黏菌主要类群营养体阶段发育特点及系统发育关系的研究
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摘要
黏菌是一类特殊的真核生物,广泛分布于自然界,其生活史十分特殊,同时具有动物和真菌的特征。在营养生长阶段,产生无细胞壁的原生质团,进行蠕动及摄食行为(类似动物)；在繁殖阶段,产生具有明显细胞壁的孢子和特定的子实体(类似真菌)。目前黏菌归属于原生动物,但是黏菌究竟是与真菌的亲缘关系近,还是与原生动物的亲缘关系更近,始终是生物分类学者热衷讨论的一个课题。本研究通过对黏菌主要类群营养阶段的发育特点及系统发育关系的探讨,从宏观到微观,从个体发育到系统发育关系,为揭示黏菌的起源与演化提供理论依据。
本研究对长春地区的黏菌进行调查并采集到标本共计2000余份,通过形态特征鉴定为5目7科23属59种。团毛菌科、绒泡菌科和发网菌科所包含的种最多,共计49种,占长春地区黏菌总种数的83.1%。团网菌属、绒泡菌属和发网菌属所包含的种最多,共计28种,占总种数的47.5%。从基质种类来看,生长在腐木上的种类最多,而且基质对黏菌的生长有影响,可以初步判断黏菌对于基质具有一定的选择性。团网菌属,绒泡菌属和发网菌属的黏菌可以适应多种基质生长,营养需求宽泛,生长范围广。
对所采集的绒泡菌目8种黏菌原质团进行了液体培养研究,根据单因素实验确定了各种的适宜培养基和培养条件。最适宜培养基为燕麦液体培养基。利用正交实验,得到了液体培养的最优条件：淡黄绒泡菌(Physarum melleum) pH9.0,26.5℃,110r/min;全白绒泡菌(P. globuliferum)pH 9.0,26.0℃,1OOr/min;扁绒泡菌(P. compressum)pH4.0,25.5℃, 100r/min;灰绒泡菌(P.cinereum) pH8.0,25.5℃,100r/min;小绒泡菌(P.pusillum) pH 5.0,26.5℃,110r/min;两瓣绒泡菌(P.bivalve) pH 6.0,25.5℃,90r/min;灰堆钙丝菌(Badhamia cinerascens) pH 7.0,26.0℃, 100r/min;黑柄钙皮菌(Didymium nigripes) pH 9.0,25.0℃,110r/min。绒泡菌目的最适的温度在25.0～26.5℃,最适pH在4.0～9.0,转速在100-110r/min之间。为黏菌原质团的液体培养奠定了基础。
对绒泡菌属4种黏菌的原质团进行液体培养,将得到的原质团使用液体培养和有饲培养相结合的方法对原质团进行了诱导孢子果的研究。使原质团在饥饿条件下,通过对光照和温度的调节,在燕麦固体培养基上获得了淡黄绒泡菌(Physarum melleum)孢子果和菌核、全白绒泡菌(P. globuliferum),扁绒泡菌(P. compressum)和小绒泡菌(P. pusillum)的孢子果。产生孢子果的最佳条件为：淡黄绒泡菌26℃,6000 1x,全白绒泡菌24℃,60001x,扁绒泡菌20℃,6000 1x,小绒泡菌22℃,6000 1x；获得菌核的最佳条件为：淡黄绒泡菌22℃,3000 1x。在燕麦液体培养基上,获得了淡黄绒泡菌(Physarum melleum)菌核,全白绒泡菌(P. globuliferum)、扁绒泡菌(P. compressum)和小绒泡菌(P. pusillum)的子实体。产生孢子果的最佳条件为：全白绒泡菌20℃,6000 1x,扁绒泡菌20℃,6000 1x,小绒泡菌22℃,6000 1x；获得菌核的最佳条件为：淡黄绒泡菌22℃,6000 1x。通过固体培养与液体培养条件的比较可以看出,在液体培养基中形成孢子果的温度要比固体培养时的温度低些,且在固体培养基上可以获得较多黏菌孢子果。
在此基础上,首次完成了小绒泡菌从孢子到孢子的全部生活史过程。孢子在松针营养液中26℃下1～2d萌发,10～12d长出原质团,20d左右青白色的原质团布满整个培养皿,将带有大量原质团的培养皿放入培养箱低温培养(20℃),并以散射光刺激孢子果的形成。成熟的小绒泡菌孢囊会在成熟后20天左右开裂,释放出的孢子具有繁殖能力,可继续萌发并形成原质团。完成从孢子到孢子的过程至少需要44d。
形态学特征在某种程度上也会反应系统进化的趋势,如原质团类型,子实体形态,孢丝和石灰结特征等。本实验结合黏菌营养阶段的发育特征,对采集到的黏菌进行核糖体小亚基、线粒体16S rDNA基因和延长因子基因的系统发育关系比较分析,探讨属及属以上分类单元的进化关系。
测定了无丝菌目、团毛菌目、绒泡菌目和发网菌目的5种黏菌(灯笼菌,金孢盖碗,煤绒菌,淡黄绒泡菌,草生发网菌)的核糖体小亚基的部分序列,并结合Genebank下载的29条序列进行了比对。用NJ法对34条序列构建系统发育树。从系统发育关系可以看出,孢子外生的鹅绒菌目单独在一个分支位于系统树的底部,绒泡菌目仍然表现出一目二科的分类体系,孢子暗色的绒泡菌目和发网菌目互为姊妹群,团毛菌目比发网菌目和绒泡菌目较早分化出来,绒泡菌目为较高级的分类类群。
对4目5科11属12种黏菌线粒体16S rDNA基因的部分序列进行了测定,并在Genebank上进行了注册,注册号为JF339204至JF339215。利用软件MEGA 5.0构建MI系统树和NJ系统树,可以看出12种黏菌被分为3大组：鹅绒菌科(Ceratiomyxaceae)、绒泡菌科(Physaraceae)和钙皮菌科(Didymiaceae)、团毛菌科(Trichiaceae)和发网菌科(Stemonitaceae)。利用形态学与系统学相结合的方法对其进行分析得出：孢子外生的鹅绒菌目独立在一个分支上,位于系统树的底部。显型原质团(绒泡菌目)与非显型原质团(发网菌目,团毛菌目及鹅绒菌目)处于不同的分支上。孢丝密集的发网菌科和团毛菌科聚在一起。这一结果与核糖体小亚基序列分析略有不同。
此外还对4目11种黏菌的肌动蛋白基因和延长因子基因的部分序列进行了测定及相关蛋白质序列的分析,共得到13条序列,其中肌动蛋白基因4条,延长因子基因9条,对延长因子基因的9条序列构建NJ系统树,绒泡菌目的进化最快,且分为2大类,高杯菌属,煤绒菌属和针箍菌属为一类,绒泡菌属和白柄菌属为一类。对肌动蛋白基因的4条序列进行ORFs的寻找及翻译,并进行BLASTp比对,其蛋白家族属于肌动蛋白家族。同时对黄垂网不同生长时期(幼孢囊和成熟孢囊时期)的肌动蛋白基因的部分序列进行比对,同源性在99%以上,具有的氨基酸数量相同,都为19种,没有色氨酸。丝氨酸含量最高为10.476(平均值),组氨酸含量最低为0.9524(平均值)。在不同生长时期的黄垂网的氨基酸含量,除丙氨酸,苯丙氨酸,异亮氨酸和脯氨酸外,其他氨基酸含量均相同。
在此基础上,对2目5种黏菌的联合基因序列(SSU基因、16S rDNA基因和EF-1c基因)以及4目6种黏菌的联合基因序列(SSU基因和16S rDNA基因)系统发育树进行了分析,从联合数据集构建的系统发育树来看,绒泡菌目的进化速率较快,结合形态学,孢子外生的鹅绒菌目单独在一个分支上,具有表面网结构的发网菌目和团毛菌目互为姊妹群。与前面单个基因所构建的发育树相比,在一些亲缘关系较近黏菌的分类地位明显地提高了分支的支持率。
以上研究为黏菌原质团的液体培养奠定了基础,并对黏菌系统起源和演化的基础资料进行了补充,由于实验材料有限,有些类群有待进一步进行研究,以揭示黏菌的属间及种间的亲缘关系。
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.
本研究对长春地区的黏菌进行调查并采集到标本共计2000余份,通过形态特征鉴定为5目7科23属59种。团毛菌科、绒泡菌科和发网菌科所包含的种最多,共计49种,占长春地区黏菌总种数的83.1%。团网菌属、绒泡菌属和发网菌属所包含的种最多,共计28种,占总种数的47.5%。从基质种类来看,生长在腐木上的种类最多,而且基质对黏菌的生长有影响,可以初步判断黏菌对于基质具有一定的选择性。团网菌属,绒泡菌属和发网菌属的黏菌可以适应多种基质生长,营养需求宽泛,生长范围广。
对所采集的绒泡菌目8种黏菌原质团进行了液体培养研究,根据单因素实验确定了各种的适宜培养基和培养条件。最适宜培养基为燕麦液体培养基。利用正交实验,得到了液体培养的最优条件：淡黄绒泡菌(Physarum melleum) pH9.0,26.5℃,110r/min;全白绒泡菌(P. globuliferum)pH 9.0,26.0℃,1OOr/min;扁绒泡菌(P. compressum)pH4.0,25.5℃, 100r/min;灰绒泡菌(P.cinereum) pH8.0,25.5℃,100r/min;小绒泡菌(P.pusillum) pH 5.0,26.5℃,110r/min;两瓣绒泡菌(P.bivalve) pH 6.0,25.5℃,90r/min;灰堆钙丝菌(Badhamia cinerascens) pH 7.0,26.0℃, 100r/min;黑柄钙皮菌(Didymium nigripes) pH 9.0,25.0℃,110r/min。绒泡菌目的最适的温度在25.0～26.5℃,最适pH在4.0～9.0,转速在100-110r/min之间。为黏菌原质团的液体培养奠定了基础。
对绒泡菌属4种黏菌的原质团进行液体培养,将得到的原质团使用液体培养和有饲培养相结合的方法对原质团进行了诱导孢子果的研究。使原质团在饥饿条件下,通过对光照和温度的调节,在燕麦固体培养基上获得了淡黄绒泡菌(Physarum melleum)孢子果和菌核、全白绒泡菌(P. globuliferum),扁绒泡菌(P. compressum)和小绒泡菌(P. pusillum)的孢子果。产生孢子果的最佳条件为：淡黄绒泡菌26℃,6000 1x,全白绒泡菌24℃,60001x,扁绒泡菌20℃,6000 1x,小绒泡菌22℃,6000 1x；获得菌核的最佳条件为：淡黄绒泡菌22℃,3000 1x。在燕麦液体培养基上,获得了淡黄绒泡菌(Physarum melleum)菌核,全白绒泡菌(P. globuliferum)、扁绒泡菌(P. compressum)和小绒泡菌(P. pusillum)的子实体。产生孢子果的最佳条件为：全白绒泡菌20℃,6000 1x,扁绒泡菌20℃,6000 1x,小绒泡菌22℃,6000 1x；获得菌核的最佳条件为：淡黄绒泡菌22℃,6000 1x。通过固体培养与液体培养条件的比较可以看出,在液体培养基中形成孢子果的温度要比固体培养时的温度低些,且在固体培养基上可以获得较多黏菌孢子果。
在此基础上,首次完成了小绒泡菌从孢子到孢子的全部生活史过程。孢子在松针营养液中26℃下1～2d萌发,10～12d长出原质团,20d左右青白色的原质团布满整个培养皿,将带有大量原质团的培养皿放入培养箱低温培养(20℃),并以散射光刺激孢子果的形成。成熟的小绒泡菌孢囊会在成熟后20天左右开裂,释放出的孢子具有繁殖能力,可继续萌发并形成原质团。完成从孢子到孢子的过程至少需要44d。
形态学特征在某种程度上也会反应系统进化的趋势,如原质团类型,子实体形态,孢丝和石灰结特征等。本实验结合黏菌营养阶段的发育特征,对采集到的黏菌进行核糖体小亚基、线粒体16S rDNA基因和延长因子基因的系统发育关系比较分析,探讨属及属以上分类单元的进化关系。
测定了无丝菌目、团毛菌目、绒泡菌目和发网菌目的5种黏菌(灯笼菌,金孢盖碗,煤绒菌,淡黄绒泡菌,草生发网菌)的核糖体小亚基的部分序列,并结合Genebank下载的29条序列进行了比对。用NJ法对34条序列构建系统发育树。从系统发育关系可以看出,孢子外生的鹅绒菌目单独在一个分支位于系统树的底部,绒泡菌目仍然表现出一目二科的分类体系,孢子暗色的绒泡菌目和发网菌目互为姊妹群,团毛菌目比发网菌目和绒泡菌目较早分化出来,绒泡菌目为较高级的分类类群。
对4目5科11属12种黏菌线粒体16S rDNA基因的部分序列进行了测定,并在Genebank上进行了注册,注册号为JF339204至JF339215。利用软件MEGA 5.0构建MI系统树和NJ系统树,可以看出12种黏菌被分为3大组：鹅绒菌科(Ceratiomyxaceae)、绒泡菌科(Physaraceae)和钙皮菌科(Didymiaceae)、团毛菌科(Trichiaceae)和发网菌科(Stemonitaceae)。利用形态学与系统学相结合的方法对其进行分析得出：孢子外生的鹅绒菌目独立在一个分支上,位于系统树的底部。显型原质团(绒泡菌目)与非显型原质团(发网菌目,团毛菌目及鹅绒菌目)处于不同的分支上。孢丝密集的发网菌科和团毛菌科聚在一起。这一结果与核糖体小亚基序列分析略有不同。
此外还对4目11种黏菌的肌动蛋白基因和延长因子基因的部分序列进行了测定及相关蛋白质序列的分析,共得到13条序列,其中肌动蛋白基因4条,延长因子基因9条,对延长因子基因的9条序列构建NJ系统树,绒泡菌目的进化最快,且分为2大类,高杯菌属,煤绒菌属和针箍菌属为一类,绒泡菌属和白柄菌属为一类。对肌动蛋白基因的4条序列进行ORFs的寻找及翻译,并进行BLASTp比对,其蛋白家族属于肌动蛋白家族。同时对黄垂网不同生长时期(幼孢囊和成熟孢囊时期)的肌动蛋白基因的部分序列进行比对,同源性在99%以上,具有的氨基酸数量相同,都为19种,没有色氨酸。丝氨酸含量最高为10.476(平均值),组氨酸含量最低为0.9524(平均值)。在不同生长时期的黄垂网的氨基酸含量,除丙氨酸,苯丙氨酸,异亮氨酸和脯氨酸外,其他氨基酸含量均相同。
在此基础上,对2目5种黏菌的联合基因序列(SSU基因、16S rDNA基因和EF-1c基因)以及4目6种黏菌的联合基因序列(SSU基因和16S rDNA基因)系统发育树进行了分析,从联合数据集构建的系统发育树来看,绒泡菌目的进化速率较快,结合形态学,孢子外生的鹅绒菌目单独在一个分支上,具有表面网结构的发网菌目和团毛菌目互为姊妹群。与前面单个基因所构建的发育树相比,在一些亲缘关系较近黏菌的分类地位明显地提高了分支的支持率。
以上研究为黏菌原质团的液体培养奠定了基础,并对黏菌系统起源和演化的基础资料进行了补充,由于实验材料有限,有些类群有待进一步进行研究,以揭示黏菌的属间及种间的亲缘关系。
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.
引文
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