Genetic variability and taxonomic position of ectomycorrhizal fungus Pisolithus from India

Eight ectomycorrhizal fungal isolates of Pisolithus associated with Eucalyptus species in different parts of India were collected and the genetic variability of these isolates was studied by ITS-RFLP and ITS sequencing. All the isolates showed same RFLP patterns with each restriction enzyme, indicating all these isolates of Pisolithus are of the same genotype. The sequence comparison of KN6 of Indian isolate showed high sequence similarities with the isolates of Pisolithus associated with Eucalyptus from Australia. Phylogeny analysis showed that all the isolates compared in this study clustered into four main groups The Indian isolate (KN6) clustered with Pisolithus albus isolates of group I, which are associated with Eucalyptus. These results suggested that Pisolithus isolates found in India are P. albus.     

Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests

Basidiocarps of Pisolithus associated with indigenous ( Afzelia quanzensis Welw.) and introduced ( Pinus caribaea Mor. and Eucalyptus camaldulensis Dehnh.) hosts in the lowland forests of the Coast Province of Kenya are morphologically distinct. Genetic variability among 52 Pisolithus basidiocarps, collected beneath the various host plants, was examined based on sequence polymorphism within the internal transcribed spacer (ITS) and intergenic spacer (IGS1) of ribosomal DNA genes. Variability in ITS and IGS1 sequences indicated that the three host-associated morphotypes were genetically different. Consensus trees generated by bootstrap analysis of sequence data of Pisolithus isolates from Australia and Kenya are polyphyletic and strongly suggest that the three different morphotypes/genotypes present in Kenya represent separate biological species. In addition, our data indicate that little genetic exchange occurs in silva between these species.     

衣藻属的系统发育分析——基于形态形状和nrDNA ITS序列

通过实验分析莱茵衣藻 ( Chlamydomonas reinhardtii) 1个种和互连网获得衣藻属 1 5个种及丝藻属 1个种 ( Ulothrix zonata) ,共 1 7个种的 nr DNA ITS序列 ,并以 U.zonata为外类群 ,采用计算机分析软件包对其进行分析及构建分子系统发育树图。同时以 1 2个传统分类性状 ,对此 1 6种衣藻构建数据矩阵 ;以 U.zonata动孢子的相应性状为外类群原始性状 ,用Wagner法在计算机上对其进行分枝分析 ;然后比较并分析分子系统树和表征性状分支分析树的异同。初步尝试以 ITS分子序列系统发育分析作为传统性状分析的补充来研究衣藻种间的亲缘关系。     

基于形态特征和ITS序列对7个鹅膏菌属菌株的分类鉴定

以采自浙江省丽水地区的7个鹅膏菌属菌株作为研究材料,在基于形态特征进行初步鉴定的基础上,对7种鹅膏菌的rDNAITS区段进行克隆测序和序列特征比较分析。进一步对ITS序列进行核酸序列数据库GenBank同源性检索比对,将从GenBank检索获得的9个最相似物种的ITS序列连同7种鹅膏菌的ITS序列一起作系统发育分析。结果表明:基于ITS序列对f6、f9和f493个菌株的分子鉴定支持了基于形态特征的鉴定结果,对f5的分子鉴定不支持形态鉴定的结果,f8为鹅膏菌属内某种,f66为鹅膏菌属内某种,并与Amanitafulva,A.atrofusca,A.orientifulva3种鹅膏菌的亲缘关系较近,f7与另外6种鹅膏菌的亲缘关系相差甚远。研究结果提示基于分子水平上的ITS序列分析不能单方面作为大型真菌分类鉴定的可靠依据,可以作为基于传统形态学分类鉴定的辅助参考依据。     

中国秋海棠属 (秋海棠科) 植物的DNA条形码评价

秋海棠属植物种类繁多,形态变异多样,导致种类的系统放置混乱,近缘种类鉴定困难。利用DNA条形码实现物种快速准确的鉴定技术具有不受形态特征约束的优势,为秋海棠属植物的分类鉴定提供了新的方法。本研究选择4个DNA条形码候选片段（rbcL,matK,trnH psbA,ITS）对中国秋海棠属26种136个个体进行了分析。结果显示：叶绿体基因rbcL,matK和trnH psbA种内和种间变异小,对秋海棠属植物的鉴别能力有限;ITS/ITS2种内和种间变异大,在本研究中物种正确鉴定率达到100%/96%,可考虑作为秋海棠属DNA条形码鉴定的候选片段。研究结果支持中国植物条形码研究组建议将核基因ITS/ITS2纳入种子植物DNA条形码核心片段中的观点。     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.     

Molecular Variation and Distribution of Anopheles fluviatilis (Diptera: Culicidae) Complex in Iran

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.     

Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia

The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species. Received September 15, 2000; accepted December 15, 2000     

Molecular variation in the Postia caesia complex

DNA sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) and small-subunit of mitochondrial ribosomal DNA (mt-rDNA) were obtained from 12 different collections initially identified as either Postia caesia or P. subcaesia based on morphological criteria. Sequences of ITS from British collections separate into three clear groups, each with identical sequences, regardless of the lignicolous host and distribution. These British collections can be distinguished morphologically as two groups, (a) thick and larger basidiomata (1.5-5.0x2.0-6.0x3.0-15 cm) with a strigose to tomentose pileus and (b) thin and smaller basidiomata (0.5-2.0x1.0-2.5x1.5-4.0 cm) with a smooth pileus. The former were all collected from hardwoods and the latter from both hardwoods and coniferous woods. Group (a) corresponds to one of the sequence groups, but group (b) displays two different sequences. Two collections from Norway, one from each of the morphological groups, exhibit further sequence variation within the ITS regions, although closer to those of British group (b). Representative sequences of mt-rDNA from each of the three British ITS sequence groups remain distinct, but those from the two Norwegian collections, however, are identical to one of the British groups. Further comparison of basidiospore size revealed no clear distinction among these groups, although the ratio of spore length to spore width is generally greater in group (a). Although there is no clear separation of these collections into two species, there is a clear tendency of variation at both morphological and molecular levels, among them. Differences in morphology and DNA sequences do not warrant species recognition, but do demonstrate high variability within the species complex.     

分离自冬虫夏草可培养真菌的多样性研究

冬虫夏草是生长于青藏高原的一种名贵中药材。天然冬虫夏草及其微环境中生活着多种真菌。作者使用常规分离培养方法对冬虫夏草的真菌区系进行研究。从天然冬虫夏草的子座、菌核和菌膜3个部位共分离到572个真菌菌株,并根据形态特征将大部分菌株鉴定到37个不同的属。这些菌株经SSCP(single-strand conformation polymorphism)分析后,再根据nrDNAITS序列的相似性(以97%为阈值)共区分出92种不同的分类单元(operational taxonomic unit,OTU)。其中,属于子囊菌的菌株数及OTU数均比接合菌和担子菌多。从菌膜分离的菌株数及OTU数都明显多于子座和菌核。分离自子座的优势真菌是产黄青霉Penicillium chrysogenum,而分离自菌核和菌膜的优势真菌均为玫红假裸囊菌Pseudogymnoascus roseus。尚未最终鉴定的部分真菌可能为新的真菌物种。     

帘蛤科贝类rDNA内转录间隔区序列的研究

根据18SrDNA、5．8SrDNA和28SrDNA保守序列设计引物,应用聚合酶链式反应（PCR）扩增了文蛤（Meretrix meretrix L．）、青蛤（Cyclina sinensis G）、硬壳蛤（Mercenaria mercenaria L．）和江户布目蛤（Protothaca jedoensis L．）4种帘蛤科贝类的第一内转录间隔区（ITS1）和第二内转录间隔区（ITS2）序列,并进行了测序。结果表明,文蛤、青蛤、硬壳蛤和江户布目蛤的ITS1扩增产物大小分别为978bp、663bp、757bp和942bp,GC含量分别为61．55％、60．78％、62．48％和64．86％～64．97％,其中ITS1序列长度分别为900bp、585bp、679bp和864bp,是迄今已报道双壳贝类中变化范围最大的,GC含量分别为61．67％、61．03％、63．03％和65．51％～65．62％,江户布目蛤种内ITS1序列有个体差异;ITS2扩增产物大小分别为644bp、618～620bp、593bp和513～514bp,GC含量分别为61．18％、61．29％～61．81％、62．73％和61．48％61．60％,其中ITS2序列长度分别为412bp、386～388bp、361bp和281～282bp,GC含量分别为65．29％、65．21％～66．06％、67．87％和67．38％～67．62％,青蛤和江户布目蛤种内ITS2序列有个体差异。4种蛤ITS1和ITS2序列种间差异很大,有明显的长度多态性,ITS2种间序列相似度73．0％～89．1％,与ITS1的种间序列相似度48．7％～81．5％相比略高。此外,在4种蛤ITS1和ITS2序列中各发现2个与rRNA加工有关的保守区。通过对ITS1和ITS2序列的组装获得了4种蛤5．8SrRNA基因完整序列,序列长度都是157bp,GC含量57．96％～58．60％,4种蛤5．8SrRNA基因相对保守,种间序列差异度0-6．0％,共有10个变异位点,其中转换4处,颠换6处,硬壳蛤和江户布目蛤5．8SrRNA基因序列完全相同。以ITS2序列（包含5．8SrRNA和28SrRNA基因部分序列）为标记,调用北极蛤科的Arctica islandica相应序列数据作外群,构建了帘蛤科贝类的系统发育树,其拓扑结构显示江户布目蛤与硬壳蛤亲缘关系最近,青蛤与其他3物种的亲缘关系最远。     

Wax plants disentangled: a phylogeny of Hoya (Marsdenieae, Apocynaceae) inferred from nuclear and chloroplast DNA sequences

Hoya (Marsdenieae, Apocynaceae) includes at least 200 species distributed from India to the Pacific Islands. We here infer major species groups in the genus based on combined sequences from the chloroplast atpB-rbcL spacer, the trnL region, and nuclear ribosomal DNA ITS region for 42 taxa of Hoya and close relatives. To assess levels of ITS polymorphism, ITS sequences for a third of the accessions were obtained by cloning. Most ITS clones grouped by species, indicating that speciation in Hoya usually predates ITS duplication. One ITS sequence of H. carnosa, however, grouped with a sequence of the morphologically similar H. pubicalyx, pointing to recent hybridization or the persistence of paralogous copies through a speciation event. The topology resulting from the combined chloroplast and nuclear data recovers some morphology-based sections, such as Acanthostemma and Eriostemma, as well as a well-supported Australian/New Guinean clade. The combined data also suggest that morphological adaptations for ant-symbiosis evolved at least three times within Hoya.     

A new species of Gauthieromyces and range extensions for other Harpellales to India

One species of each of five genera of Harpellales--Gauthieromyces, Harpella, Legeriomyces, Stachylina and Zygopolaris--are reported for the first time from India. Gauthieromyces indicus is described from the hindguts of Baetis sp. nymphs. The new species is distinguished from the only other species, G. microsporus Lichtw., by having a greater number of and smaller generative cells per fertile branchlet with smaller trichospores that bear fine, entangled appendages. Occurrence of these fungi in India supports the observation that some genera and species of Trichomycetes are widespread but with apparent disjunct distributions.     

Molecular identification of Pilobolus species from Yellowstone National Park

Pilobolus, a widely distributed coprophilous fungus, grows on herbivore dung. Species of Pilobolus traditionally are described with imprecise morphological characteristics potentially leading to misidentification. In this study we used PCR analysis of taxonomically informative sequences to provide more consistent species identification from isolates obtained in Yellowstone National Park. We collected Pilobolus park-wide from six taxa of herbivores over 9 y. Multiple transfers of single sporangium isolates provided pure cultures from which DNA was extracted. Sequence analysis of internal transcribed spacer (ITS) regions of DNA that code for rRNA genes were used to distinguish among similar species. We describe several species of Pilobolus associated with herbivores in various habitats, including two species not previously reported, P. heterosporus and P. sphaerosporus. Our results show that phylogenetic species identification of Pilobolus based on sequence analysis of pure culture isolates provides a more reliable means of identifying species than traditional methods.     

Complete mitochondrial genomes of Bos taurus and Bos indicus provide new insights into intra-species variation, taxonomy and domestication

The taurine and zebuine cattle breeds comprise the majority of the world cattle population but their taxonomic status is still controversial. The two forms of cattle are currently classified as Bos taurus and Bos indicus species and are differentiated primarily by the presence or absence of a hump. However, these two species hybridize readily, producing fully fertile offspring. We have determined and analyzed complete B. taurus and B. indicus mitochondrial genome sequences to investigate the extent of sequence divergences and to study their taxonomic status by molecular dating. The sequences encompassed 16,338 and 16,339 nucleotides, respectively, and differed at 237 positions. Estimated divergence times indicated that the two cattle lineages separated 1.7-2.0 million years ago. Combined phylogenetic analyses of 18 new and 130 previously reported extant B. taurus and B. indicus control region sequences with data from 32 archaeological specimens of the extinct wild aurochs (Bos primigenius) identified four major maternal lineages. B. primigenius haplotypes were present in all but the B. indicus lineage, and one B. taurus sequence clustered with B. primigenius P haplotypes that were not previously linked with domestic cattle. The B. indicus cluster and a recently reported new B. primigenius haplotype that represents a new lineage were approximately equidistant from the B. taurus cluster. These data suggest domestications from several differentiated populations of B. primigenius and a subspecies status for taurine (B. primigenius taurus) and zebuine (B. primigenius indicus) cattle.     

First record of Aphelinus paramali Zehavi and Rosen 1989 (Hymenoptera,Aphelinidae), parasitoid of Aphis pomi de Geer (Hemiptera,Aphididae) in Iran,and its phylogenetic position based on sequence data of ITS2 and COI genes

The occurrence of Aphelinus paramali (Zehavi & Rosen) (Hym., Aphelinidae) was evidenced from North east Iran, in association with Aphis pomi (de Geer). This species is reported from Iran for the first time, and A. pomi is introduced as a new host for this parasitoid. Detailed morphological characters were studied with scanning electron microscopy (SEM) photographs. Also, sequences of ribosomal internal transcribed spacer 2 (ITS2) and cytochrome oxidase subunit I (COI) genes were used for determining species boundaries and comparing with other Aphelinus species. Different results were obtained in phylogenetic analysis of these two regions. Analysis of COI gene supported the closer relationship of this species with Aphelinus abdominalis. This is the first data about comprehensive characterization of a parasitic wasp using morphological characters, SEM and two‐locus information from Iran.     

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

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.     

Study on the DNA Barcoding of Genus Ligularia Cass. (Asteraceae)

DNA barcoding is a new technology which can identify species rapidly based on short and standardized DNA sequences. Ligularia, a genus of Asteraceae with about 140 species, exhibits high morphological and ecological diversity, which makes the classification and species delimitation difficult, especially in the cases of closely related taxa. In this study, we tested four DNA core barcoding regions (ITS, matK, psbA trnH and rbcL) in 144 samples representing 35 species of Ligularia. The results revealed that the chloroplast regions (matK, psbA trnH and rbcL) have extremely low species identification rate due to low interspecific variation. Conversely, ITS sequence showed higher species identification rate (60%) and could discriminate the species which are difficult to identify. The combination of these four gene fragments did not improve the ability of species discrimination.