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.     

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 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.     

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     

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.     

A phylogenetic analysis of the aureoidSenecio (Asteraceae) complex based on ITS sequence data

The internal transcribed spacer region of the 18S–25S nuclear ribosomal DNA repeat was sequenced from 28 populations of the aureoidSenecio complex as well as two populations from the Lugentes group and one from the Tephroseroid group. Divergence levels for populations within the aureoid complex are very low (0.0 to 4.1%). Phylogenetic trees generated from the sequence data provide no support for the recognition of Aurei, Tomentosi and Lobati subgroups within the aureoid complex. With two Lugentes and one Tephroseroid species as outgroups,Senecio glabellus is the sister group of the rest of the aureoids. The high level of divergence between the aureoids and the three outgroup species indicates that the Lugentes and the Tephroseroids may not be closely related to the aureoids.     

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.     

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 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.     

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.     

Differentiation of two ovine Babesia based on the ribosomal DNA internal transcribed spacer (ITS) sequences

The first and second internal transcribed spacers (ITS1, ITS2) as well as the intervening 5.8S coding region of the rRNA gene for six Babesia spp. isolated from different geographic origins were characterized. Varying degrees of ITS1 and ITS2 intra- and inter-species sequence polymorphism were found among these isolates. Phylogenetic analysis of the ITS1-5.8S gene-ITS2 region clearly separated the isolates into two clusters. One held an unidentified Babesia sp. transmitted by Hyalomma anatolicum anatolicum. The second held five other isolates, which were considered to be Babesia motasi. Each Babesia species cluster possessed ITS1 and ITS2 of unique size(s) and species specific nucleotide sequences. The results showed that ITS1, ITS2 and the complete ITS1-5.8S-ITS2 region could be used to discriminate these ovine Babesia spp. effectively.     

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.     

Can genetic bar-coding be used to identify aquatic Ranunculus L. subgenus Batrachium (DC) A. Gray? A test using some species from the British Isles

Aquatic Batrachium Ranunculus species are a key component of river macrophyte communities selected for protection under European Union legislation. The group's simplified morphology and variable taxonomic interpretation often makes identification to species level very difficult. A genetic approach was trialled as an alternative, more reliable, means of identification. DNA barcoding using four markers (chloroplast and nuclear) was tested. The chloroplast sequence trnH-psbA worked best and allowed identification of three out of five species while nuclear sequences supported the identification of two hybrids. This method is amenable to simplification through techniques such as PCR-RFLP or RT-PCR. It has the potential to provide easy, rapid and inexpensive identification of Batrachium Ranunculus species.     

Chimerical nature of the ribosomal RNA gene of a Nosema species

Taxonomic resolution of the Nosema/Vairimorpha clade has been augmented with DNA sequences of the small subunit (SSU) and large subunit (LSU) ribosomal RNA (rRNA) and the arrangement of SSU and LSU. Based on the two characteristics, the clade is largely divided into two, i.e. ‘true’ Nosema sub-group and non-‘true’ Nosema sub-group within the clade. Our study shows that a novel Nosema species isolated from Pieris rapae has mixed characteristics of the ‘true’ and non-‘true’ Nosema sub-group based on the topology of SSU and LSU sequences. To our knowledge, this may be the first case of the incongruent phylogenetic placement of SSU and LSU in the Nosema/Vairimorpha clade. Additionally, the length of internal transcribed spacer (ITS) can be a diagnostic tool to distinguish ‘true’ Nosema from non-’true’ Nosema in the Nosema/Vairimorpha clade based on its nucleotide length as reported before.