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Culture and reproductive systems of 11 species of Mycetozoans

     Department of Biology, University of Kentucky, Lexington, Kentucky 40506

Edward F. Haskins ¹

     Department of Biology, Box 351330, University of Washington, Seattle, Washington 98195-1330

Steven L. Stephenson ²

     Department of Biology, Fairmont State College, Fairmont, West Virginia 26554

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

The culture and reproductive systems of 10 species (16 isolates) of myxomycetes and one species (one isolate) of protostelid were investigated. A single isolate of Ceratiomyxa fructiculosa was grown on agar and found to be nonheterothallic. This is the first report of spore-to-spore cultivation of this species and the first report of a reproductive system in the protostelids. Isolates of the myxomycetes Didymium dubium, Didymium iridis, Didymium vaccinum, Licea biforis, Perichaena vermicularis, Physarum gyrosum, Physarum pusillum (six isolates) and Semimorula liquescens all were nonheterothallic. This is the first report of culture and a reproductive system for D. vaccinum, the first report of nonheterothallism for S. liquescens and the second report of nonheterothallic isolates of D. dubium, Licea biforis, Perichaena vermicularis and P. gyrosum. The nonheterothallic isolate of D. iridis is one of many reported for this species, and the six nonheterothallic isolates of P. pusillum add to the seven nonheterothallic and two heterothallic isolates already known. In addition, five of the isolates of P. pusillum apparently represent a small form that is adapted to an ephemeral micohabitat, and the sixth is a yellow form of a species that is typically white. The Didymium ?ovoideum isolate and the two Physarum didermoides isolates have heterothallic reproductive systems. The D. ?ovoideum isolate is somewhat different from most isolates of this species in its morphology and reproductive system. It is not compatible with any of the heterothallic isolates of long-stalked Didymium, including the A0 biological species already determined for D. ovoideum; therefore, it is either a new biological species of D. ovoideum or a separate new species. The two heterothallic isolates of P. didermoides form a multiple allelic mating-type series with four alleles.

Key words: Ceratiomyxa, Didymium, Licea, Perichaena, Physarum, Semimorula

	INTRODUCTION

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Reproductive systems of myxomycetes have shown that a morphospecies may contain many local apomictic lines, as well as sexual isolates, and that these sexual isolates may belong to one or more biological species (Clark 1995, 2000). These biological species may be worldwide or regional in distribution, and they also may be allopatric or sympatric with respect to one another. Different morphospecies vary from all or mostly heterothallic, to all or mostly apogamic in their makeup. The existence of these diverse reproductive systems has raised questions regarding species definitions and evolutionary relationships within these complexes, which the study of additional species and isolates might help resolve. This paper brings together results from culture and reproductive studies on a number of species that, when taken together, contribute significantly to the body of information available on myxomycete reproduction and taxonomy.

	MATERIALS AND METHODS

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Sporangia derived from field collections or moist-chamber cultures served as the source for cultures (Table I). Spores from these sporangia were germinated on CM/2 agar (8 g Difco cornmeal agar and 8 g plain agar per L of distilled water), and the resulting myxamoebae were streaked out on fresh CM/2 plates. Amoebae then were re-isolated from areas of the streak free of fungal growth and cultured to produce plasmodia and new sporangia (Clark 1995). These sporangia then served as a source of material for morphological and genetic studies. Clonal amoebal populations were derived from isolated single spores and used to determine the reproductive system of the isolate. When the clonal populations produced plasmodia by themselves, the isolate was considered nonheterothallic, but, when the clones needed to be intermixed to produce plasmodia, the isolate was heterothallic. When two or more isolates of the same species were found to be heterothallic, tester clones representing the two mating types, from each isolate, then were crossed in all possible pairwise combinations to determine multiple alleles (plasmodia produced in cross combinations with several different mating types) or biological species (plasmodia not produced in crosses between mating types of different isolates). Myxamoebal isolates are maintained in the senior author's culture collection, and voucher specimens will be deposited in the Fairmont State College Herbarium, Fairmont, West Virginia.

	RESULTS

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A plasmodiocarpous species was identified tentatively as Didymium dubium, but positive identification was not possible because of the low level of calcareous deposits found in the fruiting body. This isolate (Idn 1) produced light yellowish-brown, short-lived plasmodia from single-spore derived amoebal populations, which makes it a nonheterothallic isolate. An earlier report of culture and nonheterothallism in Didymium dubium was given for the WV 1 isolate by Clark (1995) This isolate differed from the Idn 1 isolate of the present study, however, in that it produced more calcareous material on its plasmodiocarps and had a white instead of a light yellowish brown plasmodium, although the plasmodia of both isolates were short lived. As discussed by Martin and Alexopoulos (1969), these thin plasmodiocarpous sporangia may consist of a single variable species or of several closely related species, a situation that will require further study.

The Didymium iridis Wa 3 isolate has a typical sporangium with a dark orange-brown stalk and produces a brown phaneroplasmodium. Myxamoebal clones derived from isolated spores all produced plasmodia and, therefore, this isolate is nonheterothallic. The Didymium iridis Wa 3 isolate is one of many nonheterothallic isolates of this species (Clark 1995) and differs from most other isolates only in having a slightly darker stalk.

A dirty greenish-yellow phaneroplasmodium was isolated and fruited on agar to produce well-formed, long-stalked Didymium sp. sporangia. These sporangia are morphologically intermediate between Didymium ovoideum Nann.-Brem. and Didymium nigripes (Link) Fr. The long, dark red-orange stalked sporangia are attached, via a closed umbilicus, to the subglobose sporotheca, which has a hyaline to light brown peridium covered with small white calcareous crystals. The clavate to globose stalked columella is brown, the hyaline to light brown elements of the capillitium are dichotomously branched, and the light brown, 8–9 µm spores are covered with small warts. This isolate is heterothallic, segregating two different mating types in equal frequencies, but it is not compatible with any known biological species in the long-stalked Didymium group, including D. ovoideum. Therefore, this isolate might be a new species, or a species no longer recognized, such as Didymium proximum Berk. & M. A. Curtis, which currently is considered to be a synonym of D. iridis, or D. ovoideum (Berkeley 1873). Its plasmodium also resembles the greenish-yellow aquatic plasmodium of Didymium aquatile Gotts. & Nann.-Brem. (Gottsberger and Nannenga-Bremekamp 1971), but its longer stalk, more globose sporangium, and the presence of a columella would seem to separate the two taxa. Without more collections, this isolate (SC 1) cannot be accurately placed or identified, however, it is provisionally categorized as a possible variant of D. ovoideum.

An isolate (Mex 1) of Didymium vaccinum corresponded to the description for this species (Martin and Alexopoulos 1969), with a smooth, shell-like, white to pale ochraceous peridium, short-stalked to sessile, hemispheric to turbinate sporangia with a large white to ochraceous columella and dark brown warted spores. Clonal myxamoebal populations derived from isolated spores always produced bright yellow, slow-growing phaneroplasmodia. Therefore, this isolate of D. vaccinum is nonheterothallic. This is the first report of spore-to-spore culture and of a reproductive system for Didymium vaccinum. Our cultures indicate that this rather rare and unusual species has a stable morphology, although the large columella, characteristic of this species, is not present in all sporangia. Although more isolates are needed to be certain, it seems that this is a valid taxon that most likely is to be composed chiefly of a complex of nonheterothallic clonal lines.

The fructifications of the Wa 1 isolate of Licea biforis are small, yellow-brown, generally elongate, sessile sporangia that open by a longitudinal slit to release minutely roughened, yellow-brown spores. Isolated single spores produce myxamoebal populations that develop into hyaline to brown protoplasmodia without crossing; thus, this is a nonheterothallic isolate of L. biforis. This species previously has been grown in culture (McManus 1966, Wollman and Alexopoulos 1967), and Wollman and Alexopoulos (1967) also report nonheterothallism for the species. Therefore, the most likely reproductive system for this species is a number of related clonal lines.

The Idn 1 isolate of Perichaena vermicularis has a reticulate, dull orchraceous plasmodiocarp containing yellow, nearly smooth spores. Myxamoebal populations derived from single spores produce slow growing, yellowish-orange plasmodia without benefit of crossing, which indicates that this isolate of P. vermicularis is nonheterothallic. Both Ross (1967) and Keller (1971) reported that the plasmodium, of this species, is colorless and intermediate in character between the aphanoplasmodial and phaneroplasmodial types, however, in this isolate the plasmodium is pigmented and strongly tending toward the phaneroplasmodial form. Ross and his students (Ross 1967, Charvats et al 1973) also used this species in a number of sporangial developmental studies and found that their isolate was nonheterothallic (Ross 1967). Consequently, this is the second report of nonheterothallism for the species, which also may be composed of a swarm of clonal lines.

Two isolates of Physarum didermoides were investigated. The sporangia of both isolates are typical of this common, somewhat variable species, having cylindric to ovoid, white, sessile sporangia sometimes with short lax stalks. The capillitium, with angular to rounded nodes, was intermingled with large, dark, spiny spores. The Tx 1 isolate was determined to be heterothallic by Alexopoulos, and clones of the two mating types were deposited in the ATCC (22485, 22486). Clonal populations of myxamoebae derived from single spores of the CR 1 isolate also were heterothallic because they did not produce the white phaneroplasmodia until crossed among themselves. When the two mating-type tester clones of each of the two isolates were crossed together in all possible pair-wise combinations, we found that the two isolates were compatible (of the same biological species) and had multiple alleles at the mating locus. The Tex 1 mating types, therefore, were designated as A¹ (ATCC 22485) and A² (ATCC 22486) and the CR 1 mating types as A³ and A⁴. Although this species, which has a distinct sporangial morphology, has been grown in culture several times (Gray 1949, Polanowski 1973), the only mating system report is the report of nonheterothallism by Polanowski (1973). The heterothallic isolate collected by Alexopoulos, although deposited in ATTC, was never reported. The most likely reproductive system for this species is a single widespread biological species associated with a number of nonheterothallic clonal lines.

The PR 1 isolate of Physarum gyrosum has the typical massed rosette-like sporangial clusters with dense elastic capillitium of the species description (Martin and Alexlopoulos 1969), with the exception that its calcareous deposits (common in agar culture) on the peridium are light. Amoebal cultures derived from single spores produced white phaneroplasmodia without benefit of crossing, which indicates that this isolate is nonheterothallic. The only other mating system report for this fairly common and easily grown tropical species is the report of Clark and Collins (1976) on the UK 1 isolate, which also was nonheterothallic.

Five Physarum sp. isolates (CR 3, CR 4, CR 5, CR 6, Trin1) produced sporangia that formed poorly on agar, however, a few fruitings generally agreed with the description of Physarum pusillum (Martin and Alexopoulos 1969), except for their small size and pale stalks. Therefore, pending further studies, we consider that these isolates represent a small form of P. pusillum adapted to a ephemeral microhabitat, as also observed for Didymium iridis isolated from inflorescences (Clark et al 2001) and the small pale form of Arcyria cinerea (Bull.) Pers. found on aerial litter (Clark et al 2002). These isolates were nonheterothallic, and the white phaneroplasmodia were limited in size (rarely exceeding 20 mm in diam). A sixth isolate (Aust 1) of this species fruited well in culture, however, its sporangium was light-yellowish-brown instead of the typical white, and this coloration is correlated with the presence of a yellow instead of white plasmodium normally found in this species. Such variants could be the basis for Nannenga-Bremekamp's (1966) description of Physarum limonium Nann.-Brem. This report adds six nonheterothallic isolates to the seven nonheterothallic and three heterothallic isolates already known for this species (Clark 1995, Clark and Landolt 2001). Thus, P. pusillum seems to have a reproductive system consisting of a sexual core and numerous nonheterothallic clones.

The Wa 1 isolate of Semimorula liquescens produces small, hyaline, sessile sporangium containing a relatively small number of smooth hyaline spores. The Wa 1 isolate is nonheterothallic because single-spore derived amoebal populations, which displayed limited proliferation, always produced small hyaline protoplasmodia. The taxonomic position of the interesting species is not yet clear (Haskins et al 1983). If a mycetozoan, it may be a sessile protostelid or a very small myxomycete in the order Liceales. On the other hand, its phenetic similarity to a mycetozoan may be due to convergence.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The research reported herein provides additional support for the concept (Clark 2000) that myxomycete morphospecies generally consist of a core of sexually reproducing heterothallic isolates (which make up a varying proportion of the total population) and a swarm of genetically different asexual (presumably apomictic) clonal lines as shown by isozymes in Didymium squamulosum (Alb. & Schwein.) Fr. (ElHage et al 2000) and Physarum compressum Alb. & Schwein. (Irawan et al 2000). In addition, the sexual strains may be divided into a number of different allopatric or sympatric biological species. Together, these variations produce a complex set of reproductive possibilities that must affect our taxonomic concepts for this group, because the majority of the morphospecies, in reality, are complex species clusters or parts of clusters. Moreover, because most of the reproductively isolated subunits in these clusters do not correlate with the morphological variations present (Clark and Mires 1999), the only practical species concept for this group, at present, is a broad morphological definition of each species that include all of the variations found in the complex it represents. The Physarum pusillum isolates in this study provide an excellent example of this variation, with this common and widespread species encompassing both a color variant and a small ephemeral ecotype.

	ACKNOWLEDGMENTS

 
We wish to thank Mr. B. Irawan, Mr. M. Lizarrage, Dr. J. Landolt and Dr. N. McLetchie for providing us with the collections upon which much of this study was based. Some addition collections were obtained as a result of financing by the National Science Foundation (Grant DEB-9905464 to SLS).

	FOOTNOTES

 
¹ Corresponding author. E-mail: haskins{at}u.washington.edu

² Current address: Department of Biological Sciences, SCEN 632, University of Arkansas, Fayetteville, Arkansas 72701. E-mail: slsteph{at}uark.edu

Accepted for publication June 10, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Berkeley MJ., 1873 Notices of North American fungi. Grevillea 2:49-53

Charvats I, Ross I, Cronshaw J., 1973 Ultrastructure of the plasmodial slime mold Perichaena vermicularis. II. Formation of the peridium. Protoplasma 78:1-19[Medline]

Clark J., 1995 Myxomycete reproductive systems: additional information. Mycologia 87:779-786

———. 2000 The species problem in the myxomycetes. Stapfia 73:39-53

———, Collins OR., 1976 Studies on the mating systems of eleven species of myxomycetes. Amer J Bot 63:783-789

———, Landolt JC., 2001 Myxomycete biosystematics: various Didymium and Physarum species. Nova Hedwigia 73:437-444

———, Mires A., 1999 Biosystematics of Didymium: the non-calcareous, long-stalked species. Mycotaxon 71:369-382

———, Schnittler M, Stephenson SL., 2002 Biosystematics of the myxomycete Acryria cinerea. Mycotaxon 82:343-346

———, Stephenson SL, Landolt JC., 2001 Biosystematics of the Didymium iridis super species complex: additional isolates. Mycotaxon 79:447-454

ElHage N, Little C, Clark J, Stephenson SL., 2000 Biosystematics of the Didymium squamulosum complex. Mycologia 92:54-64

Famintzin A, Woronin M., 1873 Über zwie neue Formen von Schleimpilzen: Ceratium hydnoides und Ceratium poroides. Mém Acad Imp Sci St Petersburg VII 20:1-16

Gray WD., 1949 The laboratory cultivation and development of the Myxomycetes Physarella oblonga and Physarum didermoides. Ohio J Sci 57:69-73

Gottsberger G, Nannenga-Bremekamp NE., 1971 A new species of Didymium from Brazil. Proc K Ned Akad Wet C 74:264-268

Haskins EF, McGuinness MD, Berry CS., 1983 Semimorula: new genus with myxomycete and protostelid affinities. Mycologia 75:153-158

Irawan B, Clark J, Stephenson SL., 2000 Biosystematics of the Physarum compressum morphospecies. Mycologia 92:884-894

Keller HW., 1971 The genus Perichaena (Myxomycetes): a taxonomic and cultural study [Doctoral Dissertation]. Iowa City, Iowa: University of Iowa. 199 p

Martin GW, Alexopoulos CJ., 1969 The Myxomycetes. Iowa City, Iowa: University of Iowa Press. 561 p

McManus Sister MA., 1966 Cultivation on agar and study of the plasmodia of Licea biforis, Licea variabilis and Cribraria violacea. Mycologia 58:479-483

Nannenga-Bremekamp NE., 1966 Notes on Myxomycetes. XI. Some new species of Stemonitis, Comatricha, Badhamia, Physarum, Diderma and Didymium. Proc Kon Ned Akad Wet C 69:350-353

Olive LS., 1970 The Mycetozoa: a revised classification. Bot Rev 36:59-87

Polanowski FP., 1973 Quantitative cytochemical analysis of normal and selfing strains of three species of Myxomycetes [Doctoral Dissertation]. University Park, Pennsylvania: Penn State Univ. 108 p

Ross IK., 1967 Growth and development of the myxomycete Perichaena vermicularis. I. Amer J Bot 54:617-625

Wollman C, Alexopoulos CJ., 1967 The plasmodium of the myxomycete Licea biforis in agar culture. Mycologia 59:423-430

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Clark, J. Articles by Stephenson, S. L. Search for Related Content PubMed Articles by Clark, J. Articles by Stephenson, S. L. Agricola Articles by Clark, J. Articles by Stephenson, S. L.