| Myxomycetes (slime molds) are unique eukaryotes having both characteristics of fungi and animals. In their life cycle they have myxamoebae, swarm cells, and plasmodia like protozoa and also produce spores like fungi. Recently, they were phylogenetically included into one group of protozoa. However, myxomycetes belonged to fungi or protozoa were always the task which taxonomist discussed. Here, discussed the development of vegetative phase and phylogeny relationship. That could provide a theoretical basis for reveal the origin and evolution of myxomycete.More than 2000 specimens were collected from Changchun in 2008～2010. According to morphological classification,59 species were identified belong to 23 genus,7 families,5 orders. The species of Trichiaceae, Physaraceae and Stemonitaceae were most,49 species in all; occupied 83.1% of the total species. The speices of Arcyria, Physarum and Stemonitis were most,28 species in all, occupied 47.5% of the total species. The most species grew on decaying root, and myxomycetes have selectivity for substrates. Arcyria, Physarum and Stemonitis were adaptation to varied substrates, had broad adaptability.Cultured plasmodia of 8 Physarales species (Physarum melleum, P. globuliferum, P. compressum, P. cinereum, P. pusillum, P. bivalve, Badhamia cinerascens, and Didymium nigripes) by liquid methods, determined the suitable media and culture conditions of each species by single factor test. The suitable media was oat liquid media and the optimum conditions of P. melleum were pH 9.0,26.5℃, and 110r/min; P. globuliferum were pH 9.0, 26.0℃and 100r/min; P. compressum were pH 4.0,25.5℃and 100r/min; P. cinereum were pH 8.0,25.5℃and 100r/min; P. pusillum were pH 5.0,26.5℃and 110r/min; P. bivalve were pH 6.0,25.5℃and 90r/min; B. cinerascens were pH 7.0,26.0℃and 100r/min; D. nigripes were pH 9.0,25.0℃and 110r/min, respectively. The suitable conditions of liquid culture for Physarales were 25.0～26.5℃, pH 4.0～9.0,100～110r/min. That was provided the foundation for liquid culture of myxomycetes plasmodia.Liquid culture combined with oat-agar culture were used to culture plasmodium of Physarum in the same time.Then plasmodium was induced to obtain sporophore in lab by hunger and adjusting light (3000,6000,9000,120001x) and temperature (20,22,24,26℃). Sporophore of Physarum melleum, P. globuliferum, P. compressum and sclerotium of P. melleum were obtained from different media. On oat-agar medium, the optimum fruitification conditions of plasmodium of P. melleum were 26℃,6000 lx and formed to sclerotium at 22℃, 3000 lx. The optimum fruitification conditions of P. globuliferum were 24℃,6000 lx. The optimum fruitification conditions of P. compressum were 26℃,6000 lx. In liquid medium, the optimum cultured conditions to form sclerotium of P. melleum were 22℃,6000 lx. The optimum fruitification conditions of P. globuliferum were 20℃,6000 lx. The optimum fruitification conditions of P. compressum were 20℃,6000 lx. The optimum fruitification conditions of P. pusillum were 22℃,6000 lx. Compared agar culture with liquid culture, the temperature of agar culture was lower than liquid culture, and obtained the more sporophores.Artificial cultures of Physarum pusillum was obtained firstly and the complete life cycle was observed. Spores germinated in 2 days in nutrient solution of pine needles 26℃. Twelve days after spore inoculation, several young phaneroplasmodia were found on the oat agar, twenty days later, mass phaneroplasmodia covered the whole oat agar medium cultures and then put Petri dishes in a lower temperature incubator (20℃) with diffused light that stimulated sporophore formation. Twenty days after sporangia matured dehiscence began releasing spores with reproductive capacity. The process of spore to spore of Physarum pusillum was completed in 44 days.The characteristics of morphology reflected the trend of phylogenetic evolution, such as type of plasmodium, form of sporophores, capillitium, and lime nodes and so on. In this paper, phylogenetic analysis of myxomycetes was investigated by SSU gene,16S rDNA and EF1-a gene with their morphology, ensured the intergeneric genetic relationships.Sequenced the SSU partial sequences of Dictydium cancellatum, Perichaena chrysosperma, Fuligo septica, Physarum melleum and Stemonitis herbatica, and downloaded 29 sequences of SSU gene from Genebank, constructed the phylogenetic trees of 34 sequences based on the neighbor-joining (NJ) method. The phylogeny tree showed that Ceratiomyxales with external spores at the base of trees branched alone, dark-spore of Physarales and Stemonitales were sister group. Based on phylogenetic relationship, Trichiales were branched off earlier than Physarales and Stemonitales, Physarales was a relative developed group.Phylogenetic analysis of myxomycetes was investigated by partial 16S rDNA sequences amplified from 12 species (belonging to 5 families) firstly; the accession numbers JF339204-JF339215. Phylogenetic trees were constructed based on the neighbor-joining (NJ) and maximum-likelihood (ML) methods. The evolution trees showed that three clades: Ceratiomyxaceae, Physaraceae and Didymiaceae, Trichiaceae and Stemonitaceae. And with their morphology, the results indicated that Ceratiomyxaceae for their external spores at the base of trees separating from others, Phaneroplasmodium (Phasarales) and aphaneroplasmodium (Stemonitales and Trichiales) were not cluster together, Stemonitaceae and Trichiaceae with mass capillitium were sister group. The results different from analyzed of sequences based on SSU gene. In addition, thirteen partial sequences of 4 orders 11 species were obtained based on Actin gene and EF1-a gene, four sequences of Actin gene, nine of EF1-a gene. Phylogenetic trees of EFl-a gene were constructed based on the neighbor-joining (NJ) methods. The phylogeny tree showed that Ceratiomyxales at the base of trees branched alone, the evolutionary rate of Phasarales were quicker than others, and Phasarales were divided two classes, one included Craterium, Fuligo and Physarella, the other included Physarum and Diachea. The ORFs of four sequences of Actin gene were found, and blast the senquences the protein families were Actin superfamily. Blast the sequences of young and mature Arcyria obvelata, homology were above 99%, the numbers of amino acid were same,19 kinds in all, no Trp. The content of Ser were highest (average 10.476), His were lowest (average 0.9524). The amino acid content of young and mature Arcyria obvelata was same except Ala, Phe, Ile and Pro.Analyzed the phylogenetic trees of 2 orders 5 species of myxomycetes based on combine three gene (SSU gene,16S rDNA and EF1-a gene) and 4 order 6 species based on combine two gene (SSU gene and 16S rDNA). The phylogenetic trees were showed that the evolution rate were more quickly, and combined with their morphology, Ceratiomyxales with external spores at the base of trees branched alone, Trichiaceae and Stemonitaceae were sister group for their surface net structure. Compared with the phylogenetic trees of single gene, the support rate of each branch was increase of some species with close genetic relationship.The results established the foundation of liquid culture for plasmodium of myxomycetes, and supplied the data to systematic evolutional. However, due to the experimental material limited, some group needing to move forward research for revealed the intergeneric and interspecific evolution relationship of myxomycetes.
|
PDF Full Text Request