AJ287210	AF157301
P202b	Umbelopsis ramanniana	NRRL5844	X89435	AF113463	AJ287166	AF157258
P202	Umbelopsis sp.	FSU10157	JQ014049	JN939141	na	na
P189	Utharomyces epallocaulus	NRRL3168	AF157168	AF157222	AJ287211	AF157302
P201	Zychaea mexicana	NRRL6237	AF157169	AF157223	AJ287212	AF157303
P180a	Zygorhynchus heterogamus	NRRL1489	AF157170	AF157224	AJ287213	AF157304
	Neocallimastigomycota
P252	Neocallimastix sp.	AFTOL638	DQ322625	DQ273822	na	na
	Zoopagomycotina
P230	Kuzuhaea moniliformis	NRRL13723	AB016010	DQ273796	na	na
P234	Piptocephalis corymbifera	ATCC12665	AB016023	AY546690	na	DQ282619

na = not available; 1 = estExt_Genewise1Plus.C_200172.

Sequence alignment, phylogeny, distance matrices, CBC

Multiple sequence alignments were generated using MAFFT v. 6.901b (server: mafft.cbrc.jp/alignment/server/) or v. 6.822 as implemented at the CIPRES portal (//www.phylo.org/; Miller et al. 2010). Alignments comprised 201 taxa and 1 586 characters for 18S rDNA, 358 characters for 28S rDNA, 807 characters for actin and 1 092 characters for translation elongation factor 1-alpha. Phylogenetic trees were calculated using RAxML v. 7.3.0 and MrBayes v. 3.1.2 from the CIPRES portal under the default settings with the following adjustments: RAxML was run choosing rapid bootstrapping (GTRCAT) and GTRGAMMA for final tree inference with 1 000 bootstrap iterations. Bayesian inference was run setting the number of substitution types to 6 (GTR), with among-site rate variation set to invgamma. Analysis was run with four chains each in two runs for 5 million generations. 5 001 trees were sampled, and 2 501 trees were analysed discarding the first 50 % of the samples as burnin. Bootstrapping was done with 1 000 iterations. Dataset was partitioned for both analyses. Alignments and phylogenetic trees are deposited in TreeBase2 under TB2:S13469. Distances were calculated using distMat from the EMBOSS suite (Rice et al. 2000; http://emboss.sourceforge.net/) with the alignments as input. Distances are expressed as substitutions per 100 bases or amino acids. CBC analyses were done as described previously (Pawłowska et al. In press).

RESULTS AND DISCUSSION

Species recognition is an essential step to higher level classification. Yet, morphology and/or mating behaviour played a major role in traditional fungal species concepts. Depending on the experience of the mycologist and on experimental conditions, morphology and mating behaviour could profoundly vary, and today, both methods were shown to be unsuitable to define mucoralean species if they are not combined with DNA data. Additional concepts have been surveyed and evaluated for fungi (Mayden 1997) with the genealogical concordance phylogenetic species recognition (GCPSR, Taylor et al. 2000) being the most likely one to recognize natural species. Phylogenetic species recognition (PSR) already revealed more species within originally identified species using morphological or biological species recognition (e.g., Hibbett et al. 1995, Taylor et al. 1999). The underlying problems of interbreeding and geographic/allopatric speciation were extensively discussed by Taylor et al. (2000). Following the discovery of phylogenetic species, additional biological and morphological characters were revealed that supported those species (reviewed by Taylor et al. 2000).

In Mucorales, the application of GCPSR resulted in the detection of several new species (Álvarez et al. 2010a, b, Alastruey-Izquierdo et al. 2010, Hermet et al. 2012) but on the other hand several taxa were synonymized based on comparisons of ITS sequences (Abe et al. 2006, Álvarez et al. 2010a, Walther et al. 2013).

In contrast to the naturally existing species there are no concepts for the recognition of higher or lower taxonomic levels. Traditionally, certain morphological features (Table 2) that were regarded as synapomorphies were used to define families (Zycha et al. 1969, Hesseltine & Ellis 1973, Benjamin 1979, Benny 1982, von Arx 1982). Later they were adapted based on results of molecular phylogeny. Undoubtedly higher taxa should represent monophyletic groups but the taxonomic rank that a group deserves remains a subjective decision. Genetic distances are helpful in this decision but they cannot be translated directly into higher level taxonomy because of dramatic difference in the phylogenetic age in fungal groups.

Even though studies implementing molecular data are still very rare for Mucorales compared to other fungal groups, the number of sequences submitted to GenBank is constantly increasing (Fig. 7). Yet, sequences deposited are predominantly sequences of the rDNA cluster, (Fig. 9). Protein coding sequences are still under represented. This may be due to the lack of appropriate primers which are able to work over a broad range of isolates and an often encountered problem of direct sequencing of the amplificates (Schoch et al. 2012) and the frequent presence of paralogs (Alastruey-Izquierdo et al. 2010). Studies which do apply this kind of molecular data and which are predominantly focused on mucoralean phylogeny count far below 100 if searching ISI Web of Science and NCBI. Furthermore, most of these studies using only one marker for the analyses (Fig. 8). If different sequences are combined in an analysis, it is often rDNA and the genomically linked ITS region, but also rDNA combined with protein coding genes (Fig. 8).

The phylogenetic analysis in Fig. 10 consists of combined sequences coding for LSU, SSU, actin and translation elongation factor. At least one member of all accepted genera is included with a total of 201 isolates, 151 belonging to the Mucorales, and 103 unique species representing around half of all described species in the order. Species were included if at least two loci were present in the alignment.

Fig. 10.

Bayesian analysis of combined sequences coding for actin, translation elongation factor 1-alpha, 18S rDNA and 28S rDNA. Bootstrap values and posterior probabilities are given for branches supported with equal or higher than 75 % in maximum likelihood (RAxML) and Bayesian analysis (see legend within figure for explanation of the symbols). Strain numbers are given in parts to distinguish different isolates (compare with Table 5). Furthermore, a rough outline about the historical family structures and changes are given on the right site including benchmark studies since 1969 (Zycha et al. 1969, Hesseltine & Ellis 1973, Benjamin 1979, Benny 1982, von Arx 1982, Benny et al. 2001, Voigt & Wöstemeyer 2001, O’Donnell et al. 2001, Kirk et al. 2008). Families accepted here, are colour coded over the whole tree branches.

A distance matrix was calculated for each locus. The order-wide distance analyses were based solely on the isolates in the illustrated tree (Fig. 10). Species-specific variations for each locus were not considered. The inclusion and analyses of all available sequences from GenBank would constitute a separate research project that goes beyond the scope of this study.

As expected, distance matrices derived from protein coding genes vary less if based on amino acids instead of nucleic acids. Based on the underlying data, amino acid sequences of actin are more conserved within the Mucorales with relatively similar distances over the whole order versus the situation for the translation elongation factor. When comparing all distance matrices, three major groups can be distinguished (Fig. 11):

Fig. 11.

Distance matrices for all applied loci based on nucleic acid and amino acid sequences. The range of distances is given for each locus. Families are coded according to Fig. 10.

• i) Low to moderate distances for the most derived clade of the ‘Mucorineae’ including the Mucoraceae, Mycotyphaceae, Choanephoraceae, Pilobolaceae, Rhizopodaceae and Backusellaceae. All matrices show the lowest distances for Mucoraceae (incl. Mycotyphaceae).

  All other groups and clades in the tree show no low distance values to any other group. Shortest distances exist only with-in each group whereas distances to all other groups are more or less similar. Clades included here are:

• ii) the Cunninghamellaceae. Within this family, the shortest distances are between the genera Absidia, Halteromyces, Chlamydoabsidia and Cunninghamella (except for translation elongation factor, where distances between Gongronella/Hesseltinella and Absidia/Halteromyces/Chlamydoabsidia are shorter than to the embedded Cunninghamella.

• iii) Lichtheimiaceae/Syncephalastraceae/Lentamycetaceae/Umbelopsidiaceae/Radiomycetaceae/Phycomyceteaceae.

  High distance values for the more ancient clades of the phy-logenetic tree result from the different evolutionary times of origin which gives the more basal groups more time to evolve separately.

In the following, clades of the phylogenetic tree will be discussed including proposed/necessary changes in nomenclature or family delimitation.

A) Well-established and supported clades:

Aa) Umbelopsidaceae W. Gams & W. Mey.

Species of this family were thought to belong most likely to the Mortierellales, rather than to the Mucorales, mainly because of the highly reduced columella (nearly ‘acolumellatae’, Fig. 4a) and non-mucoralean colony morphology. The colony mycelium is very dense and velvety as opposed to floccose. And unlike the colonies formed by species of Mortierella, those of Umbelopsis are reddish, brownish or ochraceous and lack a typical garlic-like odour. This distinction and a relationship to Mucorales are surveyed in detail by Meyer & Gams (2003, including a detailed description of the family). With those slight morphological differences compared to all other mucoralean fungi, this group is currently regarded as the most basal in this order. The family is a monogeneric group with a clade support (CS, bootstrap support from the Likelihood analysis and Posterior Probabilities from the Bayesian analysis) of at least 99 %, and a clear distinction from the core Mucorales (CS ≥ 99 %) (Fig. 10).

Ab) Phycomycetaceae Arx

This clade (CS ≥ 99 %) includes only two genera with different life styles. Species of Phycomyces are saprobic, whereas those of Spinellus are facultatively parasitic on the basidioma of Agaricomycotina (Fig. 2a). Species of Phycomyces are model organisms for studies of phototropism and geotropism as well as carotenoide synthesis, carotenoide degradation and zygosporogenesis.

Ac) Pilobolaceae Corda

The Pilobolaceae is one of the few families recognized from the pre-genomics era with one taxonomic change. The genus Pilaira (Fig. 5e, f), thought to be a member of the family due to morphological characteristics, was placed in the Mucoraceae and is related most closely to Helicostylum, Thamnidium, Pirella and Mucor mucedo.

Fig. 5.

Pilobolaceae. a. Substrate mycelium with trophocysts; b. sporangium of Utharomyces epallocaulus with subsporangial swelling; c. colony morphology of Pilobolus sp. on horse dung. Sporangia are phototrophic; d. sporangiophores with subsporangial swelling and the black sporangium. Light is focused through the swelling towards carotenoids at the base of the vesicle, the ocellus (orange colour); e. colony morphology of Pilaira sp. Sporangiophores are also light sensitive; f. sporangium and columellae of Pilaira sp. — Scale bar = 50 μm.

Main characteristics of this family are the formation of trophocysts (Fig. 5a), the mode of spore release and the growth on dung of herbivores and rodents (Fig. 5c, d). Both included genera possess a vesicle/swelling below the sporangium, which functions in Pilobolus during active discharge of the sporangium (Page 1964, Zycha et al. 1969). In Utharomyces (Fig. 5b) spores are passively released. Pilobolus is especially difficult to cultivate on artificial media over several generations, resulting in changes in morphology and eventually in death of the culture. Based on analyses of molecular data, only the size and shape of the sporangiospores is retained as of relevance in species delimitation since this feature is the only one that correlates with molecular phylogenies (Foos et al. 2011).

Ad) Choanephoraceae J. Schröt.

This clade (CS ≥ 99 %) includes species producing only sporangia (Poitrasia, Gilbertella; Fig. 3d, e), or also sporangiola (Choanephora, Blakeslea; Fig. 3f). Sporangia and sporangiola are produced on separate sporangiophores. The wall of the sporangium is persistent. At maturity the wall ruptures at preformed sutures to release sporangiospores with hyaline, hair-like polar appendages representing a synapomorphy of this family. The species are saprobes or fruit and vegetable inhabiting parasites, sometimes occurring as major post-harvest pathogens in tropical and subtropical regions (Fig. 2c). The newly introduced subfamilies Gilbertelloideae (MycoBank IF550022) and Choanephoroideae (MycoBank IF550021) are distinguished by the characters of the zygospore, e.g. suspensors opposed or apposed, zygosporangium ornamented or smooth (Voigt 2012).

Ae) Cunninghamellaceae Naumov ex R.K. Benj.

Although this clade is highly supported (CS ≥ 99 %), it is one family that should be studied in more detail. While two recent studies dealt with the genus Cunninghamella and incorporated the largest number of isolates studied so far, the sister genera lack such a profound study. The authors evaluated all available information ranging from morphology to growth temperatures, mating experiments and molecular data (Liu et al. 2001, Zheng & Chen 2001). Currently, only Absidia and Cunninghamella are well sampled; Gongronella, and especially Hesseltinella, Halteromyces and Chlamydoabsidia, definitely need more isolates to study. Since Chlamydoabsidia is always nested within Absidia, its status as a distinct genus should be evaluated; this might also be extended to Halteromyces. The distances between sequences are very high in this family representing one of the highest variabilities when compared to other clades (Fig. 11).

Af) Lentamycetaceae K. Voigt & P.M. Kirk — MycoBank IF550009

Since the first analyses including species of the genus Lentamyces (formerly Absidia) it was obvious, that these species should be separated. And since there are no other species of the Mucorales closely related to this genus, a separate family is introduced (Voigt 2012). Species of the Lentamycetaceae (Fig. 4b, c) are homothallic and mycoparasitic, although the mycoparasitic potential of L. zychae was lost during cultivation (Zycha et al. 1969). Kwaśna & Nirenberg (2008a, b) introduced the genus Siepmannia that included the two Lentamyces species besides the new species S. pineti and S. lariceti. A correct classification of these taxa is still unclear because only ITS sequences and no living material are available from S. pineti and S. lariceti. A resampling of strains of Siepmannia is necessary to perform multilocus studies and to determine their mycoparasitic potential.

Ag) Backusellaceae K. Voigt & P.M. Kirk — MycoBank IF550011

Species included here originally were placed in the Mucoraceae or Thamnidiaceae. Like other described families once included in the Mucoraceae (e.g. Pilobolaceae, Choanephoraceae), this clade should also be distinguished from the Mucoraceae. The monogeneric Backusellaceae are characterised by transitorily recurved sporangiophores and the tendency to produce sporangiola in addition to the sporangia. Several Mucor species owning these characters were transferred to Backusella. Clade support for the Backusellaceae is ≥ 99 % (Fig. 10) and it contains three species: Backusella lamprospora, B. circina, B. recurva. The members of the Backusellaceae seem to be saprotrophs found e.g. in soil, on wood and fallen leaves (Walther et al. 2013).

Ah) Rhizopodaceae K. Voigt & P.M. Kirk — MycoBank IF550010

Like the Backusellaceae, the Rhizopodaceae forms a well-supported clade, distinct from the Mucoraceae (CS ≥ 99 %). Within this clade, a trifurcation is observed (each with a CS ≥ 99 %), with one Rhizopus microsporus-clade containing predominantely thermotolerant fungi (growth up to 45 °C), a sub-thermotolerant R. arrhizus-group (37–40 °C) and a meso-philic group containing R. stolonifer, Sporodiniella, and Syzygites. This was already observed applying morphology and growth temperatures (Schipper & Stalpers 1984), establishing a classification accepted as standard for many decades. The application of molecular data and biochemistrical features (e.g. production of lactic acids) supported those three major clades, but revealed also new/cryptic species (Abe et al. 2006, 2007). The implementation of GCPSR, including different genetic markers, resulted in the publication of a new, reliable Rhizopus classification (Abe et al. 2010). Yet, the final clustering in the Rhizopodaceae (Fig. 10) remains unresolved, because some species (R. caespitosus, R. homothallicus, R. lyococcus, R. schip-perae, R. sexualis) were not included because of missing data. But the thermotolerant species R. caespitosus, R. homothallicus and R. schipperae, seem to be closely related to the R. microsporus clade (rDNA analysis, Abe et al. 2006). In this study, R. sexualis (mesophilic) is related to R. stolonifer and R. lyococcus (mesophilic) appears as a very basal species (Abe et al. 2006). All species of the Rhizopodaceae are reported to be pathogenic to other organisms. Whereas Syzygites is a parasite of Dikarya (Kovacs & Sundberg 1999), Sporodiniella is a parasite of insect larvae (Evans & Samson 1977, Chien & Huang 1997), and species of Rhizopus are pathogens of plants and opportunists of animals, including humans.

Ai) Radiomycetaceae Hesselt. & J.J. Ellis & Saksenaeaceae Hesselt. & J.J. Ellis

The Radiomycetaceae contains only one genus with three species (Benny & Benjamin 1991). Radiomyces is coprophilous and pathogenic to mice (experimental infections, Kitz et al. 1980). The unispored or multispored sporangia are produced on pedicels, which originate from a vesicle. The Saksenaeaceae contain two genera, Saksenaea and Apophysomyces are saprobic in soil and compost. Some species are also known to infect animals and humans (Álvarez et al. 2010a, b).

B) Moderately supported clades:

Ba) Mycotyphaceae Benny & R.K. Benj.

The Mycotyphaceae currently contains only one genus (Benny & Benjamin 1976). Although the inclusion of adjacent species is proposed (Voigt 2012), the results of molecular phylogenetics are still controversial (Fig. 10). Furthermore, CS is ≥ 99 % for Mycotyphaceae, but strong support for the separation from Mucoraceae is only given for Bayesian analysis (CS ≥ 90 %). Although molecular distances (Fig. 11) of Mycotypha are similar to those of the Mucoraceae, the Mycotyphaceae is maintained as the sister family to Mucoraceae also because of the exceptional sporangiophores bearing terminal, elongate, cylindrical vesicles (Fig. 4k). The unispored sporangiola are of two types, an inner layer that consists of globose spores and an outer layer of spores that are either obovoid or more or less cylindrical.

Bb) Lichtheimiaceae Kerst. Hoffm., G. Walther & K. Voigt & Syncephalastraceae Naumov ex R.K. Benj.

Species of the genus Absidia growing well at elevated temperatures were transferred to the genus Lichtheimia based on both molecular and physiological data (Hoffmann et al. 2007, 2009). Lichtheimia has appeared as a well-supported sister taxon to Dichotomocladium in many phylogenetic analysis (e.g. O’Donnell et al. 2001, White et al. 2006) requiring an emendation of the Lichtheimiaceae. Dichotomocladium has been included in the Chaetocladiaceae (Benny & Benjamin 1993) based on morphological structures such as sterile spines, unispored sporangiola and branched, tree-like sporangiophores (Fig. 4d–f, l, m). Molecular data, however, revealed that these morphological features are of no phylogenetic significance. A shared feature of Lichtheimia and Dichotomocladium is their tolerance of higher temperatures. Species of Lichtheimia are consistently able to grow at and above 37 °C (Hoffmann et al. 2007), the species of Dichotomocladium tolerate 35 °C and some species, namely D. hesseltinei, D. floridanum and D. robustum are even able to grow at 37 °C (unpubl. data). The subfamilies Lichtheimioideae (MycoBank IF550086) and Dichotomocladioideae (MycoBank IF550087) are proposed for the Lichtheimiaceae (Voigt 2012). Based on a smaller set of sequences a third subclade within the Lichtheimiaceae was suggested: namely the Rhizomucoroideae (MycoBank IF550085) (Voigt 2012) but this classification could not be verified in this study.

Syncephalastrum (Syncephalastraceae) is the only genus in the Mucorales producing sporangiola with the spores arranged in a linear series (merosporangia, Fig. 4g). Whether other genera (e.g. Protomycocladus) should be included in this family needs to be studied in more detail because of the low phylogenetic branch support (Fig. 10), leaving Syncephalastrum the only genus in this family. The final position of Protomycocladus could not be resolved unquestionable due to low branch support in this study as well as other publications (e.g. O’Donnell et al. 2001, Voigt & Wöstemeyer 2001, White et al. 2006, Walther et al. 2013).

Closely related to the families, Lichtheimiaceae and the Syncephalastraceae, are three additional clades: i) Protomycocladus faisalabadensis; ii) Rhizomucor/Thermomucor; iii) Fennellomyces/Circinella/Thamnostylum/Zychaea/Phascolomyces. Clades i) and ii) include thermotolerant species with growth temperature maxima at 45 °C for Protomycocladus (Schipper & Samson 1994), and thermophilic species with growth temperature maxima at 55–57 °C for Rhizomucor (de Hoog et al. 2000) or above 60 °C for Thermomucor (Subrahamanyam et al. 1977). Clade iii) contains species that are predominantly mesophilic, not growing at elevated temperatures. Furthermore, this clade is characterised by circinate (strong or less pronounced) elements in the sporangiophores (Fig. 4p, q).

For a reliable placement of clades i–iii, in relation to the Lichtheimiaceae and Syncephalastraceae, additional data are needed, since the relationships of the former clades are not significantly supported in any published analyses. Therefore, these clades gain the status incertae sedis till their relationships could be solved unambiguously.

In order to test the taxonomic stability in the newly delimitated Lichtheimiaceae, ITS2 sequences of all isolates were searched for compensatory base changes (CBC) as indicators for species boundaries. A comprehensive study on CBC suggests that with a reliability of 93.11 % one CBC is present in two specimens belonging to two different species. But the lack of CBCs does not indicate that two specimens do belong to the same species (Müller et al. 2007). Applying CBC analyses to several clades within the Lichtheimiaceae/Syncephalastraceae, CBC is widely concordant with species concepts in Rhizomucor (Fig. 12), Lichtheimia (Fig. 13, except L. corymbifera and L. ornata), Dichotomocladium (Fig. 14), Zychaea and Thamnostylum (Fig. 15).

Fig. 12.

CBC analyses of ITS2 sequences from the genus Rhizomucor. Numbers of detected CBCs are given.

Fig. 13.

CBC analyses of ITS2 sequences from the genus Lichtheimia. Numbers of detected CBCs are given.

Fig. 14.

CBC analyses of ITS2 sequences from the genus Dichotomocladium. Numbers of detected CBCs are given.

Fig. 15.

CBC analyses of ITS2 sequences from the clade Circinella / Phascolomyces / Zychaea / Fennellomyces / Thamnostylum. Numbers of detected CBCs are given.

There are few species in the analyses which could not be clearly separated from others, which is due to the lack of CBCs (e.g. Dichotomolcadium hesseltinei and D. floridanum, Fennellomyces heterothallicus, Thamnostylum repens). However, no CBCs at all were detected in the genera Syncephalastrum and Circinella showing that CBC analyses cannot be used generally as a tool for species recognition in Mucorales. CBC analyses between different genera remains difficult if not impossible (especially in the ancient clades of the Mucorales) due to highly diverse ITS2 sequences and thus secondary structure. If differing too much, no comparison of the secondary structure is possible, which results in no detectable CBCs. CBC analyses are in parts suitable for distinguishing species that are highly similar in their morphology (e.g. Lichtheimia ramosa and L. corymbifera) and could assist in supporting molecular phylogenies.

Bc) Mucoraceae Dumort.

The Mucoraceae is undoubtly the largest family and presumably the most derived in the Mucorales (Fig. 10). Traditionally all species lacking features for classification within any other family where assigned to the Mucoraceae making the family polyphyletic. This study has circumscribed a monophyletic Mucoraceae with highly diverse features that characterise different species and genera. All species are saprobes except Dicranophora, Parasitella and Chaetocladium which are facultative mycoparasites (Dicranophora on Agaricomycetes, Parasitella and Chaetocladium on Mucorales). A few species are also described as opportunistic pathogens causing deep and systemic mycoses. Species are either homothallic or heterothallic, the zygospores form a warty to smooth zygosporangial wall with naked (without appendages) opposed suspensors. Sporangia are borne on branched or unbranched, sometimes phototrophic sporangiophores, sporangiola are rare and the sporangia are ± lageniform, ± apophysate and columellate.

SUMMARY AND CONCLUDING REMARKS

Traditional classification in Mucorales was done, as in all Eumycetes, mainly by using morphological characters. Already eleven years ago large deficiencies in the morphology-based system were revealed by molecular data. The distinctly extended dataset of the current study gives now a clearer picture of the family structure in the Mucorales. Our phylogeny based on four markers and contains 14 clades that we interpret as families: 1) Umbelopsidaceae; 2) the newly erected monogeneric Lentamyetaceae; 3) Syncephalastraceae presumably including Protomycocladus; 4) Lichtheimiaceae containing Lichtheimia and Dichotomocladium; 5) Phycomycetaceae; 6) Saksenaeaceae; 7) Radiomycetaceae; 8) Cunninghamellaceae inclusively Absidia s.str.; 9) the newly erected monogeneric Backusellaceae; 10) Pilobolaceae; 11) the newly erected Rhizopodaceae including the genera Rhizopus, Sporodiniella and Syzygites; 12) Choanephoraceae; 13) Mycotyphaceae; and 14) Mucoraceae. Most of these family clades were well supported. Only the delimitation between the Mucoraceae and the Mycotyphaceae as well as the Lichtheimiaceae and the Syncephalastraceae could not be defined doubtlessly, few subclades are classified as incertae sedis. The Mucoraceae, Mycotyphaceae and Cunninghamellaceae involve several taxonomic deficiencies and a detailed study of the phylogenetic relationships in these families is needed.