Speciation ofBotryobasidium subcoronatum (Basidiomycota) collected in Taiwan: Morphology, mating tests, and molecular data

Taiwan is a phytogeographically isolated area, with a rate of ca. 40% endemism in higher plants. In this study, the strong tendency towards genetic isolation and speciation ofBotryobasidium subcoronatum (Basidiomycota) from Taiwan is evaluated in comparison with European specimens. Mating tests amongB. subcoronatum specimens collected in Taiwan and Europe were performed. Mon-mon and di-mon matings confirm different intersterility groups or biological species. The biological species concept was compared with results from morphological comparison and molecular data from mtSSU rDNA and the nuclear ITS2 region. Three intersterility groups have been detected comprising European and Taiwanese strains from temperate and subtropical areas. MtSSU rDNA and the ITS2 rDNA sequences confirm consepecifity ofB. subcoronatum.     

Presence of a short repeat sequence in internal transcribed spacer (ITS) 1 of the rRNA gene of <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae) from geographically different populations in Asia

Nucleotide sequences of the internal transcribed spacer 1 (ITS1)–5.8S–ITS2 region of the nuclear ribosomal RNA gene were determined in the white-backed planthopper (WBPH) Sogatella furcifera (Horváth) to detect molecular variation among regional populations in Asia. We analyzed 932 sequences from 172 individuals (4–9 clones per individual) of 33 populations collected in 1987–2008 from six countries, Japan, China, Taiwan, Vietnam, Philippines, and Papua New Guinea. WBPH showed intra-individual variation in ITS1, which is mainly attributable to the frequency (0–10) of the 66-bp repeat sequence in ITS1. Among the examined clones, the sequences of 5.8S were mostly identical and those of ITS2 were similar. A single planthopper had a maximum of 6 different variants in the number of ITS1 repeats, suggesting highly varied repeat numbers in individual planthoppers. The ITS1 with four repeats was the most frequently (64%) detected. Such a repeat was not observed in two other economically important planthopper species, Nilaparvata lugens (Stål) and Laodelphax striatellus (Fallén). The ITS nucleotide sequences in the WBPH populations in Asia were genetically close and some variations in the sequences were not related to regional populations, indicating that the nucleotide sequences of the ITS region are not useful for geographical discrimination of the WBPH. This closeness seems to be caused by long distance migration and genetic exchange among populations.     

Characterization and Sequence Variation in the rDNA Region of Six Nematode Species of the Genus Longidorus (Nematoda)

Total DNA was isolated from individual nematodes of the species Longidorus helveticus, L. macrosoma, L. arthensis, L. profundorum, L. elongatus, and L. raskii collected in Switzerland. The ITS region and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The sequences obtained were aligned in order to investigate sequence diversity and to infer the phylogenetic relationships among the six Longidorus species. D1-D2 sequences were more conserved than the ITS sequences that varied widely in primary structure and length, and no consensus was observed. Phylogenetic analyses using the neighbor-joining, maximum parsimony and maximum likelihood methods were performed with three different sequence data sets: ITS1-ITS2, 5.8S-D1-D2, and combining ITS1-ITS2+5.8S-D1-D2 sequences. All multiple alignments yielded similar basic trees supporting the existence of the six species established using morphological characters. These sequence data also provided evidence that the different regions of the rDNA are characterized by different evolution rates and by different factors associated with the generation of extreme size variation.     

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei)

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.     

Species identification of <Emphasis Type="Italic">Ixodes granulatus</Emphasis> (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences

To investigate the genetic specificity of Ixodes granulatus ticks collected from Taiwan, the genetic identities and phylogenetic relationships were analyzed by comparing the sequences of the internal transcribed spacer 2 (ITS2) region obtained from 27 strains of ticks representing twelve species of Ixodes. Five major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks collected from Taiwan and Japan were genetically affiliated to a monophyletic group with highly homogeneous sequences (95.8–99.5% similarity), and can be discriminated from other species and subgenera of Ixodes ticks with a sequence divergence ranging from 13.6% to 62.9%. Moreover, interspecific analysis revealed that four distinct lineages are evident between Ixodes ticks, and all these I. granulatus ticks collected from Taiwan and Japan belong to the same lineage. Our results provide the first investigation on the genetic specificity of I. granulatus ticks, and demonstrate that all these I. granulatus ticks represent a unique lineage distinct from other species and subgenera of Ixodes ticks. The feasibility of ITS2-based genetic analysis for species-specific identification of I. granulatus ticks around East Asia was highly anticipated.     

Phylogenetic Analysis of Leymus (Poaceae: Triticeae) Inferred from Nuclear rDNA ITS Sequences

To investigate the phylogenetic relationships of polyploid Leymus (Poaceae: Triticeae), sequences of the nuclear rDNA internal transcribed spacer region (ITS) were analyzed for 34 Leymus accessions representing 25 species, together with three Psathyrostachys species (Ns genome), two Pseudoroegneria (St genome) species, Lophopyrum elongatum (E(e) genome), and Thinopyrum bessarabicum (E(b) genome). The phylogenetic analyses (maximum likelihood and Bayesian inference) supported two major clades, one including 21 Leymus species and three Psathyrostachys species, the other with nine Leymus species and four diploid species. The ITS RNA secondary structure of the Leymus species was compared with that of their putative diploid donor. It is suggested that (1) the species from the same areas or neighboring geographic regions are closely related to each other; (2) L. coreanus, L. duthiei, L. duthiei var. longearistatus, and L. komarovii are closely related to other Leymus species, and it is reasonable to transfer these species from the genus Hystrix to Leymus; (3) the ITS sequences of Leymus are evolutionarily distinct; (4) the different Leymus species and different distribution of a species derived their Ns genome from different Psathyrostachys species; and (5) there is a close relationship among Leymus, Pseudoroegneria, Lophopyrum, and Thinopyrum, but it is difficult to presume that the St, E(e), and E(b) genome may be the Xm genome donor of the Leymus species.     

ATLANTIC KELP SPECIES LAMINARIA LONGICRURIS AND L. SACCHARINA (LAMINARIALES) ARE CONSPECIFIC

The identification of two Atlantic kelp species, Laminaria longicruris and L. saccharina , has been controversial. In order to know if these two species are conspecific, plastid-encoded RuBisCo spacer and nuclear rDNA ITS sequences were analyzed from twelve individuals of L. longicruris , five of L. saccharina , from Connecticut and Nova Scotia. Four individuals of L. digitata from the above coasts were also analyzed as reference in this study. All RuBisCo spacer sequences from L. longicruris and L. saccharina were exactly identical except for one individual with three different nucleotides. Zero to five different nucleotides of ITS sequences, inculding four polymorphic sites, were found in two species. However, their RuBisCo spacer and ITS sequences are quite different from those of L. digitata. These results strongly suggest that L. saccharina (L.) Lamour. 1813 is conspecific with L. longicruris De La Pylaie 1824 and the different local populations, despite various morphotypes, may be genetically very similar.     

The evolution and utility of ribosomal ITS sequences in Bambusinae and related species: divergence, pseudogenes, and implications for phylogeny

Ribosomal internal transcribed spacer (ITS) sequences are commonly used for phylogenetic reconstruction because they are highly reiterated as components of rDNA repeats, and hence are often subject to rapid homogenization through concerted evolution. Concerted evolution leads to intragenomic uniformity of repeats even between loci on nonhomologous chromosomes. However, a number of studies have shown that the ITS polymorphism within individuals is quite common. The molecular systematics of Bambusinae and related species were recently assessed by different teams using independently generated ITS sequences, and the results disagreed in some remarkable features. Here we compared the ITS sequences of the members of Bambusa s. l., the genera Dendrocalamus, Dinochloa, Gigantochloa, Guadua, Melocalamus, Monocladus, Oxytenanthera, Thyrsostachys, Pleioblastus, Pseudosasa and Schizostachyum.We have reanalysed the ITS sequences used by different research teams to reveal the underlying patterns of their different results. After excluding the sequences suspected to represent paralogous loci, a phylogenetic analysis of the subtribe Bambusinae species were performed using maximum parsimony and maximum-likelihood methods. The implications of the findings are discussed. The risk of incorporating ITS paralogues in plant evolutionary studies that can distort the phylogenetic signal should caution molecular systematists.     

Intrastrain internal transcribed spacer heterogeneity in Ganoderma species

Intrastrain internal transcribed spacer (ITS) heterogeneity is first reported from Ganoderma, a fungal genus within Basidiomycetes. ITS amplification products from 4 strains, representing 4 Ganoderma species, were cloned and sequenced. Two to five different ITS types were found within a single strain. The clone sequences were analyzed along with other sequences from Ganoderma retrieved from GenBank. The results show that sequence variation within strains varies considerably with species and the heterogeneity may occur in the 3 parts (ITS1, ITS2, and 5.8S) of the ITS region.     

An ITS phylogeny of Leccinum and an analysis of the evolution of minisatellite-like sequences within ITS1

Phylogenetic relationships of the European species of Leccinum (Boletales, Boletaceae) were investigated by maximum parsimony, Bayesian and likelihood analyses of nrITS1-5.8S-ITS2 and 28S sequences. The separate gene trees inferred were largely concordant, and their combined analysis indicates that several traditional sectional and species-level taxonomic schemes are artificial. In Leccinum, the nrITS region ranges in size from 694 to 1480 bp. This extreme length heterogeneity is localized to a part of the ITS1 spacer that contains a minisatellite characterized by the repeated presence of CTATTGAAAAG and CTAATAGAAAG core sequences and mutational derivatives thereof. The number of core sequences present in the minisatellite varied from 12 to 36. Intra-individual sequence variation of the minisatellite was always smaller than between different species, indicating that concerted evolution proceeds rapidly enough to retain phylogenetic signal at the infraspecific level. In contrast, the evolutionary pattern exhibited by the major ITS1 repeat types found was homoplastic when mapped onto the species lineages inferred from the combined 5.8S-ITS2 sequences. The minisatellite therefore appears not to be useful for phylogeny reconstruction at or above the species level.     

Comparative study on DNA sequences of ribosomal DNA and cytochrome c oxidase subunit 1 of mitochondrial DNA among five species of gnathostomes

The nucleotide sequences of partial 18S, complete internal transcribed spacer region 1 (ITS1), complete 5.8S, complete ITS2 and partial 28S of ribosomal DNA (rDNA) and cytochrome c oxidase subunit 1 of mitochondrial DNA (MCOI) from five species of gnathostomes (G. spinigerum, G. doloresi, G. nipponicum, G. hispidum and G. binucleatum with the former four species being distributed in Japan and Asia) that cause human gnathostomiasis were compared by direct polymerase chain reaction cycle-sequencing. The nucleotide sequences of each region of the18S (613 bp), 5.8S (158 bp) and 28S (598 bp) rDNA from the five species were almost identical. The ITS1 region was different in length for the five species. The nucleotide sequences of each region of ITS2 and partial MCO1 regions were different among the five species. Therefore, these two regions can be used as genetic markers for identification of worms.     

米尔顿姬小蜂rDNA ITS1和ITS2的序列分析及其分子鉴定

本研究测定了米尔顿姬小蜂Anselmella miltoni Girault的rDNA ITS1和ITS2序列,以探讨其分子鉴定方法。米尔顿姬小蜂的ITS1和ITS2侧翼区（18S和5.8S）序列相对稳定,ITS1和ITS2序列存在种间差异。根据18S rDNA部分序列,利用DNAMAN的Maximum Likelihood方法构建了与膜翅目其它科的系统发育树。根据米尔顿姬小蜂ITS1和ITS2序列设计了特异性引物,应用特异性引物对样品进行了PCR扩增,扩增效果理想,采用上述特异性引物可从单头米尔顿姬小蜂稳定地扩增出明显的目的DNA条带。因此,可以采用ITS1和ITS2区的特异性对米尔顿姬小蜂进行快速的分子鉴定。     

Molecular characteristics of Camallanus Spp. (Spirurida: Camallanidae) in fishes from China based on its rDNA sequences

In the paper, we explored the intra- and interspecific evolutionary variation among species of Camallanus collected from different fish species in various regions of China. We determined the internal transcribed spacers of ribosomal DNA (ITS rDNA) sequences of these nematodes. The divergence (uncorrected p-distance) of ITS1, ITS2, and ITS rDNA data sets confirmed 2 valid species of Camallanus in China, i.e., C. cotti and C. hypophthalmichthys. The 2 species were distinguished not only by their different morphologies and host ranges but also by a tetranucleotide microsatellite (TTGC)n present in the ITS1 region of C. cotti. Phylogenetic analyses of the nematodes disclosed 2 main clades, corresponding to different individuals of C. cotti and C. hypophthalmichthys from different fish species in various geographical locations, although the interior nodes of each clade received poor support.     

金耳与其近似种的rDNA-ITS序列分析

对金耳(Tremella aurantialba)的担子果、酵母状分生孢子培养物和菌丝培养物的核糖体DNA内部转录间隔区(ITS)序列进行PCR扩增和测序。结果表明金耳担子果的ITS区PCR产物均为碱基数不同的两条带,片段长度和序列与酵母状分生孢子培养物、菌丝培养物一致。通过对ITS1和ITS2联合进行系统发育分析表明金耳酵母状分生孢子培养物归属于银耳属的金耳,参与组成担子果的寄主菌丝为毛韧革菌(Stereum hirsutum)。结合GenBank中登录的金黄银耳、脑状银耳、橙黄银耳等近似种构建了的系统发育树,结果支持形态学证据,表明金耳是一个独立种。     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

A secondary structural common core in the ribosomal ITS2 (internal transcribed spacer) of Culexspecies from diverse geographical locations

In the present study, sequence and structural analysis of ITS2 region (the spacer segment between 5.8S and 28S rRNA of mature rRNA sequences) of 7 Culex species belonging to 5 different geographical locations was carried out. Alignment of the ITS2 sequence from the 7 species revealed 8 homologous domains. Four species namely C. vishnui, C. annulus, C. pipiens, C. quiquefasciatusshowed high sequence (98-100%) and RNA secondary structure similarity. The ITS2 similarity among different species is high despite their varying geographical locations. Several common features of secondary structure are shared among these species, with some of them supported by compensatory changes, suggesting the significant role by ITS2 as an RNA domain during ribosome biogenesis.