Phylogeny of the Malus (Apple Tree) Species,Inferred from the Morphological Traits and Molecular DNA Analysis

Some debated issues of the genus Malus (apple) taxonomy were examined using a variety of species from the collection of the Maikop Experimental Station, Vavilon Research Institute of Plant Industry (Krasnodar krai). Phylogenetic relationships among these species were studied using traditional analysis of morphological traits, RAPD, and complete sequencing of the 5"- internal transcribed spacer (ITS1), 5.8S rRNA, 3"- internal transcribed spacer (ITS2) (constituting a cluster of the rRNA genes), and the terminal fragment of the matK gene encoding chloroplast maturase. The results showed that the Sorbomalussection was polyphyletic; the American apple M. fusca was closely related to the species contributing to the East Asian center of the genus origin, and the American speciesM. angustifolia, M. coronaria, and M. ioensis were closely related to the M. trilobata relict species, whose assignment to the genus Malus is debated by some authors. Molecular analysis of the species relationships showed that the Middle Asian apple M. sieversii is the species from which apple domestication started.     

Re-evaluation of the phylogenetic relationship among species of the genus Tricholoma

The internal transcribed spacer sequences spanning the regions between the 17S and 25S rRNAs (ITS1 and ITS2) and including the complete sequence of the 5.8S rRNA were used for phylogenetic analyses. This approach to define phylogenetic relationships within the genus Tricholoma was tested using different isolates of T. terreum. Fruitbodies identified in nature were analysed in order to allow use of morphology for taxonomy. The isolates from different locations were closely related as could be expected for one species. Thus, the method could be applied to different Tricholoma species. Three clusters within the genus Tricholoma can be distinguished with four additional species not included in any of these clusters. Molecular analyses of two Cortinarius species confirm a phylogenetically distinct genus.     

16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences analysis of the genus Myxococcus.

Phylogenetic relationships of the species belonging to the genus Myxococcus were elucidated based on the sequences of 16S rRNA genes and 16S-23S rRNA gene internal transcribed spacer (ITS) regions. The Myxococcus species were consequently classified into four distinct groups. The type strain of Myxococcus coralloides occupied an independent position (Group 1); it has been recently reclassified as Corallococcus coralloides. Group 2 comprised the type strains of both Myxococcus virescens and Myxococcus xanthus, and some strains assigned to Myxococcus flavescens. The type strain of M. flavescens was contained in Group 3 along with the strains of Myxococcus fulvus. Group 4 included the strains belonging to C. coralloides, M. fulvus, and M. stipitatus. The type strain of M. fulvus that was allocated outside Group 4 in the 16S rRNA gene tree belonged to Group 3 in the ITS tree. These results strongly suggest that the morphological characteristics of Myxococcus species are not consistent with the phylogenetic relationships. The Myxococcus species must therefore be redefined according to the phylogenetic relationships revealed in this study.     

Phylogenetic relationships among species of Hypochaeris (Asteraceae, Cichorieae) based on ITS, plastid trnL intron, trnL-F spacer, and matK sequences

Nuclear internal transcribed spacer (ITS) regions and chloroplast trnL intron and trnL/trnF spacer and matK sequences were used from 86 accessions to assess relationships among 31 European and South American species of Hypochaeris plus 18 representatives of related genera of tribe Cichorieae. The ITS tree shows high resolution compared to that of the maternally inherited trnL intron, trnL/F spacer, and matK sequences. The ITS and the combined tree reveal clades that agree well with sections of the genus established previously on morphological and cytological grounds, except for H. robertia, which groups with Leontodon helveticus and L. autumnalis. Monophyly of species of Hypochaeris from South America is strongly supported by both ITS and the joint matrix of ITS, trnL, and matK data. European species lie basal to South American taxa, which suggests that species in South America evolved from a single introduction from European progenitors and not from H. robertia as suggested previously. Low levels of sequence divergence among South American taxa suggest a pattern of rapid speciation, in contrast to much greater divergence among European representatives. Different species of Leontodon form two different clades that are also supported by chromosome numbers and morphology. Both nuclear and chloroplast markers suggest that Helminthotheca, Leontodon, and Picris are closely related to each other as well as to Hypochaeris.     

Sequence analysis of the ribosomal DNA internal transcribed spacer region in some scallop species (Mollusca: Bivalvia: Pectinidae).

The internal transcribed spacer (ITS) region of the ribosomal DNA from the European scallops Aequipecten opercularis, Mimachlamys varia, Hinnites distortus, and Pecten maximus was PCR amplified and sequenced. For each species, three or five clones were examined. The size ranged between 636 and 713 bp (ITS1, 209-276 bp; 5.8S rRNA gene, 157 bp; ITS2, 270-294 bp) and GC content ranged between 47 and 50% (ITS1, 43-49%; 5.8S rRNA gene, 56-57%; ITS2, 44-49%). Variation within repeats was minimal; only clones from M. varia and P. maximus displayed a few variable sites in ITS2. Among scallops, including Chlamys farreri whose ITS sequence appears in databases, significant variation was observed in both ITS1 and ITS2. Phylogenetic analysis using ITS1, ITS2, or both spacer sequences always yielded trees with similar topology. Aequipecten opercularis and P. maximus grouped in one clade and the other three scallops (C. farreri, M. varia, and H. distortus) in another, where M. varia and H. distortus are the more closely related species. These results provide new insights into the evolutionary relationships of scallop species and corroborate the close evolutionary relationship between the tribes Aequipectinini and Pectinini previously deduced from 18S rDNA sequences.     

Compensatory base changes illuminate morphologically difficult taxonomy

Compensatory base changes (CBCs) in the ribosomal RNA (rRNA) internal transcribed spacer 2 (ITS2) secondary structures have been used to successfully verify the taxonomy of closely related species. CBCs have never been used to distinguish morphologically indistinct species. Under the hypothesis that CBCs will differentiate species in higher eukaryotes, novel software for CBC analysis was applied to morphologically indistinguishable insect species in the genus Altica. The analysis was species-specific for sympatric Altica beetles collected across four ecoregions and concordant with scanning electron microscopy data. This research shows that mining for CBCs in ITS2 rRNA secondary structures is an effective method for eukaryotic taxon analysis.     

Molecular phylogenetic analysis of ixodid ticks based on the ribosomal DNA spacer, internal transcribed spacer 2, sequences

An internal transcribed spacer (ITS2) sequence between the 5.8S and 28S rRNA genes was used to estimate the phyletic relationships among Ixodes spp. tick vectors of Lyme disease-causing Borrelia spirochetes. Analysis indicates that Borrelia burgdorferi sensu lato species associated with Lyme disease are found mainly in ticks of the Ixodes ricinus species complex. Other closely related tick species are not known to transmit the Borrelia-that cause Lyme disease in humans, but they appear to have a specific association with other closely related Borrelia species. There is a high degree of concordance in the phylogenetics of Borrelia taxa and the phylogenetic relationships among Ixodes ticks.     

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.     

Patterns of molecular and morphological differentiation in Fagus (Fagaceae): phylogenetic implications

To study phylogenetic relationships among species of Fagus, the internal transcribed spacer regions ITS1 and ITS2 of the nuclear ribosomal DNA and morphological data were analyzed. Both molecular and morphologically based phylogenies suggest that Eurasian species of Fagus subgenus Fagus are basal to the North American Fagus grandifolia. The subgenus Fagus is a paraphyletic group basal to three East Asian species forming the subgenus Engleriana. Due to a considerably large amount of DNA polymorphism, relationships among basal species of Fagus could not be entirely resolved when analyzing ITS sequences with standard methods. Morphological trees helped to resolve more clearly relationships within the subgenus Fagus. The East Asian F. hayatae is suggested to be basal to the rest of the genus. This hypothesis is further supported by distinctive patterns of nucleotide variability found for ITS regions, allowing for basic and derived types to be distinguished. The high degree of ITS polymorphism within Fagus can be explained by (1) the complex evolutionary behavior of this marker, (2) the stenoecious ecological characteristic of Fagus with respect to its continuous geographic range throughout much of the Cenozoic, and (3) the absence of major radiations into further habitats as occurred in other Fagaceae.     

中国野生稻及栽培稻核糖体DNA第一转录间隔区序列分析及其系统学意义

用 PCR技术从产于我国的 3种野生稻和亚洲栽培稻的 2个亚种中特异地扩增和测序了 r DNA的第一转录间隔区。普通野生稻 (Oryza rufipogon)、药用野生稻 (O.officinalis)、疣粒野生稻 (O.granu-lata)和栽培稻的两个亚种 (O.sativa ssp.indica,O.sativa ssp.japonica)的 ITS1序列为 1 93bp、1 94bp、2 1 8bp、1 94bp和 1 94bp,它们的 G/ C含量为 69.3%～ 72 .7% ,序列中位点趋异率为 1 .5%～ 1 0 .6%。序列的相似性比较和简约性分支分析的结果表明 ,普通野生稻与栽培稻的两个亚种之间的亲缘关系最为密切 ;药用野生稻与普通野生稻和与栽培稻的两个亚种的相似性都为 82 % ,说明它与 AA基因组有一定的亲缘关系 ;疣粒野生稻与普通野生稻、药用野生稻和栽培稻两个亚种的亲缘关系相对较远 ,它在稻属中可能是一个系统地位较独特的类群。以 ITS1序列构建的 3种野生稻和 2个栽培稻亚种的系统发育关系与前人用同工酶、叶绿体 DNA、线粒体 DNA和核 DNA资料重建的稻属的系统发育关系基本一致     

Contrasting nifD and ribosomal gene relationships among Mesorhizobium from Lotus oroboides in northern Mexico

PCR screens for length variation in a 5' portion of 23S ribosomal RNA and in the 3' end of the 16S rRNA-23S rRNA internal transcribed spacer (ITS) region indicated that nodule bacteria from a Mexican population of Lotus oroboides were diverse on a local scale. Three 23S rRNA length variants and five ITS length variants were detected among the 22 isolates. Sequencing of nearly full-length 16S rRNA genes in three isolates indicated that they fell into the genus Mesorhizobium, but comprised two distinct groups. Two isolates were closely related to M. loti LMG 6125T, while the other isolate clustered with an assemblage of Mesorhizobium taxa that included M. amorphae, M. plurifarium and M. huakuii. However, a phylogenetic tree based on 715 bp of the nitrogenase alpha-subunit (nifD) gene was significantly discordant with the relationships inferred from rRNA sequences. Two isolates that were nearly identical for 16S rRNA had nifD genes that varied at 2% of sites, and one of these nifD sequences was identical to that of another isolate with a strongly divergent 16S rRNA gene. A plasmid screen followed by Southern hybridization indicated that only one of these strains harbored a plasmid-borne nifD gene. These results imply that gene transfer events have altered the distribution of nifD sequences among lineages within this natural population of Mesorhizobium strains.     

Molecular systematics of the genus Megoura (Hemiptera: Aphididae) using mitochondrial and nuclear DNA sequences

To construct the molecular systematics of the genus Megoura (Hemiptera: Aphididae), DNA based-identifi-cation was performed using four mitochondrial and three nuclear DNA regions: partial cytochrome c oxidase I (COI), partial tRNA-leucine + cytochrome c oxidase II (tRNA/COII), cytochrome b (CytB), partial 12S rRNA + tRNA-valine + 16S rRNA (12S/16S), elongation factor-1 alpha (EF1 alpha), and the internal transcribed spacers 1 and 2 (ITS1, ITS2). Pairwise sequence divergences between taxa were compared, and phylogenetic analyses were performed based on each DNA region separately, and the combined datasets. COI, CytB, EF1 alpha, ITS1, and ITS2 were relatively effective in determining species and resolving their relationships. By contrast, the sequences of tRNA/COII and 12S/16S were not able to separate the closely related species. CytB and EF1alpha gave better resolution with higher average sequence divergences (4.7% for CytB, 5.2% for EF1 alpha). The sequence divergence of COI (3.0%) was moderate, and those of the two ITS regions (1.8% for ITS1, 2.0% for ITS2) were very low. Phylogenetic trees were constructed by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic analyses. The results indicated that the phylogenetic relationships between Megoura species were associated with their host preferences. Megoura brevipilosa and M. lespedezae living on Lespedeza were closely related, and M. nigra, monophagous on Vicia venosa, was rather different from M. crassicauda, M. litoralis, and M. viciae, which are oligophagous on Lathyrus and Vicia. The three populations of M. crassicauda formed a clade separated from M. litoralis and M. viciae. Nevertheless M. litoralis and M. viciae, which are morphologically similar, were not separated due to negligible sequence divergence. We discuss the phylogenetic relationships of the Megoura, and the usefulness of the seven DNA regions for determining the species level phylogeny of aphids.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

Phylogeny of Rozites, Cuphocybe and Rapacea inferred from ITS and LSU rDNA sequences

Phylogenetic relationships of Rozites, Cuphocybe, and Rapacea were assessed using molecular phylogenetic approaches. These three genera are placed in Cortinariaceae and have been regarded as closely related to Cortinarius. Rozites includes more than 20 species, which are characterized by having both a membranaceous partial veil in the form of a persistent annulus and a membranaceous universal veil. Cuphocye (4 species) lacks an annulus or cortina, but has pigmented veil fibrils or scales. The monotypic genus Rapacea accommodates a distinct taxon with pale, nearly smooth and thick-walled basidiospores. We analyzed 56 sequences of the internal transcribed spacer region (ITS1, ITS2, and the intervening 5.8S rRNA gene) for nine species of Rozites, three species of Cuphocybe, 28 species of Cortinarius, Rapacea mariae and Protoglossum luteum. Two species of Hebeloma were used as outgroup. Large subunit (LSU) rDNA sequences from selected taxa were also analyzed. The results clearly demonstrate that Rozites species are nested within the clade/Cortinarius, and that Rozites is polyphyletic, suggesting that membranaceous veils have evolved several times in the genus Cortinarius. Also Rapacea and Cuphocybe are nested within Cortinarius, making the latter genus paraphyletic. Based on phylogenetic studies, Rozites, Cuphocybe and Rapacea are artificial genera and do not reflect natural relationships.     

Unusual compact rDNA gene arrangements within some members of the Ascomycota: evidence for molecular co-evolution between ITS1 and ITS2.

The internal transcribed spacers of the ribosomal DNA tandem repeat were examined in members of the ascomycetous genus Sphaeronaemella. Species of Sphaeronaemella and its mitotic counterpart Gabarnaudia, have a compact rDNA gene arrangement due to unusually short internal transcribed spacer (ITS) regions. Examination of these regions from phylogenetically related taxa, Cornuvesica, Gondwanamyces, and Ceratocystis, showed that their ITS1 and ITS2 regions could be folded into central hairpin-like structures with the size reduction in species of Sphaeronaemella being due to length reduction of the main-hairpin and the loss of smaller hairpin-like structures that emanate from the main hairpin. A databank compilation, combined with newly obtained sequences, provided an ITS data set that includes sequences of 600 species belonging to the Ascomycota. Correlation analysis revealed that the sizes of ITS1 and ITS2 show a strong positive correlation, suggesting that the 2 rDNA regions have co-evolved. This supports biochemical evidence indicating that the ITS1 and ITS2 segments interact to facilitate the maturation of the rRNA precursor.     

Phylogeny and quaternary history of the European montane/alpine endemic Soldanella (Primulaceae) based on ITS and AFLP variation

Soldanella contains 16 species of herbaceous perennials that are endemic to the central and south European high mountains. The genus is ecogeographically subdivided into forest/montane and alpine species. Evolutionary relationships and large-scale biogeographic patterns were inferred from parsimony analyses of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA, and genetic distance analyses based on amplified fragment length polymorphism (AFLP) markers. The ITS region proved useful for examining subgeneric relationships and testing hypotheses on genus-wide divergence times, whereas the AFLP markers were suitable for studying relationships among closely related taxa and biogeographic patterns of divergence. Neither ITS nor AFLP data supported sectional delimitations, particularly those related to the grouping of S. alpina (sect. Soldanella) with S. pusilla (sect. Tubiflores), which may be the result of hybridization. Additional results and conclusions drawn are (1) Soldanella is derived from an ancestor of Asian origin with a montane ecology; (2) estimates of divergence times suggest a late Quaternary origin of the genus; (3) alpine species of sect. Tubiflores diverged from within a paraphyletic sect. Soldanella of mainly montane species; (4) alpine and montane species of Soldanella experienced different cycles of range expansion and contraction during late Quaternary climatic changes, resulting in differential patterns of geographic distribution; and (5) AFLP divergence among montane species from eastern Europe was lower than between alpine species; we hypothesize that the latter differentiated in allopatric regions of expansion during glacials, while the former experienced secondary contact at lower elevations in more southern refugia.     

Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species

Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production. The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia. In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods. The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium. These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium. Three strains were closely related to B. elkanii and the rest of the strains were related to the phylogenetic group constituted by B. japonicum, B. liaoningense, B. yuanmingense and B. betae. These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.     

Characterization of Euplotes lynni nov. spec., E. indica nov. spec. and description of E. aediculatus and E. woodruffi (Ciliophora,Euplotidae) using an integrative approach

Four species belonging to the genus Euplotes have been investigated, namely: E. lynni nov. spec., E. indica nov. spec., E. aediculatus, and E. woodruffi. All populations are from India and were investigated using morphological and molecular markers. The phylogenetic relationships were inferred from small subunit ribosomal rRNA gene (SSU rRNA), internal transcribed spacer (ITS) region, and mitochondrial cytochrome c oxidase subunit I (COI) gene. Predicted secondary structure models for two new species using the hypervariable region of the SSU rRNA gene and ITS2 region support the distinctness of both species. Morphological characters were subjected to principal component analysis (PCA) and genetic variations were studied in-depth to analyze the relatedness of the two new species with their congeners. An integrative approach combining morphological features, molecular analysis, and ecological characteristics was carried out to understand the phylogenetic position of the reported species within the different clades of the genus Euplotes.     

ITS1 Sequences of Nuclear Ribosomal DNA in Wild Rices and Cultivated Rices of China and Their Phylogenetic Implications

he first internal transcribed spacer (ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3%to 72.7%. In pairwise comparison among the five taxa, sequence site divergence ranged from 1.5 % to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well-resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) of the genus Oryza.