Phylogenetic analysis of Culicoides species from France based on nuclear ITS1-rDNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play important roles in the transmission of viral diseases affecting wild and domestic ruminants and horses, including Bluetongue (BT) and African horse sickness (AHS) respectively. In southern Europe, BT has been largely transmitted by the classical Afro-Asian vector Culicoides imicola Kieffer. However, other species such as C. obsoletus Meigen, C. scoticus Downs & Kettle and C. pulicaris Linné may also be involved in BTV transmission. As a consequence of the discovery of C. imicola followed by BTV-2 outbreaks on the island of Corsica in October 2000, further studies on these biting midges have been carried out. To better characterize the evolution and phylogenetic relations of Culicoides, molecular analysis in parallel with a morphology-based taxonomic approach were performed. Phylogenetic analyses of French Culicoides species were undertaken using the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) as a molecular target. This region was shown to be useful in understanding evolutionary and genetic relationships between species. Construction of several trees showed that molecular phylogeny within the genus Culicoides correlates not only with morphological-based taxonomy but also with ecological patterns.     

Phylogenetic analysis of Allium subg. Melanocrommyum infers cryptic species and demands a new sectional classification

Allium subgenus Melanocrommyum (Alliaceae) from Eurasia comprises about 150 mostly diploid species adapted to arid conditions. The group is taxonomically complicated with different and contradictory taxonomic treatments, and was thought to include a considerable number of hybrid species, as the taxa show an admixture of assumed morphological key characters. We studied the phylogeny of the subgenus, covering all existing taxonomic groups and their entire geographic distribution. We analyzed sequences of the nuclear rDNA internal transcribed spacer region (ITS) for multiple individuals of more than 100 species. Phylogenetic analyses of cloned and directly sequenced PCR products confirmed the monophyly of the subgenus, while most sections were either para- or polyphyletic. The splits of the large sections are supported by differences in the anatomy of flower nectaries. ITS data (i) demand a new treatment at sectional level, (ii) do not support the hypotheses of frequent gene flow among species, (iii) indicate that multiple rapid radiations occurred within different monophyletic groups of the subgenus, and (iv) detected separately evolving lineages within three morphologically clearly defined species (cryptic species). In two cases these lineages were close relatives, while in Allium darwasicum they fall in quite different clades in the phylogenetic tree. Fingerprint markers show that this result is not due to ongoing introgression of rDNA (ITS capture) but that genome-wide differences between both lineages exist. Thus, we report one of the rare cases in plants where morphologically indistinguishable diploid species occurring in mixed populations are non-sister cryptic species.     

Species identity and phylogenetic relationship of the pearl oysters in Pinctada Röding, 1798 based on ITS sequence analysis

Analysis of ITS 1 and ITS 2 sequences in the pearl oysters Pinctada albina, Pinctada chemnitzi, Pinctada fucata, Pinctada fucata martensii, Pinctada imbricata, Pinctada margaritifera, Pinctada maxima, Pinctada nigra and Pinctada radiata was carried out. Homogeneity test of substitution patterns suggests that GC contents are highest in P. margaritifera and P. maxima and chromosomal rearrangements occurred in P. chemnitzi. These observations indicate that P. margaritifera and P. maxima are primitive species and P. chemnitzi is a recent species. Phylogenetic analysis shows that the pearl oysters studied constitute three clades with P. margaritifera and P. maxima forming the basal clade, congruent with results revealed by the substitution pattern test. The second clade consists of P. fucata, P. fucata martensii and P. imbricata. Low genetic distances among these taxa indicate that they may be conspecific. The remaining species make up the third clade and low genetic divergence between P. albina and P. nigra suggests that they may represent the same species. The ITS 1 sequence of P. radiata in GenBank is almost identical to that of P. chemnitzi determined in the present study and we suspect that the specimen used for the P. radiata sequence was misidentified.     

Phylogenetic analysis of two putative Nosema isolates from Cruciferous Lepidopteran pests in Taiwan

In this study, a new microsporidian, PX2, was isolated from the diamondback moth, Plutella xylostella, and then compared with another isolate (PX1), and with Nosema spodopterae and N. bombycis. Sequence data showed that the rRNA gene organizations of PX1 and PX2 exhibited a typical Nosema-specific organization: 5'-LSUrRNA (large subunit ribosomal RNA)-ITS (internal transcribed spacer)-SSUrRNA-IGS (intergenic spacer)-5S-3'. Phylogenetic analysis (maximum likelihood, neighbor joining, maximum parsimony, and Bayesian analysis) of the LSUrRNA and SSUrRNA gene sequences, and the sequences of the alpha-tubulin, beta-tubulin, and RPB1 (DNA dependent RNA polymerase II largest subunit) genes found that PX1 was closer to N. bombycis and N. spodopterae than to PX2. Comparison of the identities of the rRNA domains and of the other three genes showed a high divergence in the sequences of the rRNA spacer regions (ITS and IGS). This is consistent with the hypothesis that PX2, if not PX1, might represent a new Nosema species.     

PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in<Emphasis Type="Italic"> Musa</Emphasis> L.

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the diploid ancestors of modern bananas that are mostly diploid or triploid cultivars with various combinations of the A and B genomes, including AA, AAA, BB, AAB and ABB. The objective of this study was to identify molecular markers that will facilitate discrimination of the A and B genomes, based on restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes. The ITS regions of seven M. acuminata and five M. balbisiana accessions were each amplified by PCR using specific primers. All accessions produced a 700-bp fragment that is equivalent in size to the ITS of most plants. This fragment was then digested with ten restriction enzymes (AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI and TaqI) and fractionated in 2% agarose gels, stained with ethidium bromide and visualized under UV light. The RsaI digest revealed a single 530-bp fragment unique to the A genome and two fragments of 350-bp and 180-bp that were specific to the B genome. A further 56 accessions representing AA, AAA, AAB, AB and ABB cultivars, and synthetic hybrids, were amplified and screened with RsaI. All accessions with an exclusively A genome showed only the 530-bp fragment, while accessions having only the B-genome lacked the 530-bp fragment but had the 350-bp and 180-bp fragments. Interspecific cultivars possessed all three fragments. The staining intensity of the B-genome markers increased with the number of B-genome complements. These markers can be used to determine the genome constitution of Musa accessions and hybrids at the nursery stage, and, therefore, greatly facilitate genome classification in Musa breeding.Communicated by H.F. Linskens     

核糖体转录间隔子2应用于鱼类种属的鉴别

为了防止珍稀鱼类的非法捕捞和销售, 鱼类种属的鉴别就成为非常关键的问题, 特别是形态学方法无法区分的样品(如鱼苗、鱼鳞、鱼卵、鱼肉及其加工产品等)。为了帮助珍稀鱼类资源的管理和保护, 文章报道了一种利用核糖体基因的转录间隔子2鉴别鱼类种属的分子遗传学方法: (1) 利用同一目鱼类5.8S rRNA和28S rRNA基因的保守性, 设计出扩增鲤形目鱼类这两个基因间转录间隔子2 DNA片段, 测序获得它们的碱基排列顺序; (2) 再根据不同鱼类转录间隔子2序列的差异, 设计出每种鱼的种属特异引物、种属鉴别标准物, 构建鱼类分子分类图谱, 利用PCR复合扩增技术鉴别鱼类种属。通过对国内不同地方采集的5种鲤形目鱼类的210个单一品种样本和40个混合样本的鉴别检验, 该方法能够准确、灵敏和快速鉴别这5种鱼, 可用于鱼类资源保护和评估、管理和开发, 特别是在渔业管理人员渔业执法、海关打击珍稀鱼类走私、防止商业欺诈和外来有害生物入侵等方面非常有用     

ITS polymorphism within a single strain ofSclerotium rolfsii

Two morphologically distinct strains, 63–76 and 63H1, were isolated from a protoplast and a hyphal tip of the parentalSclerotium rolfsii strain S-63, respectively. Strains 63–76 and 63H1 showed reduced mycelial growth and lacked clamp connections on hyphae. The two strains also differed from each other and from their parent in RAPD patterns generated by several primers, suggesting that 63–76 and 63H1 were homokaryons isolated from the hetereokaryon S-63. Whereas the parent S-63 belonged to ITS-RFLP group 1, RFLP patterns of internal transcribed spacer (ITS) regions of rDNA of 63–76 and 63H1 were similar to those of ITS-RFLP groups 5 and 3, respectively. The sequence similarity of ITS regions were more than 99% between 63–76 and group 5 strains, 100% between 63H1 and the group 3 strain, and 96.3% between 63–76 and 63H1. Direct sequencing failed in the parental strain S-63. S-63 was considered to contain ITS types of groups 5 and 3.     

Phylogenetic analysis of eastern Asian and eastern North American disjunct Lespedeza (Fabaceae) inferred from nuclear ribosomal ITS and plastid region sequences

Lespedeza (tribe Desmodieae, Fabaceae) follows a disjunct distribution in eastern Asia and eastern North America. Phylogenetic relationships among its species and related taxa were inferred from nuclear ribosomal internal transcribed spacer (ITS) and plastid sequences (trnH‐psbA, psbK‐psbI, trnK‐matK and rpoC1). We examined 35 species of Lespedeza, two of Kummerowia and one of Campylotropis, the sole constituents of the Lespedeza group. An analysis of these data revealed that the genus Campylotropis is sister to the other two genera. However, we were unable to resolve the relationships between Kummerowia and Lespedeza in the strict consensus trees of parsimony analyses based on plastid and combined DNA data. In the genus Lespedeza, the Old World subgenus Macrolespedeza is monophyletic, whereas the transcontinental subgenus Lespedeza is paraphyletic. Monophyly of eastern Asian species and of North American species is strongly supported. Although inconsistent with the traditional classification, this phylogenetic finding is consistent with seedling morphology. Three subgroups recognized in subgenus Macrolespedeza were unresolved in our phylogenetic trees. An incongruence length difference (ILD) test indicated that the two partitions (nuclear ITS and plastid sequences) were significantly incongruent, perhaps because of hybridization between species in Lespedeza. Most of the primary clades of tribe Desmodieae are Asian, implying that the relatively few New World ones, such as those in Lespedeza, are more recently derived from Asia. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 221–235.     

Molecular phylogeny of ectomycorrhizal <Emphasis Type="Italic">Pisolithus</Emphasis> fungi associated with pine,dipterocarp, and eucalyptus trees in Thailand

The phylogenetic relationships among 135 Pisolithus basidiomes and two isolates collected from three pine forests, a pine-dipterocarp forest, two dipterocarp forests, and 29 eucalyptus plantations in Thailand were investigated. Internal transcribed spacer (ITS) polymorphism analyses, including terminal RFLP, divided them into 26 groups. The ITS in a representative basidiome of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggested that at least three Pisolithus species are present in Thailand. Pisolithus basidiomes collected in the pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5 as previously described by Martin et al. in 2002. Those collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from the eucalyptus plantations were all identified as Pisolithus albus.     

Phylogenetic analysis of the genus Hexachlamys (Myrtaceae) based on plastid and nuclear DNA sequences and their taxonomic implications

Myrtaceae are one of the most species‐rich families of flowering plants in the Neotropics. They include several complex genera and species; Hexachlamys is one of the complex genera. It has not been recognized as a distinct genus and has been included in Eugenia, based on morphological grounds. Therefore, molecular systematic studies may be useful to understand and to help to solve these relationships. Here, we performed a molecular phylogenetic analysis using plastid and nuclear data in order to check the inclusion of Hexachlamys in Eugenia. Plastid (accD, rpoB, rpoC1, trnH‐psbA) and nuclear (ITS2) sequence data were analysed using Bayesian and maximum parsimony methods. The trees constructed using ITS2 and trnH‐psbA were the best able to resolve the relationships between species and genera, revealing the non‐monophyly of Hexachlamys. The molecular phylogenetic analyses were in agreement with previous morphological revisions that have included Hexachlamys in Eugenia. These results reinforce the importance of uniting knowledge and strategies to understand better issues of delimitation of genera and species in groups of plants with taxonomic problems. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 532–543.     

Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.     

Use of rpoB gene sequence analysis for phylogenetic identification of Cronobacter species

Here, we report the RNA polymerase beta-subunit gene (rpoB) as a new molecular marker for the identification of the Cronobacter species. The results indicated that members of the Cronobacter genus are more easily discriminated by rpoB sequencing than 16S rRNA sequencing, and reliable identification could be achieved by rpoB gene sequence comparison.     

Variation of ITS sequences in a natural Japanese population of Pleurocybella porrigens

Genetic relationships in a natural Japanese population of Pleurocybella porrigens were determined based on ITS sequence data. In a UPGMA similarity tree, all sequences of 23 specimens from 13 different geographic origins were grouped into two distinct clusters (groups A and B). Sequence variation of the ITS region between groups A and B consisted of 33–40 nucleotides, corresponding to 5%–6% of their total length, and specific nucleotide variations characterizing groups A and B were found. Although these results did not show correlation with differences of substrates for fruiting and geographic origins of the specimens, it was suggested that P. porrigens distributed in Japan include at least two genetically different populations. Contribution no. 376 of the Tottori Mycological Institute     

Phylogenetic relationships ofPseudopanax species (Araliaceae) inferred from parsimony analysis of rDNA sequence data and morphology

Sequence data from internal transcribed spacer (ITS) regions of rDNA and data from morphology, cytology and wood anatomy are used to study phylogenetic relationships inPseudopanax. The molecular and non-molecular data are analysed as independent data sets and in combination using parsimony. Results supported the conclusion that the genusPseudopanax is polyphyletic.Pseudopanax species emerge in two major monophyletic groups. The Anomalus group containsPseudopanax anomalus, P. edgerleyi, andP. simplex; these species share a common ancestor withCheirodendron trigynum and more distantly withPseudopanax gunnii. The second major group consists of two smaller groups: the Arboreus group, includingPseudopanax arboreus, P. colensoi, P. kermadecensis, P. laetus, andP. macintyrei, and the Crassifolius/Discolor group, includingP. chathamicus, P. crassifolius, P. discolor, P. ferox, P. gilliesii, P. lessonii, andP. linearis. Meryta species are close relatives of thePseudopanax Arboreus and Crassifolius/Discolor groups.     

Phylogenetic selection of target species in Amaryllidaceae tribe Haemantheae for acetylcholinesterase inhibition and affinity to the serotonin reuptake transport protein

We present phylogenetic analyses of 37 taxa of Amaryllidaceae, tribe Haemantheae and Amaryllis belladonna L. as an outgroup, in order to provide a phylogenetic framework for the selection of candidate plants for lead discoveries in relation to Alzheimer's disease and depression. DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) and the plastid trnL-F regions were used. Maximum parsimony analyses provide increased support for the sister relationship of Haemanthus and Scadoxus. Within Haemanthus, a well supported clade (89% BS) corresponds to a summer rainfall group (mainly Eastern Cape) with white-pale pink flowers. A second strongly supported clade (100% BS) corresponds to a winter rainfall group (mainly Western Cape) with red-pale pink flowers. Haemanthus montanus, which is from the summer rainfall region, is sister to the winter rainfall group. Alkaloid profiles and bioactivity profiles were investigated for 16 taxa using gas chromatography-mass spectrometry (GC-MS) and assays measuring acetylcholinesterase (AChE) inhibition and affinity to the serotonin reuptake transport protein (SERT). No alkaloids were detected by GC-MS in extracts of the two species of Gethyllis included in the present study suggesting that Gethyllis (and possibly Apodolirion) species may not produce the alkaloids characteristic for the family. AChE inhibitory activity was found in all investigated clades except the Apodolirion-Gethyllis clade, which can be explained by the apparent lack of alkaloids in this clade. In spite of infra-specific variability of alkaloid profiles observed, dose-dependent SERT activity appears to be pronounced and restricted to the genus Haemanthus within tribe Haemantheae. Three of eight Haemanthus species tested had IC₅₀ < 10 μg/ml. Two of the most active extracts in the present study contained primarily montanine type alkaloids which have not been tested for SERT affinity previously. Simultaneous evaluation of bioactivity and alkaloid profiles in a phylogenetic framework can potentially be used to select candidate species for phytotherapy and drug discovery.     

Molecular-genetic analyses reveal cryptic species of trematodes in the intertidal gastropod, Batillaria cumingi (Crosse)

Cryptic species of the digeneans, Cercaria batillariae (Heterophyidae) and an undescribed philophthalmid, were detected using polymerase chain reaction-based restriction fragment-length polymorphism methodology and sequence analyses. These digeneans were all collected from the same species of gastropod first intermediate host, Batillaria cumingi (=Batillaria attramentaria). The mitochondrial cytochrome oxidase subunit 1 gene (approximately 800bp) and nuclear internal transcribed spacer 1 gene (approximately 400bp) were used for species level discrimination. Restriction fragment-length polymorphism analyses of cytochrome oxidase subunit 1 gene showed that C. batillariae included 10 distinguishable fragment patterns, and the philophthalmid included five patterns. On the basis of subsequent sequence analyses, the restriction fragment length polymorphism patterns of C. batillariae were grouped into eight phylogenetically distinct lineages and those of the philophthalmid into three phylogenetically distinct lineages. There was no evidence of gene flow among the different lineages due to the lack of heterozygosity within the observed internal transcribed spacer 1 gene fragment patterns. This suggests that all of these lineages are different species. Most of these species were widespread, but some exhibited restricted geographic distributions. We discuss the implications of these findings for host specificity of these trematodes. These results demonstrate the utility of genetic analysis to distinguish species of morphologically similar trematodes. Hence, trematode species diversity may often be underestimated when species identifications are limited to morphological features.     

Discrimination of <Emphasis Type="Italic">Tricholoma</Emphasis> species by species-specific ITS primers

The ITS region of ectomycorrhizal fungi was analyzed, and species-specific PCR primers were designed for 8 ectomycorrhizal Tricholoma species. Although a high degree of intraspecific homology was observed, interspecific variation was sufficient to design species-specific primers based on sequence of the ITS region. PCR amplification with the specific primers generated fragments of the expected sizes from DNA extracted from the strains of each species but gave no amplified products from the strains of the other 16 species in eight genera. These results suggest that sequence of the ITS region is appropriate to be used for species-level identification of ectomycorrhizal fungi.     

Phylogenetic relationship of Phytophthora cryptogea Pethybr. & Laff and P. drechsleri Tucker

The phylogeny and taxonomy of Phytophthora cryptogea and Phytophthora drechsleri has long been a matter of controversy. To re-evaluate this, a worldwide collection of 117 isolates assigned to either P. cryptogea, P. drechsleri or their sister taxon, Phytophthora erythroseptica were assessed for morphological, physiological (pathological, cultural, temperature relations, mating) and molecular traits. Multiple gene phylogenetic analysis was performed on DNA sequences of nuclear (internal transcribed spacers (ITS), ß-tubulin, translation elongation factor 1α, elicitin) and mitochondrial (cytochrome c oxidase subunit I) genes. Congruence was observed between the different phylogenetic data sets and established that P. drechsleri and P. cryptogea are distinct species. Isolates of P. drechsleri form a monophyletic grouping with low levels of intraspecific diversity whereas P. cryptogea is more variable. Three distinct phylogenetic groups were noted within P. cryptogea with an intermediate group providing strong evidence for introgression of previously isolated lineages. This evidence suggests that P. cryptogea is an operational taxonomic unit and should remain a single species. Of all the morphological and physiological traits only growth rate at higher temperatures reliably discriminated isolates of P. drechsleri and P. cryptogea. As a homothallic taxon, P. erythroseptica, considered the cause of potato pink rot, is clearly different in mating behaviour from the other two species. Pathogenicity, however, was not a reliable characteristic as all isolates of the three species formed pink rot in potato tubers. The phylogenetic evidence suggests P. erythroseptica has evolved from P. cryptogea more recently than the split from the most recent common ancestor of all three species. However, more data and more isolates of authentic P. erythroseptica are needed to fully evaluate the taxonomic position of this species.