Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies.

羊草遗传多样性分析和赖草属的分子系统发育

羊草（Leymus chinensis（Trin.）Tzvel.或Aneurolepidium chinense Trin.），隶属禾本科（Gramineae），大麦族（Hordeae），小麦亚族（Triticieae），赖草属（Leymus），兼具重要的生态价值和经济价值。面向国家重大需求，本论文选择羊草作为主要研究对象，开展了两方面的研究工作，即我国羊草种质资源的遗传多样性评价和赖草属物种的系统发育分析。 羊草是一种多年生根茎型禾草，根据叶色可以划分为黄绿型和灰绿型两种生态型。其分布范围横穿亚欧草原的东部，包括朝鲜和蒙古的西部，以及西伯利亚的西北部，集中分布于我国的东北部。羊草是我国典型草原植物群落中的优势种。它可以在多种土壤和气候条件下正常生长，如松嫩平原、内蒙古草原和黄土高原。从另一个角度讲，不同类型的生境也造就了羊草丰富的遗传多样性。 自上世纪90年代中期开始，我们陆续从我国东北部6个省市收集了293份羊草种质，包括205份灰绿型羊草和88份黄绿型羊草。经过连续3年的观测记录，通过37个重要农艺性状，对293份羊草的遗传多样性进行了评估。依据10个质量性状和27个数量性状，统计了羊草不同性状和不同地域羊草的Shannon遗传多样性指数，同时还使用了主成分分析和通径分析做了相关统计。结果显示：（1）与黄绿型羊草相比，灰绿型羊草具有更高的遗传变异（P<0.05）。结合这两个趋异型羊草的分布范围，可以得出两种类型羊草之间存在稳定的遗传差异;（2）通径分析显示，羊草营养生长性状和遗传多样性两者的组合效应可以解释羊草生殖特性中20.6％的遗传变异;（3）在124-128ºE这一区域，羊草的遗传多样性指数最高（H＝2.252），表明这一地区具有最丰富的羊草种质资源。 赖草属（Leymus Hochst.）是一个异源多倍体属，约有34个物种，该属未知基因组的起源一直争论不休。其倍性范围从四倍体（2n=4x=28）、八倍体（2n=8x=56）一直到十二倍体（2n=12x=84）。新麦草属（Psathyrostachys Nevski）只含有Ns一个基因组，约有9个物种。这两个属都是多年生牧草，具有抗旱、抗病和耐盐碱等生物学特性。 应用核糖体ITS（Internal Transcribed Spacer）序列和叶绿体trnL-F序列，我们对13个赖草属物种、小麦族18个属（40份二倍体材料）、以及Elymus californicus和Bromus catharticus，共计57份材料进行了系统发生分析。ITS序列分析表明，赖草属分别与新麦草属和小麦族中一个未知属在进化上具有紧密的关系。ITS谱系树表明赖草属内部存在大量的分化，以及赖草属物种具有多次起源的特征。trnL-F序列分析表明，赖草属物种的母本，部分来自Ns基因组，部分来自Xm基因组，这可能与赖草属物种的地理分布有关，分布于亚欧大陆的物种其母本是新麦草属，而分布于北美的大部分物种其母本是Xm基因组。trnL-F序列分析还表明，E. californicus和赖草属未知的基因组具有紧密的关系。以上研究结果表明：（1）从分子层面证明，赖草属的未知基因组并非来自薄冰草属，或是一个修正的新麦草基因组，其基因组组成应是NsNsXmXm;（2）赖草属物种的母本，部分来自Ns基因组，部分来自Xm基因组，这可能与赖草属物种的地理分布有关;（3）E. californicus的母本是Xm基因组，父本是Ns基因组，该物种应从披碱草属转移至赖草属。

大白花杜鹃菌根真菌多样性的研究

杜鹃花属(Rhododendron L.)植物分布广泛，研究发现所有的杜鹃花科植物都能形成一种特殊的菌根——杜鹃花类菌根(Ericoid Mycorrhiza)。杜鹃花类菌根对杜鹃花科植物在营养胁迫的环境下生长起到重要的作用。近几年，对杜鹃花类菌根的生物学和生态功能的研究越来越重视。我国是杜鹃花属植物资源最为丰富的国家，因此研究杜鹃花属植物菌根真菌多样性，充分利用杜鹃花特有的菌根资源，促进杜鹃花迁地保护成功具有重大的意义。 本研究以分布较广并且是中国特有的杜鹃花属植物——大白花杜鹃(Rhododendron decorum Franch.)的野生植株为研究对象，应用直接扩增根中真菌ITS区的分子鉴定方法和T-RFLP(末端限制性片段长度多态性)的分析方法，来研究其菌根真菌的多样性；并结合生态化学计量学特征分析、宿主遗传相似性及其群落组成分析等内容，探讨大白花杜鹃的菌根真菌－宿主植物－根际土壤三者之间的关系。主要结果如下： (1)通过用直接扩增真菌ITS区序列，揭示了大白花杜鹃根部真菌的多样性，本研究发现，野生大白杜鹃根部的真菌种类比较丰富，至少有26个ITS-taxa，包括子囊菌和担子菌共5个真菌目：Helotiales、Lecanorales(≡Agyriales)、Onygenales、Sebacinales和Thelephorales，其中包括典型的ERM真菌——树粉孢属Oidiodendron sp.(Myxotrichaceae)真菌。另外还发现了黑色有隔内生菌(Dark septate endophyte，DSE)以及一些未命名的子囊菌。担子菌在本研究中占有较大比例，尤其是蜡壳耳菌目真菌；此外还有较典型的外生菌根真菌——革菌目真菌。 (2)大白花杜鹃野生植株与栽培植株在菌根真菌种类组成上，有一定的相似性；在忽略种源差异等条件下相较而言，前者的物种丰富度远高于后者。 (3)大白花杜鹃菌根真菌多样性和丰富度同它的根际土壤与叶片的C、N、P含量以及C/N、N/P、土壤pH值、宿主的海拔高度等都没有显著的相关关系。 (4)在大白花杜鹃的菌根真菌群落组成方面，整体上保持了相当程度的相似性，同时还保持了一定水平的差异；大白杜鹃菌根真菌的种类是丰富的，优势度指数表明其多样性水平很高。 (5)大白花杜鹃的遗传距离与其菌根真菌群落组成结构有极显著的相关关系，宿主的种内遗传差异可能对菌根真菌群落物种组成产生选择偏好。 (6)大白花杜鹃的群落组成与其菌根真菌群落组成有极为显著的关联性，伴生种的菌根类型可能会影响宿主植物菌根真菌的物种组成结构。

Molecular Phylogeny of Geographical Isolates of Bursaphelenchus xylophilus: Implications on the Origin and Spread of this Species in China and Worldwide

The genetic diversity and phylogeny of 26 isolates of Bursaphelenchus xlophilus from China, Japan, Portugal and North America were investigated based on the D2/3 domain of 28S rDNA, nuclear ribosomal Internal Transcribed Spacer (ITS) sequences, and random amplified polymorphic DNA (RAPD) analysis. The genetic diversity analysis showed that the D2/3 domain of 28S rDNA of isolates of B. xlophilus from China, Portugal, Japan and the US were identical and differed at one to three nucleotides compared to those from Canada. ITS sequences of isolates from China and Portugal were the same; they differed at one or two nucleotides compared to those of Japanese isolates and at four and 23 nucleotides compared to those front the US and Canada, respectively. The phylogenetic analysis indicated that Chinese isolates share a common ancestor with one of the two Japanese clades and that the Canadian isolates form a sister group of the clade comprised of isolates from China, Portugal,Japan, and the US. The relationship between Japanese isolates and those from China was closer than with the American isolates. The Canadian isolates were the basal group of B. xylophilus. This suggests that B. xlophilus originated in North America and that the B. xylphilus that occurs in China could have been first introduced from Japan. Further analysis based on RAPD analysis revealed that the relationship among isolates from Guangdong, Zhejiang, Shandong, Anhui provinces and Nanjing was the closest, which suggests that pine wilt disease in these Chinese locales was probably dispersed from Nanjing, where this disease first occurred in China.

Heterorhabditidoides chongmingensis gen. nov., sp nov (Rhabditida : Rhabditidae), a novel member of the entomopathogenic nematodes

During a recent soil sample survey in Eastern China, a new entomopathogenic nematode species, collected from the Chongming Islands in the southern-eastern area of Shanghai, was discovered. Morphological characteristics of different developmental stages of the nematode combined with molecular data showed that this nematode is a new genus of Rhabditidae, and described as Heterorhabditidoides chongmingensis gen. nov., sp. nov., for that it shares more morphological characteristics with heterorhabditids than with ste-inernematids. For males, the papillae formula of bursa is 1, 2, 3, 3, with constant papillae number in the terminal group, stoma tubular-shaped and about 1.5 head width; cheilorhabdions cuticularized, esophageal collar present and long, median bulb present. For infective juveniles, EP = 90 (80-105) mu m, ES = 104 (92-120) mu m, tail length = 111 (89-159) mu m, and a = 19.1 (15-21). The percentages of the nucleotides A, T, C and G in the ITS1 regions of the new species are significantly different from those of heterorhabditids and other rhabditids. Molecular phylogenetic trees based on 18S rDNA and the internal transcribed spacer (ITS) sequences data revealed that the new entomopathogenic nematode species forms a monophyletic group, which is a sister group of the clade comprised of some genera of Rhabditidae. (c) 2008 Elsevier Inc. All rights reserved.

松口蘑与假松口蘑 ITS 序列测定和分析比较

对松口蘑和假松口蘑进行ITS 序列测序，通过DNAStar 软件比较分析，发现松口蘑与 假松口蘑的5.8S rDNA 序列完全一致，ITS1 和ITS2 呈现不同程度的多态性。松口蘑ITS 序列 长度为601bp，假松口蘑ITS 序列长度为563bp。设计了扩增松口蘑和假松口蘑ITS1 的特异性 引物，能够快速地区别松口蘑与假松口蘑。

Population genetic structure of the parasitic nematode Camallanus cotti inferred from DNA sequences of ITS1 rDNA and the mitochondrial COI gene

The population genetic structure of fish parasitic nematode, Camallanus cotti, collected from the Yangtze River, Pearl River and Minjiang River in China was investigated. From these parasites, the similar to 730 bp of the first internal transcribed spacer of ribosomal DNA (ITS1 rDNA) and the 428 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene were sequenced. For the ITS1 rDNA data set, highly significant Fst values and low rates of migration were detected between the Pearl River group and both the Yangtze River (Fst = 0.70, P < 0.00001; Nm = 0.21) and Minjiang River (Fst = 0.73, P < 0.00001; Nm = 0.18) groups, while low Fst value (Fst = 0.018, P > 0.05) and high rate of migration (Nm = 28.42) were found between the Minjiang and the Yangtze rivers. When different host/locality populations (subpopulations) within each river were considered, subpopulations between the Yangtze River and Minjiang River had low Fst values (<= 0.12) and high Nm values (>3.72), while Pearl River subpopulations were significantly different from the Yangtze River and Minjiang River subpopulations (Fst >= 0.59; Nm < 1). The COI gene data set revealed a similar genetic structure. Both phylogenetic analyses and a statistical parsimony network grouped the Pearl River haplotypes into one phylogroup, while the Yangtze River and Minjiang River haplotypes formed a second group. These results suggested that the Yangtze River and Minjiang River subpopulations constituted a single reproductive pool that was distinct from the Pearl River subpopulations. In addition, the present study did not find host-related genetic differentiation occurring in the same drainage. (C) 2009 Published by Elsevier B.V.

Morphological and molecular phylogenetic analysis of two Saprolegnia sp (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates under-went repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (1) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species. (C) 2009 Published by Elsevier Ltd on behalf of The British Mycological Society.