ABSTRACT

Mortierellales constitutes one of the largest groups of zygomycetes. Among them, some species have biotechnological significance as producers of polyunsaturated fatty acids, while others have been applied in various regio- and stereospecific biotransformations. One species, Mortierella wolfii, is reported as animal pathogen being casual agent of bovine mycotic abortion, pneumonia and systemic mycosis. The aim of the present study was to infer a comprehensive phylogeny from nuclear ribosomal as well as protein-coding sequence data. Therefore, the complete ITS region together with the LSU and SSU genes and partial regions of the EF-1α and RPB1 protein-coding genes were sequenced in a large number of type and reference strains of the genera Mortierella, Dissophora, Gamsiella and Lobosporangium. Using these data, phylogenetic analyses involving several partitioned and mixture models were performed. The resulting phylogeny substantially deviates from the traditional, morphology-based division of the order raising the need for a new classification of Mortierellales. Although Mortierellales proved to be monophyletic, the genera Dissophora, Lobosporangium and Gamsiella were found to be nested within the genus Mortierella indicating the paraphyly of the latter genus. Mortierella longicollis was found in a basal position forming a sister group of the core Mucorales. It was also observed that phenotypic traits of mortierellalean fungi strongly depend on the culturing conditions. The need to standardized descriptions for the reporting of the phenotype in taxonomic studies will also be discussed.

RESULTS

At the genus level, two large clades can be recognized, Umbelopsis (BPP: 1.00) and Mortierella (BPP: 0.99). As expected, Umbelopsis, including representatives of Micromucor, clusters with Rhizopus oryzae, a representative of the Mucorales. The genera Dissophora, Lobosporangium and Gamsiella are nested within the genus Mortierella with significant support. With the exception of Mortierella longicollis, a monophyletic Mortierellaceae can be recognized if the above–mentioned genera are included. M. longicollis is placed closest to the outgroup taxon R. oryzae (Mucorales).

Within the Mortierella clade, twelve large clades were distinguished and named after a representative or well–known species.

The /selenospora clade (BPP: 1.00) includes the M. wolfii strain CBS 614.70 and the type strain of M. selenospora.

The position of the other isolates of M. wolfii suggests that strain CBS 614.70 was misidentified, which is reinforced by the mesophilic nature of this strain with an optimal growth temperature of 24 °C.

The thermophilic M. wolfii isolates were found in the /wolfii clade (BPP: 1.00) together with M. capitata.

The /alpina clade (BPP: 1.00) contains three closely related taxa, M. alpina, M. amoeboidea and M. antarctica, all with preference for low temperatures.

The /parvispora clade (BPP: 1.00) contains six taxa, of which M. cystojenkinii, M. elongatula, M. turficola and M. pulchella form a closely related subclade.

The /verticillata–humilis clade (BPP: 1.00) includes ten taxa, among which isolates of M. verticillata and M. humilisform one species–level group with low support (BPP: 0.68) species–level group. M. minutissima and M. horticola also seem to be very closely related (BPP: 1.00).

The /strangulata clade (BPP: 1.00) contains M. rostafinskii and M. strangulata.

The eight Mortierella species in the well–defined /lignicola clade (BPP: 1.00) are distributed in five subclades where all but one are well supported. Lobosporangium transversale is also found within this clade with significant support (BPP: 1.00).

The well supported /angusta clade (BPP: 1.00) contains the two described species of the genus Dissophora.

The third non-mortierellalean species, Gamsiella multidivaricata is included in the also significantly supported /stylospora clade (BPP:0.99).

The /globulifera clade (BPP: 1.00) is closely related to the latter two clades.

The type species of the genus Mortierella, M. polycephala, is included in the /polycephala clade (BPP: 1.00), together with M. bisporalis, M. polygonia, M. indohii, M. hypsicladia and M. hyalina.

The most species–rich and heterogeneous group is the /gamsii clade (BPP: 1.00), consisting of several species and subgroups.

The majority rule consensus tree computed from 105 trees sampled after stationarity. BPP values are shown at the branches.

MATERIALS AND METHODS

Strains and cultivation. Strains involved in the study were obtained from Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands). For DNA extraction, fungal strains were cultured in malt extract broth in a rotary shaker for 3-7 days at the corresponding temperature.

DNA isolation and PCR. Genomic DNA was prepared from mycelia disrupted with pestle and mortar in liquid nitrogen and purified with the MasterPure Yeast DNA Purification Kit (Epicentre). The ITS1 and ITS2 regions, including the 5.8S rDNA were amplified by PCR using the standard primers ITS1 and ITS4. Amplification of the nrLSU and nrSSU fragments was done by using the primers LR0R-LR7 (LSU) and NS1-NS4 (SSU) (1). The protein-coding genes were amplified with the primer pairs 983F-2218R (EF-1α) and RPB1F-RPB1R (RPB1).

Phylogenetic analysis. A total of 107 strains were involved in this study. Sequence alignments were computed with ClustalX (2) and the Probalign algorithm (3). Bayesian MCMC analyses were performed on the concatenated alignment by using the parallel version of the MrBayes software (4). 107 generations were run, every 100th state being saved. For each partition, the model selected by jModelTest (5) (GTR+ Γ) was invoked. For the indel matrix, a one–state Markov model was used, with a correction for invariant sites not included in the matrix. The burn–in value was determined by checking likelihood and topological convergence. The convergence of likelihood values was checked by using Tracer 1.4 (6), while topological convergence was inspected by using AWTY (7). To obtain posterior probabilities, post–burn–in trees of the two runs were pooled and a 50% Majority Rule phylogram was generated. Clades receiving Bayesian posterior probabilities (BPPs) ≥0.95 were considered significant.

REFERENCES

1. White TJ, Bruns T et al. 1990 In: PCR Protocols Academic Press, San Diego, USA pp. 315-322.2. Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc

Bioinformatics Chapter 2: Unit 2 3. Roshan U, Livesay DR (2006) Probalign: Multiple sequence alignment using partition function posterior probabilities.

Bioinformatics 22: 2715-2721.4. Altekar, G., S. Dwarkadas, J. P. Huelsenbeck, and F. Ronquist. 2004. Parallel Metropolis Coupled Markov chain Monte Carlo

for Bayesian phylogenetic inference. Bioinformatics 20(3):407-415. 5. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25: 1253-1256.6. Rambaut A, Drummond A (2008) Tracer v 1.4.1. Software distributed by the authors at http://beast.bio.ed.ac.uk/.7. Wilgenbusch JC, Warren DL, Swofford DL (2004) AWTY: a system for graphical exploration of MCMC convergence in

Bayesian phylogenetic inference. http://cebcsitfsuedu/awty.

The SOILMAP project is co-financed by the European Union through the Hungary-Romania Cross-Border Co-operation Programme 2007-2013 (HURO/0901/058/2.2.2). This research was partly supported by an international cooperation grant of the German and Hungarian Research Foundations (DFG Vo 772/9–1 and OTKA NN75255).

Mortierellaceae, a typical fungal group of soil samples

Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary

Tamás Petkovits, Stella A. Kovács, Ildikó Nyilasi, László G. Nagy,Csaba Vágvölgyi and Tamás Papp