同宗配合蜜环菌与欧美异宗配合蜜环菌狭义种的分子系统学关系*

从亚洲、欧洲和北美收集18个蜜环菌狭义种菌株,PCR扩增其rDNA的IGS和ITS区域,用AluI、HaeIII、HinfI和TagI四种限制性内切酶进行酶切,同时用随机扩增微卫星(RAMS)多态性,对蜜环菌狭义种的进行了遗传多样性和分子系统学分析。结果蜜环菌狭义种的IGS和ITS-RFLP类型比以前报道的更多,亚洲、欧洲和北美菌株都具有比较明显的特征片段,通过ITS和IGS图谱可将三个大陆的菌株区分开,其中IGS-HinfI图谱能完全区分3个大陆的菌株。RFLP数据的系统学分析表明,该种存在明显的大陆遗传分化,亚、欧、北美的群体分属三个相互独立的进化系,北美群体IGS变异程度较小,ITS变异程度极高;而欧洲群体IGS的变异程度较大,多态性高,ITS变异程度非常小。RAMS系统分析表明,中国、日本和非洲的同宗配合蜜环菌属于一个系统发育系,该发育系同欧洲和北美的异宗配合种的两个发育系分属3个不同的独立进化分支。据此建议欧洲和北美的异宗配合蜜环菌应作为蜜环菌的两个亚种。     

同宗配合蜜环菌与欧美异宗配合蜜环菌狭义种的分子系统学关系

从亚洲、欧洲和北美收集18个密环菌狭义种菌株,PCR扩增其rDNA的IGS和ITS区域,用AluI、HaeIII、HinfI和TagI四种限制性内切酶进行酶切,同时用随机扩增微卫星（RAMS）多态性,对密环菌狭义种的进行了遗传多样性和分子系统学分析。结果密环菌狭义种的IGS和ITS-RFLP类型比以前报道的更多,亚洲、欧洲和北美菌株具有比较明显的特征片段,通过ITS和IGS图谱可将三个大陆的菌株区分开,其中IGS-HinfI图谱能完全区分3个大陆的菌株。RFLP数据的系统学分析表明,该种存在明显的大陆遗传分化,亚、欧、北美的群体分属三个相互独立的进化系,北美群体IGS变异程度较小,ITS变异程度极高;中国、日本和非洲的同宗配合密环属于一个系统发育系,该发育系同欧洲和北美的异宗配合种的两个发育系分属3个不同的独立进化分支。据此建议欧洲和北美的异宗配合密环菌应作为密环菌的两个亚种。     

中国与北美大陆的高卢蜜环菌系统发育分析

在中国和北美大陆分别收集53和15株高卢蜜环菌Armillaria gallica菌株,并用ISSR（inter-simple sequence repeat）分子标记对这些菌株进行了亲缘关系及系统发育分析。结果表明：中国和北美大陆的A. gallica因地理隔离产生明显的遗传分异,在系统发育树上分别形成了各自的进化分支;北美大陆的分支内部遗传分化尚不明显,而中国的进化分支遗传分化程度相对较大且更为古老,中国菌株可能是两个大陆A. gallica的祖先株系。     

中国与欧洲高卢蜜环菌的遗传多样性

高卢蜜环菌(Armillaria gallica)为北半球广布种,不同大陆间的菌株遗传相似性和多样性水平能反映出该种在洲际大陆尺度上的地理遗传变异关系。作者用ISSR(inter-simple sequence repeat)分子标记技术,对从中国和欧洲收集到的高卢蜜环菌79个菌株进行了遗传多样性分析。用6个ISSR引物扩增得到210个位点,其中多态性位点(频率<0.95)为202个,占96.2%,平均每个引物多态位点多达33.6个,表明ISSR标记在蜜环菌中存在较高的多态性。根据非加权类平均法(UPGMA)聚类分析,中国53个菌株中的49个在0.773的相似性水平上聚成了中国类群(China group);而欧洲26个菌株遗传分化较大,分别在0.775和0.763的相似性水平上聚成了欧洲类群A(Europe group A)和B(Europe group B);2个欧洲类群间的相似性水平仅为0.738,而欧洲类群A与中国类群间的相似性却达到了0.770;两个大陆均有少数菌株表现出较为明显的遗传分化,个别菌株的种内遗传相似性甚至低于蜜环菌种间的遗传相似性。结果表明,中欧两个大陆间的A.gallica菌株因地理隔离已经表现出明显的遗传分化,处于异域物种形成过程中;欧洲大陆的菌株遗传分化更为明显,可能是两个大陆A.gallica菌株的起源地。     

天麻栽培用蜜环菌的遗传多样性、生长和水解酶

蜜环菌Armillaria是一种广泛分布的担子菌,是栽培中药天麻所必须的共生菌。为揭示我国天麻栽培用和鄂西地区部分野生蜜环菌资源的遗传多样性和生物学特性,本文收集了9个省市EF-1-α的19个蜜环菌商品菌株和鄂西地区20株野生蜜环菌菌株,以IGS、ITS和elongation factor-1-alpha（EF-1-α）为分子标记对这些菌株进行系统发育与遗传多样性分析,并对影响与天麻共生的生物学特性（菌索发育和胞外水解酶活性）进行对比研究。结果表明,这19株蜜环菌商品菌株分别归属于2个clade。其中,有18个菌株聚在cladeⅢ,说明不同来源的蜜环菌商品菌株之间系统发育关系很近;通过对蜜环菌的系统发育、菌索生长以及胞外水解酶活性的研究,总体上商品种优于鄂西的野生蜜环菌生物种。从鄂西野生蜜环菌中筛选出4个具有潜在栽培价值的菌株。本研究为天麻栽培生产中蜜环菌菌种的选择提供参考。     

大兴安岭和长白山地区蜜环菌生物种的研究

从大兴安岭和长白山采集到３０号蜜环菌标本,选其中有代表性的１０个号的担子果获得单孢株。交配试验表明每一担子果都具有双因子异宗配合系。不同子实体交配型之间交配结果表明,在大兴安岭和长白山地区蜜环菌目前至少存在５个生物种,分别称为生物种Ａ、Ｂ、Ｃ、Ｄ和Ｅ。将这５个生物种的单孢菌种与欧洲５个蜜环菌生物种的单孢菌株进行配合,生物种Ｂ与欧洲Ａ．ｇａｌｌｉｃａ,生物种Ｅ与欧洲Ａ．ｏｓｔｏｙａｅ亲和交配,因此将     

大兴安岭和长白山地区蜜环菌生物种的研究

从大兴安岭和长白山采集到30号蜜环菌(Armillaria mellea)标本,选其中有代表性的10个号的担子果获得单孢株。交配试验表明每一担子果都具有双因子异宗配合系。不同子实体交配型之间交配结果表明,在大兴安岭和长白山地区蜜环菌目前至少存在5个生物种,分别称为生物种A、B、C、D和E。将这5个生物种的单孢菌种与欧洲5个蜜环菌生物种的单孢菌株进行配合,生物种B与欧洲A.gallica,生物种E与欧洲A.ostoyae亲和交配,因此将生物种B和E分别定为A.gallica和A.ostoyae。生物种A、C和D则不与任何欧洲生物种交配。     

中国蜜环菌的新生物种

在调查研究蜜环菌生物种的过程中,先后在东北、华北、华中和西南地区,发现了四个新的中国蜜环菌生物种,CBS F、CBS G、CBS H和CBS I。研究表明,CBS G属于典型的同宗配合种,其余三个为异宗配合。CBS H与CBS C之间存在部分互交可育,比例高达17.8%,在同一个国家的两个种之间出现如此高的互交可育比例还是首次报道。上述三个生物种同欧美的已知蜜环菌种均互交不育,为亚洲特有种。CBS I为无菌环的假蜜环菌A.tabescens (Scop. ) Emel,它与欧洲的假蜜环菌互交可育,属于同种。但同中国其它各生物种互交不育。同时讨论了蜜环菌生物种的群体观点。     

用ISSR标记研究高卢蜜环菌系统发生学的尝试

用ISSR(Inter-Simple Sequence Repeat)标记对黑龙江省牡丹江地区高卢蜜环菌的系统发生学进行了研究,用6个引物对35个菌株的DNA模板进行扩增。结果表明该地区的35个菌株分属3个不同的发育系,并且3个发育系在地理分布上是交错在一起的。同时表明ISSR标记是研究蜜环菌系统发育关系的理想的分子标记手段。     

鄂西地区蜜环菌生物种的遗传多样性研究

蜜环菌属Armillaria真菌是世界范围内广泛分布的担子菌,既是引起森林病害的重要病原菌,也是天麻和猪苓生长的必须共生真菌,具有重要的经济价值。本文对我国鄂西天麻主产区即神农架林区、大老岭自然保护区、宜昌市所属的夷陵区和五峰县、恩施自治州等地进行了系统的调查采样,共获得45份蜜环菌子实体样本及其二倍体菌株,其中通过孢子印分离获得约320个单孢菌株。在此基础上,采用IGS、ITS和elongation factor-1 alpha（EF1-α）分子标记对45份二倍体菌株进行测序和系统发育分析,对有单孢株的样本采用遗传互交不育性实验进行生物种鉴定。结果表明,鄂西地区的蜜环菌分为4个clade,共有6个不同的蜜环菌生物种,分别为CBS J,CBS L,CBS N,CBS O,CBS F和Armillaria mellea,具有丰富的遗传多样性。其中,24个样本鉴定为CBS L,是鄂西地区的优势种;9个样本鉴定为CBS J;9个样本鉴定为CBS N;另有3个样本分别鉴定为CBS O,CBS F和Armillaria mellea。其中CBS L、CBS N、CBS O首次在湖北地区报道,扩大了这3个生物种的分布范围。本研究为了解鄂西地区蜜环菌的资源情况提供基础数据,对天麻生产蜜环菌菌种选育具有重要的参考价值。     

Species delimitation in the African Armillaria complex by analysis of the ribosomal DNA spacers

Thirteen Armillaria isolates, collected from various geographical areas in tropical Africa and previously characterized by cultural morphology, pairing tests and isozyme analysis, were evaluated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). DNA regions corresponding to the intergenic spacer (IGS) and internal transcribed spacer (ITS) were amplified and analyzed by restriction enzyme digestion. The IGS amplification products were about 875 bp long and uniform in length among the isolates. The amplified-ITS region showed two different lengths corresponding to two groups. The first group included the isolates believed to belong to A. mellea ssp. africana and two Kenyan isolates (K11 and K12) belonging to a yet unnamed biological species. The second group included isolates identified as A. heimii and a Tanzanian isolate (T7). Each length variant of the ITS showed distinct RFLP banding patterns. Digestion with EcoRI confirmed the two polymorphic groups while the endonucleases AluI and NdeII discriminated the A. mellea isolates from the Kenyan isolates K11 and K12. In addition, the latter enzyme showed a slight dissimilarity between the A. heimii isolates from Western and Eastern Africa (C1 and Z1). Digestion with HinfI cleaved the isolates of A. heimii into two sub-groups corresponding to the heterothallic and homothallic forms. This endonuclease also indicated that the isolate T7, originating from Tanzania, was clearly similar to the heterothallic species A. heimii. Data presented support the maintenance of three distinct species of Armillaria in tropical Africa with A. heimii as a variable species, the isolates of which were separated in accordance with their sexual system. The results indicate that PCR-RFLP can be used as a simple and speedy taxonomical tool for the ecological studies of Armillaria species.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

Evidence of natural hybridization among homothallic members of the basidiomycete Armillaria mellea sensu stricto

Populations of Armillaria mellea (Basidiomycota, Agaricales) across much of its range are heterothallic; homothallic populations occur only in Africa (A. mellea ssp. africana), China (China Biological Species CBS G), and Japan (A. mellea ssp. nipponica). Monosporous isolates of heterothallic A. mellea are haploid and their mating behaviour is consistent with the requirement of two different alleles at two mating-type loci (tetrapolar mating system) to create a diploid individual. In contrast, monosporous isolates of homothallic A. mellea are putatively diploid; they bypass the haploid phase by undergoing karyogamy in the basidium (a unique type of secondary homothallism/pseudohomothallism). In order to determine the genetic origin of this homothallism, we analyzed genetic variation of 47 heterothallic isolates from China, Europe, and North America, and 14 homothallic isolates from Africa, China, and Japan. Gene trees and mutational networks were constructed for partial mitochondrial gene ATP synthase subunit 6 (ATP6) and for the following nuclear genes: actin (ACTIN), elongation factor subunit 1-alpha (EFA), glyceraldehyde 3-phosphate dehydrogenase (GPD), and the RNA polymerase subunit II (RPB2). Homothallic isolates from Africa and Japan shared a common mitochondrial ATP6 haplotype with homothallic isolates from China, and are likely introductions. Homothallic isolates from China that shared a common mitochondrial haplotype with all European isolates did not share European nuclear haplotypes, as revealed by median-joining networks, but instead clustered with haplotypes from China or were intermediate between those of China and Europe. Such mitochondrial-nuclear discordance in homothallic isolates from China is indicative of hybridization between lineages originating from China and Europe.     

The root rot fungus Armillaria mellea introduced into South Africa by early Dutch settlers

Dead and dying oak (Quercus) and numerous other woody ornamental trees and shrubs showing signs and symptoms of Armillaria root rot were identified in the Company Gardens, Cape Town, South Africa, which were established in the mid-1600s by the Dutch East Indies Trading Company. Nineteen isolates from dying trees or from mushrooms were collected and analysed to identify and characterize the Armillaria sp. responsible for the disease. The AluI digestion of the amplified product of the first intergenic spacer region (IGS-1) of the rRNA operon of 19 isolates from the Company Gardens was identical to that of some of the European isolates of A. mellea s. s. The IGS-1 region and the internal transcribed spacers (ITS) were sequenced for some of the Cape Town isolates. Phylogenetic analyses placed the Cape Town isolates in the European clade of A. mellea, which is distinct from the Asian and North American clades of this species. Identification based on sexual compatibility was conducted using A. mellea tester strains in diploid-haploid pairings, which showed some compatibility between the Cape Town isolates and testers from Europe. Somatic compatibility tests (diploid-diploid pairings) and DNA fingerprinting with multilocus, microsatellite probes indicated that the Cape Town isolates were genetically identical and may have resulted from vegetative (clonal) spread from a single focus in the centre of the original Company Gardens (c. 1652). The colonized area is at least 345 m in diameter. Assuming a linear spread rate underground of 0.3 m/year to 1.6 m/year, the genet (clone) was estimated to be between 108 and 575 years old. These data suggest that A. mellea was introduced into Cape Town from Europe, perhaps on potted plants, such as grapes or citrus, planted in the Company Gardens more than 300 years ago.     

Biogeographical patterns in Artomyces pyxidatus

Artomyces pyxidatus (Auriscalpiaceae) is a lignicolous, coralloid basidiomycete found throughout temperate regions of the Northern Hemisphere. Previous studies established that populations from the eastern United States, Sweden, and China were conspecific based on mating compatibility and enzyme profiles. In this study, mating compatibility was extended to include collections from Russia, Costa Rica, Mexico, and Utah. The molecular diversity of A. pyxidatus was examined by DNA sequence and restriction site analyses of the nuclear ribosomal internally transcribed spacer region (ITS1-5.8S-ITS2). A phylogenetic analysis of twelve isolates based on ITS sequences revealed a broad geographical pattern in which Eurasian isolates comprise a sister clade to North American isolates. North American isolates appear to be further subdivided into northeastern and southwestern clades. A survey of 255 A. pyxidatus isolates using restriction enzymes revealed variable RFLP patterns that follow similar geographical patterns.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

Molecular phylogenetic analysis of Grifola frondosa (maitake) reveals a species partition separating eastern North American and Asian isolates

A phylogenetic analysis was performed on 51 isolates of the commercially valuable basidiomycete, Grifola frondosa (maitake), using sequences from the Internal Transcribed Spacers and 5.8S region of the nuclear ribosomal DNA (rDNA) and a portion of the β-tubulin gene. The β-tubulin gene provided more than twice as many variable characters as the ITS/5.8S regions. The isolates analyzed comprised 21 from eastern North America, 27 from Asia, one from Europe, and two of unknown geographic origin, one of which was the major US commercial production strain in use. Grifola sordulenta was used as an outgroup. Combined and separate analysis of both genes showed a partition separating Asian versus eastern North American isolates. Bootstrap analysis showed strong support for these clades in the β-tubulin data alone and in the combined data. The major commercial isolate of unknown geographic origin is apparently of Asian descent based on its grouping within the Asian clade. The single European isolate analyzed was distinct from both the eastern North American and Asian clades. These results indicate strong support for a species partition separating eastern North American and Asian isolates of G. frondosa, despite previous studies indicating no morphological distinction between them.     

Molecular phylogeny and historical biogeography of the Holarctic wetland leaf beetle of the genus Plateumaris

Leaf beetles of the genus Plateumaris inhabit wetlands across the temperate zone of the Holarctic region. To explore the phylogeographic relationships among North American, East Asian, and European members of this genus and the origin of the species endemic to Japan, we studied the molecular phylogeny of 20 of the 27 species in this genus using partial sequences of mitochondrial cytochrome oxidase subunit I (COI) and the 16S and nuclear 28S rRNA genes. The molecular phylogeny revealed that three species endemic to Europe are monophyletic and sister to the remaining 11 North American and six Asian species. Within the latter clade, North American and Asian species did not show reciprocal monophyly. Dispersal-vicariance analysis and divergence time estimation revealed that the European and North America-Asian lineages diverged during the Eocene. Moreover, subsequent differentiation occurred repeatedly between North American and Asian species, which was facilitated by three dispersal events from North America to Asia and one in the opposite direction during the late Eocene through the late Miocene. Two Japanese endemics originated from different divergence events; one differentiated from the mainland lineage after differentiation from the North American lineage, whereas the other showed a deep coalescence from the North American lineage with no present-day sister species on the East Asian mainland. This study of extant insects provides molecular phylogenetic evidence for ancient vicariance between Europe and East Asia-North America, and for more recent (but pre-Pleistocene) faunal exchanges between East Asia and North America.