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Mycologia, 95(3), 2003, pp. 474-479.
© 2003 by The Mycological Society of America

Primers are designed for amplification and direct sequencing of ITS region of rDNA from Myxomycetes


María P. Martín 1
Carlos Lado

     Real Jardín Botánico, C.S.I.C., Plaza de Murillo 2, 28014 Madrid, Spain

Steinar Johansen

     Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Four new primers were designed, based on comparison of Physarum polycephalum sequences retrieved from Genbank (primers PHYS-5 and PHYS-4) and our own sequences (primers PHYS-3 and PHYS-2), to amplify the ITS regions of rDNA, including the 5.8S gene segment from Lamproderma species. Sequencing analysis shows that Lamproderma contains ITS1-5.8S-ITS2 regions of approximately 900 bp, which is similar in size to most eukaryotes. However, the corresponding region in another common myxomycete, Fuligo septica, is more than 2000 bp due to the presence of large direct-repeat motifs in ITS1. Myxomycete rDNA ITS regions are interesting both as phylogenetic markers in taxonomic studies and as model sequences for molecular evolution.

Key words: DNA amplification, Lamproderma, ribosomal DNA, sequencing


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Myxomycetes, or plasmodial slime molds, are eukaryotic organisms characterized by two morphologically distinct assimilative stages. One of the stages consists of uninucleate amoebae with or without flagella, and the other consists of a distinctive multinucleate structure, the plasmodium, and a static reproductive phase, the sporophore, which is minute (0.1–2 mm) (Lado and Pando 1997Citation). Relationships among members of the myxomycetes, as well as the other two groups of slime molds (dictyostelids and protostelids), are not well defined, and morphological and biochemical data have not provided conclusive evidence to support one phylogenetic tree (Rusk et al 1995Citation). Based on small-subunit ribosomal-DNA (SSU rDNA) sequence phylogeny, myxomycetes are included in the Protozoa (Cavalier-Smith 1993Citation). However, only one myxomycete species (Physarum polycephalum Schwein.) was included in the analysis. Based on gene analysis of the elongation factor EF-1{alpha}, Baldauf (1999)Citation concluded that the clade formed by Physarum, Dictyostelium and Planoprotostelium is the sister group of Animalia and Fungi. Furthermore, it was stressed that analyses based on any single molecule appear to be inaccurate in reconstructing all higher-order taxonomic relationships. In general, literature on the origin and evolution of myxomycetes, based on molecular methods, is scarce. The primary problem is the difficulty of isolating DNA from a single sporophore in sufficient quantity and quality to amplify and obtain sequences from the target regions. DNA extractions from myxomycetes have been done from plasmodia in culture (Rusk et al 1995Citation, Baldauf and Doolittle 1997Citation). However, the plasmodium of many species remains unknown, or the plasmodia do not grow well and form no sporophores, which makes it impossible to establish the identity of the species. The two objectives of this study were to develop an easy and reliable method to isolate DNA from, a single sporophore for amplification and sequencing and to develop primers by using published sequence data from conserved rDNA genes of related species. By this approach, it would be possible to create a sequence database from single sporophore to identify the myxomycetes and to establish the relationship with its plasmodial phase. Three species from the genus Lamproderma (Stemonitales) were chosen for this study. Lamproderma species previously have not been included in DNA sequencing studies, despite the fact that they represent a significant taxonomic problem among myxomycetes (Kowalski 1970Citation, 1975Citation). Significant morphological variability is observed among Lamproderma species in extreme climatic habitats, such as those near melting snow in alpine areas (nivicolous species). Such climate conditions might include strong, rough and fast changes in temperature, humidity or UV radiation during fructification.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Myxomycete isolates – Specimens were obtained from wood or plant material as denoted in Table I. The specimens are: Lamproderma atrosporum Meyl. (MA-Fungi 47896; MA-Fungi 47897; MA-Fungi 47898; MA-Fungi 47899), L. ovoideum Meyl. (MA-Fungi 47900) and L. sauteri Rostaf. (MA-Fungi 47894; MA-Fungi 47895).


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  		TABLE I. Specimens studied

 
SEM technique – The critical-point dried technique was used in scanning electron microscopy (SEM) preparations, and the specimens were examined by a Jeol T 330 A scanning electron microscope at 10–15 kV.

DNA extractions – DNA was extracted using the methods of Whiting et al (1997), modified according to Martín and Winka (1997), and a E.Z.N.A Fungi DNA miniprep kit (Omega Biotech), following the instructions of the manufacturer but without adding RNase and ß-mercaptoethanol to the lysis buffer. In both protocols, an overnight incubation was done in the lysis buffer. DNA was resuspended in 100 µL prewarmed sterile water (FLUKA, Ref. 95305).

DNA amplification and sequencing – DNA amplification was performed with two protocols: a) standard procedure described in White et al (1990) in a total reaction volume of 20 µL and b) Ready-To-Go® PCR Beads (Amersham-Pharmacia Biotech) in individual reactions to a final volume of 25 µL, as described in Winka et al (1998)Citation. The primers ITS1F, ITS5, ITS1, ITS4, ITS3 and ITS2 (Gardes and Bruns 1993Citation, White et al 1990) were tested to use in PCR amplifications of the ITS regions, including the 5.8S rDNA. However, no PCR products were obtained due to differences between the primers and the target regions. Four new primers were designed, based on comparison of P. polycephalum sequences retrieved from GenBank (primers PHYS-5 and PHYS-4) and our own sequences (primers PHYS-3 and PHYS-2) to amplify ITS regions of rDNA included the 5.8S (Table II). Thirty-five cycles were conducted in a PE-9700 thermocycler: 94 C for 1 min, 52 C for 2 min, 72 C for 3 min, with a final extension at 72 C for 10 min. PCR products were separated on 2.0% agarose gels, stained with ethidium bromide and viewed under UV light. Amplification products were cleaned with the E.Z.N.A. Clean kit (Omega Biotech), and both strands were sequenced separately with primers PHYS-5, PHYS-4, PHYS-3 and PHYS-2 at the Automatic Sequencing Service (CIB-CSIC, Madrid).


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  		TABLE II. Primers designed in this study

 
Sequence alignment and phylogenetic analysis – Sequence NavigatorTM Sequence Comparison for pairwise comparisons and SEQAPP software for multiple sequences were used to search for the best alignment. Where ambiguities in the alignment occurred, the alignment chosen was the one generating the fewest potentially informative characters. Alignment gaps were marked "–" and unresolved nucleotides or unknown sequences were indicated with "N". Parsimony and maximum-likelihood analyses were performed with the computer program PAUP 4.0b* (Phylogenetic Program Using Parsimony) of Swofford (1996)Citation. Branch robustness was estimated by bootstrap analysis (Felsenstein 1985Citation) of 10 000 heuristic replicates using the fast stepwise-addition option.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The capillitium of L. atrosporum, as seen by SEM (Fig. 2A, C), has slightly expanded tips and is attached to fragments of the peridium in a funnel shape. In L. ovoideum (Fig. 2E) and L. sauteri (Fig. 2G), the peridium does not remain attached to the capillitium, the tips are acute, sometimes bifurcate and without expansions. The spores of L. atrosporum are subreticulate with some dispersed warts or reticulate when observed by SEM (Figs. 2A, B), but in some collections (MA-Fungi 47899) the reticulum is very well developed (Fig. 2D) with a more open mesh and prominent muri with some holes; this ornamentation is defined by Rammeloo (1975) as "reticulum formed by muri and muri perforati". In L. ovoideum and L. sauteri, the spores are densely baculate (Figs. 2F, H), many bacula then become slightly pilate, and occasionally in L. ovoideum some bacula can be fused.



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  		FIG. 2. Scanning-electron micrograph of Lamproderma species. A–B: Lamproderma atrosporum (MA-Fungi 47897). A. Capillitium, the tips expanded into a funnel-shape and attached to the peridium. B. Spore subreticulate with some dispersed warts. C–D: L. atrosporum cf. (MA-Fungi 47899). C. Capillitium, the tips expanded into a funnel-shape and attached to the peridium. D. Spore reticulate. E–F: L. ovoideum (MA-Fungi 47900). E. Capillitium, the tips acute and not attached to the peridium. F. Spore densely baculate, some bacula fused. G–H: L. sauteri (MA-Fungi 47894). G. Capillitium, the tips are scanty, flexuose, acute and not attached to the peridium. H. Spore densely baculate or slightly pilate. Scale bars: A, C, E, G = 10 µm, B, D, F, H = 5 µm

 
The best PCR products were obtained from DNA isolated with E.Z.N.A. Fungal DNA miniprep kit (Omega Biotech, Doraville, USA) (Martín and García-Figueres 1999Citation), which yielded a DNA total concentration of 2–20 ng/µL, and using Ready-To-Go® PCR Beads (Amersham-Pharmacia Biotech) (Winka et al 1998Citation).

The new primers were used to sequence both strands from species of the genus Lamproderma. ITS-1 and ITS-2 were found to be approximately 325 bp and 400 bp in size, respectively (http://www.fagmed.uit.no/info/imb/amb/), which is similar to most other eukaryotes analyzed.

Sequences have been lodged in the EMBL database with accession numbers: L. atrosporum (AJ302665, MA-Fungi 47896; AJ302666, MA-Fungi 47897; AJ302667, MA-Fungi 47898; AJ302668, MA-Fungi 47899), L. ovoideum (AJ302669, MA-Fungi 47900) L. sauteri (AJ302663, MA-Fungi 47894; AJ302664, MA-Fungi 47895); Fuligo septica (AJ312113).

The alignment of ITS rDNA sequences of Lamproderma species produces no ambiguous regions and 1005 characters were included in the analyses. The alignment is available in TreeBASE http://herbaria.harvard.edu/treebase/. Table III shows the uncorrected "p" distance matrix. From the characters, 158 were parsimony informative. Three most-parsimonious trees (MPT) were obtained by an exhaustive search (tree length = 271; consistency index CI = 0.9779; retention index RI = 0.9653; rescaled consistency index = 0.9439). The strict consensus tree is shown in Fig. 1. Similar tree topology was generated from maximum-likelihood analysis (not shown) under Felsenstein (1985)Citation settings. Both analyses agree, placing L. sauteri in different clades from L. atrosporum and L. ovoideum.


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  		TABLE III. Uncorrected ("p") distance matrix (PAUP* 4.0) between Lamproderma collections (MA-Fungi) mentioned in this study

 


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  		FIG. 1. Strict-consensus tree of three MPTs using PAUP 4.b* under exhaustive search based on 158 informative characters. Bootstrap values are indicated above branches. Lamproderma atrosporum, L. ovoideum and L. sauteri

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The reported primers also are compatible to rDNA ITS regions of more distantly related myxomycetes.

Lamproderma ITS sequences did not contain any obvious repetitive features. The 5.8S segment, which is an integrated region of the large subunit (LSU) rDNA, was found to be almost identical among the different Lamproderma species and 86%, 83% and 81% similar to 5.8S rDNA of the Physarales myxomycetes Didymium iridis (Johansen et al 1992Citation), P. polycephalum (Otsuka et al 1983Citation), and Fuligo septica, respectively. Some myxomycetes belonging to the order Physarales appear to contain unusually large ITS regions. ITS-1 in both F. septica and D. iridis (Johansen et al 1997Citation) are approximately 1.5 kb in size. Both ITS-1 regions consist of a complex, but unrelated, pattern of direct repeat motifs. Sequence and repeat organization of F. septica ITS-1 is shown at http://www.fagmed.uit.no/info/imb/amb/ and contains three different direct-repeat motifs, named A (26 bp), B (ca 95 bp) and C (ca 235 bp), with copy numbers of 3, 5 and 2, respectively. Individual repeats are not 100% identical to each other. In D. iridis, we found that ITS-1 repeat-copy numbers were fixed within a particular natural isolate or strain (Johansen et al 1997Citation), somewhat similar to nuclear microsatellites, and probably informative in evaluating relationships among closely related species or strains.

The four L. atrosporum collections do not form a monophyletic clade because one of the collections (MA-Fungi 47899) forms a group with L. ovoideum, with a high bootstrap value. As shown in Fig. 2 and mentioned in Table I, L. atrosporum collections that form a clade have subreticulate spores, whereas the collection not included in the clade has reticulate spores. According to Clark and Haskins (1998)Citation, the observed morphological differences in other myxomycetes are related to the reproductive systems expressed. Here, a biological species appears to have larger spores than those from an apomitic isolate. Clark (2000)Citation considers myxomycete morphospecies, in general, to be a complex of heterothallic sexual isolates and apomitic independent lines, allowing independent evolution, which thus could result in the accumulation of morphological variations. Because Lamproderma species cannot be grown in culture, we do not have data concerning the reproductive systems of collections in this study. However, the differences, observed both microscopically and in comparing the sequence of ITS rDNA, suggest that L. atrosporum MA-Fungi 47899 is an independent taxon from L. atrosporum s.e. Based on DNA isolate from a single sporophore, sequences of the ITS regions, including the 5.8S rDNA, have been obtained from a number of myxomycetes to create a sequence database.


   ACKNOWLEDGMENTS
 
We thank M. Schnittler for helpful advice during the elaboration of this work and K. Haugli for technical assistance in sequencing F. septica rDNA. This work has been financially supported by DGES, under the research project Flora Micológica Ibérica PB98-0538-C04-01 and the postdoctoral grant from the Comunidad de Madrid to MPM. Thanks to A. A. Diaz, G. Porras and S. Carbajo (Automatic Sequencing Service, CIB-CSIC, Madrid).


   FOOTNOTES
 
1 Corresponding author. E-mail: maripaz{at}ma-rjb.csic.es Back

Accepted for publication November 25, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Baldauf SL., 1999 A search for the origins of animals and fungi: comparing and combining molecular data. Amer Nat 154:S178-S188[Medline]

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Cavalier-Smith T., 1993 Kingdom Protozoa and its 18 Phyla. Microbiol Rev 57:953-994[Abstract/Free Full Text]

Clark J., 2000 The species problem in the Myxomycetes. Stapfia 73:39-53

———, Haskins EF., 1998 Heterothallic mating systems in the Echinostelium complex. Mycologia 90:382-388

Felsenstein J., 1985 Confidence limits on phylogenesis: an approach using the bootstrap. Evolution 39:783-791

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Johansen S, Elde M, Vader A, Haugen P, Haugli K, Haugli F., 1997 In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis. Mol Microbiol 24:737-745[Medline]

———, Johansen T, Haugli F., 1992 Extrachromosomal ribosomal DNA of Didymium iridis: sequence analysis of the large subunit ribosomal RNA gene and sub-telomeric region. Curr Genet 22:305-312[Medline]

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Lado C, Pando F., 1997 Flora micológica Ibérica. Vol. 2. Myxomycetes, I. Ceratomyxales, Echinosteliales, Liceales, Trichiales. J. Cramer. Madrid, Berlin, Stuttgart

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———, Winka K., 2000 Alternative methods of extracting and amplifying DNA from lichens. Lichenologist 32:189-196

Otsuka T, Nomiyama H, Yoshida H, Kukita T, Kuhara S, Sakaki Y., 1983 Complete nucleotide sequence of the 5.8S rRNA gene of Physarum polycephalum: its significance in the gene evolution. Proc Natl Acad Sci USA 80:3163-3167[Abstract/Free Full Text]

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