Secondary Structure Analyses of the Nuclear rRNA Internal Transcribed Spacers and Assessment of Its Phylogenetic Utility across the Brassicaceae (Mustards)

The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.     

Defining orthologous groups among multicopy genes prior to inferring phylogeny,with special emphasis on the Triticeae (Poaceae)

Sequence information from multicopy genes has been widely used for phylogenetic inference. Among those sequences analyzed, nuclear 5S rRNA genes, the two internal transcribed spacer regions (ITS1 and ITS2) of the 18S-26S rDNA genes, and the intergenic spacer (IGS) regions of the same 18S-26S rDNA genes have all been used at the specific, generic, familial and tribal levels. Many investigations have used direct sequencing of PCR products to generate sequence data. The merits of an alternate approach, namely, cloning prior to sequencing followed by careful alignment of numerous cloned sequences to discern groups of putative orthologous sequences that may then be useful for the inference of relationships among species and genera, are examined and discussed. This process discerns patterns resulting from several cycles of careful alignment followed by manual editing conducted by eye--an exacting operation especially when sequences are unequal in length due to the presence of additions/deletions. Based upon examples taken from our work on the sequencing of individual 5S rDNA clones from several wheat and barley species (Triticum and Hordeum respectively), and the re-analysis of data of others taken from several studies using the nuclear genes mentioned above, we are able to identify groups of putative orthologous sequences that we have named "unit classes". Furthermore, comparisons between provisional orthologous sequences isolated from different species are required for the inference of phylogenetic relationships between them. Paralogous sequences from different unit classes can be compared to infer evolutionary relationships among repeat types only, i.e. among unit classes. In several cases, the analysis of the sequence diversity obtained from different clones permitted the assignment of unit classes to specific haplomes.     

Phylogenetic relationships within the cyst-forming nematodes (Nematoda, Heteroderidae) based on analysis of sequences from the ITS regions of ribosomal DNA

The ITS1, ITS2, and 5.8S gene sequences of nuclear ribosomal DNA from 40 taxa of the family Heteroderidae (including the genera Afenestrata, Cactodera, Heterodera, Globodera, Punctodera, Meloidodera, Cryphodera, and Thecavermiculatus) were sequenced and analyzed. The ITS regions displayed high levels of sequence divergence within Heteroderinae and compared to outgroup taxa. Unlike recent findings in root knot nematodes, ITS sequence polymorphism does not appear to complicate phylogenetic analysis of cyst nematodes. Phylogenetic analyses with maximum-parsimony, minimum-evolution, and maximum-likelihood methods were performed with a range of computer alignments, including elision and culled alignments. All multiple alignments and phylogenetic methods yielded similar basic structure for phylogenetic relationships of Heteroderidae. The cyst-forming nematodes are represented by six main clades corresponding to morphological characters and host specialization, with certain clades assuming different positions depending on alignment procedure and/or method of phylogenetic inference. Hypotheses of monophyly of Punctoderinae and Heteroderinae are, respectively, strongly and moderately supported by the ITS data across most alignments. Close relationships were revealed between the Avenae and the Sacchari groups and between the Humuli group and the species H. salixophila within Heteroderinae. The Goettingiana group occupies a basal position within this subfamily. The validity of the genera Afenestrata and Bidera was tested and is discussed based on molecular data. We conclude that ITS sequence data are appropriate for studies of relationships within the different species groups and less so for recovery of more ancient speciations within Heteroderidae.     

Genetic analysis of housekeeping genes of members of the genus Acholeplasma: phylogeny and complementary molecular markers to the 16S rRNA gene

The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

A Primer to Molecular Phylogenetic Analysis in Plants

Reconstructing a tree of life by inferring evolutionary history is an important focus of evolutionary biology. Phylogenetic reconstructions also provide useful information for a range of scientific disciplines such as botany, zoology, phylogeography, archaeology and biological anthropology. Until the development of protein and DNA sequencing techniques in the 1960s and 1970s, phylogenetic reconstructions were based on fossil records and comparative morphological/physiological analyses. Since then, progress in molecular phylogenetics has compensated for some of the shortcomings of phenotype-based comparisons. Comparisons at the molecular level increase the accuracy of phylogenetic inference because there is no environmental influence on DNA/peptide sequences and evaluation of sequence similarity is not subjective. While the number of morphological/physiological characters that are sufficiently conserved for phylogenetic inference is limited, molecular data provide a large number of datapoints and enable comparisons from diverse taxa. Over the last 20 years, developments in molecular phylogenetics have greatly contributed to our understanding of plant evolutionary relationships. Regions in the plant nuclear and organellar genomes that are optimal for phylogenetic inference have been determined and recent advances in DNA sequencing techniques have enabled comparisons at the whole genome level. Sequences from the nuclear and organellar genomes of thousands of plant species are readily available in public databases, enabling researchers without access to molecular biology tools to investigate phylogenetic relationships by sequence comparisons using the appropriate nucleotide substitution models and tree building algorithms. In the present review, the statistical models and algorithms used to reconstruct phylogenetic trees are introduced and advances in the exploration and utilization of plant genomes for molecular phylogenetic analyses are discussed.     

Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae)

The internal transcribed spacer 2 (ITS2) is a small non-coding region located inside the nuclear ribosomal DNA cluster. ITS2 sequence variability is thought to be appropriate to differentiate species and for phylogenetic reconstructions analyses, which can be further improved if structural information is considered. We evaluated the potential of ITS2 as a molecular marker for phylogenetic inference in Calliphoridae (Diptera: Brachycera) using a broad range of inference methods and different substitution models, accounting or not for structural information. Sequence analyses revealed a hierarchically organized pattern of sequence variation and a small level of nucleotide substitution saturation. Intragenomic variation due to small sequence repeats was found mainly in the most variable domain (IV), but it has no significant impact on the phylogenetic signal at the species level. Inferred secondary structures revealed that GC pairs are more frequently found flanking bulges and loops regions in more conserved domains, thus ensuring structure stability. In the phylogenetic analyses, the use of substitution models accounting for structural information significantly improves phylogenetic inference in both neighbour-joining and Bayesian analyses, although the former provides limited resolution for dealing with highly divergent sequences. For Bayesian analyses, a significant improvement in likelihood was observed when considering structure information, although with small changes in topology and overall support, probably reflecting better evolutionary rates estimates. Based on these findings, ITS2 is a suitable molecular marker for phylogenetic analyses in Calliphoridae, at both species and generic level.     

Phylogeny reconstruction in the gap between too little and too much divergence: the closest relatives of Senecio jacobaea (Asteraceae) according to DNA sequences and AFLPs

The species composition and molecular phylogeny of Senecio sect. Jacobaea (Asteraceae; Senecioneae) were studied to identify the closest relatives of Senecio jacobaea. Maximum parsimony and Bayesian inference analyses of DNA sequence data of the plastid (the trnT-L igs, the trnL intron, two parts of the trnK intron, and the psbA-trnH igs) and nuclear genome (ITS1, 5.8S, and ITS2) showed these markers to be suitable to assess the species composition of sect. Jacobaea, identifying 24 species as members of this section. Of these, nine species were not previously assigned to the section. The selected DNA sequence regions, however, showed too little sequence divergence to be optimal for phylogenetic inference within sect. Jacobaea. In contrast, AFLPs proved to be too variable to be used to study relationships between the basal lineages in sect. Jacobaea. Nonetheless, these markers are very useful to study the phylogeny of S. jacobaea and its closest relatives. The combined use of DNA sequence data and AFLPs allowed us to take a major step towards resolving phylogenetic relationships in sect. Jacobaea, identifying Senecio alpinus, Senecio pancicii and Senecio subalpinus (using DNA sequence data) or Senecio chrysanthemoides (using AFLPs) as the closest relatives of S. jacobaea.     

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.     

Chloroplast genome sequences from total DNA for plant identification

Chloroplast DNA sequence data are a versatile tool for plant identification or barcoding and establishing genetic relationships among plant species. Different chloroplast loci have been utilized for use at close and distant evolutionary distances in plants, and no single locus has been identified that can distinguish between all plant species. Advances in DNA sequencing technology are providing new cost‐effective options for genome comparisons on a much larger scale. Universal PCR amplification of chloroplast sequences or isolation of pure chloroplast fractions, however, are non‐trivial. We now propose the analysis of chloroplast genome sequences from massively parallel sequencing (MPS) of total DNA as a simple and cost‐effective option for plant barcoding, and analysis of plant relationships to guide gene discovery for biotechnology. We present chloroplast genome sequences of five grass species derived from MPS of total DNA. These data accurately established the phylogenetic relationships between the species, correcting an apparent error in the published rice sequence. The chloroplast genome may be the elusive single‐locus DNA barcode for plants.     

Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of Ribosomal DNA in Winteraceae

The internal transcribed spacers and the 5.8S coding region of nuclear ribosomal DNA were sequenced and analyzed to address questions of generic relationships in Winteraceae. The molecular data generated a single tree that is congruent with one based on morphological data. The sequences of ITS 1 in the family range from 235 to 252 bases in size and of ITS 2 from 213 to 226 bases. The size of the 5.8S coding region is 164 bases. The range of ITS 1 and ITS 2 sequence divergence between pairs of genera within Winteraceae is relatively low in comparison to other plant families. Two types of ITS 1 and ITS 2 sequences were observed in the same individual for some taxa. Sequence variations between the two arrays are 4.7%–6.3% for ITS 1 and 5.1%–7.0% for ITS 2. Both arrays of sequences, however, generate the same phylogenetic relationships. Rates of nucleotide substitutions for the internal transcribed spacers are 3.2–5.2 × 10^-10 substitution per site per year estimated in ITS 1 and 3.6–5.7 × 10^-10 in ITS 2.     

Phylogenetic utility of the external transcribed spacer (ETS) of 18S-26S rDNA: congruence of ETS and ITS trees of Calycadenia (Compositae)

The 3' region of the external transcribed spacer (ETS) of 18S-26S nuclear ribosomal DNA was sequenced in 19 representatives of Calycadenia/Osmadenia and two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3' end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheae sensu lato was designed to amplify and sequence directly the 3' ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheae sensu lato from approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor of Calycadenia/Osmadenia. Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats in Calycadenia/Osmadenia have evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.     

Identification of astigmatid mites using the second internal transcribed spacer (ITS2) region and its application for phylogenetic study

The second internal transcribed spacer (ITS2) of nuclear ribosomal DNA from 73 specimens of Astigmata was analyzed by PCR amplification and DNA sequencing. The length of the ITS2 region varied from 282 to 592 bp. The interspecific variation based on consensus sequences was more than 4.1%, while the intraspecific or intra-individual variation was from 0 to 5.7%. The variation between geographically separated populations (0–3.2%) was almost the same as the variation within strains. The sequences of the ITS2 region of Astigmata were concluded to be species-specific. The phylogenetic tree inferred from the ITS2 region supported Zachvatkins morphological classification in the subfamily Rhizoglyphinae. The species-specific ITS2 sequence is useful for the species identification of astigmatid mites and for studying low-level phylogenetic relationships.Chemical Ecology of Astigmatid Mites LXXVThis revised version was published online in May 2005 with a corrected cover date.     

Molecular confirmation of the hybrid origin of Eupatorium godfreyanum (Asteraceae)

Analysis of nuclear ribosomal ITS sequence data was used to assess the relationships of Eupatorium godfreyanum, an agamospermous polyploid species of putative hybrid origin. A data set of ITS sequences that included representatives of all but two of the North American species of Eupatorium was compiled from a combination of previously published and newly obtained results. Assessment of the data showed that each species was relatively distinctive, although the results from parsimony analysis suggested that there was little phylogenetic structure within the data beyond a basal split between members of the dog fennel group ("Traganthes") and the remainder of the genus. Cloning was required to obtain readable ITS sequence from E. godfreyanum, and analysis of individual clones produced sequences that matched closely those of either E. rotundifolium or E. sessilifolium. The ITS sequence data thus supported the hypothesis that Eupatorium godfreyanum is of hybrid origin from a combination of E. rotundifolium and E. sessilifolium.     

DNA序列在悬钩子属植物分子系统学研究中的应用进展

悬钩子属植物种类繁多,类群复杂,而且多为多倍体和杂种。该文就近年来国内外有关DNA序列在悬钩子属植物分子系统学研究中的应用现状和进展进行了综述,并对中国悬钩子属植物系统发育研究进行了展望。研究认为:叶绿体DNA序列多应用非编码区,且多与ITS序列联合分析;核基因组中ITS序列应用最为广泛,主要用于研究悬钩子属空心莓组与木莓组的进化关系、栽培品种间亲缘关系及部分杂种和多倍体的起源等;在该属植物中发现了ITS个体内多态性,但未进行ITS假基因检测,其系统学应用价值需重新评价;低拷贝核基因只有GBSSI和LEAFY有相关应用。同时认为,悬钩子属植物系统学研究中应用的DNA序列及研究类群均较少,缺乏对整个悬钩子属全面而系统的研究。指出应进一步选择具有代表性的样本、筛选合适的DNA片段,并结合形态学、孢粉学和细胞学等手段对中国悬钩子属植物系统关系进行深入研究。     

Better the devil you know? Guidelines for insightful utilization of nrDNA ITS in species-level evolutionary studies in plants

The internal transcribed spacers (ITS) of the nuclear ribosomal 18S-5.8S-26S cistron continue to be the most popular non-plastid region for species-level phylogenetic studies of plant groups despite the early warnings about their potential flaws, which may ultimately result in incorrect assumptions of orthology. It has been gradually realized that the alternative target regions in the nuclear genome (low-copy nuclear genes, LCNG) are burdened with similar problems. The consequence is that, to date, developing useful LCNG for non-model organisms requires an investment in time and effort that hinders its use as a real practical alternative for many labs. It is here argued that ITS sequences, despite drawbacks, can still produce insightful results in species-level phylogenetic studies or when non-anonymous nuclear markers are required, provided that a thoughtful use of them is made. To facilitate this, two series of guidelines are proposed. One helps to circumvent problems of ITS amplification from the target organism, including spurious results from contaminants, paralogs and pseudogenes, as well as detection of sequencing artifacts. The other series helps to find out causes for unresolved clades in phylogenetic reconstruction, to integrate gene phylogenies, to distinguish horizontal transfer from lineage sorting, and to reveal if ITS phylogeny is not a good estimate of organism phylogeny.     

Phylogeny of Populus (Salicaceae) based on nucleotide sequences of chloroplast TRNT-TRNF region and nuclear rDNA

The species of the genus Populus, collectively known as poplars, are widely distributed over the northern hemisphere and well known for their ecological, economical, and evolutionary importance. The extensive interspecific hybridization and high morphological diversity in this group pose difficulties in identifying taxonomic units for comparative evolutionary studies and systematics. To understand the evolutionary relationships among poplars and to provide a framework for biosystematic classification, we reconstructed a phylogeny of the genus Populus based on nucleotide sequences of three noncoding regions of the chloroplast DNA (intron of trnL and intergenic regions of trnT-trnL and trnL-trnF) and ITS1 and ITS2 of the nuclear rDNA. The resulting phylogenetic trees showed polyphyletic relationships among species in the sections Tacamahaca and Aigeiros. Based on chloroplast DNA sequence data, P. nigra had a close affinity to species of section Populus, whereas nuclear DNA sequence data suggested a close relationship between P. nigra and species of the section Aigeiros, suggesting a possible hybrid origin for P. nigra. Similarly, the chloroplast DNA sequences of P. tristis and P. szechuanica were similar to that of the species of section Aigeiros, while the nuclear sequences revealed a close affinity to species of the section Tacamahaca, suggesting a hybrid origin for these two Asiatic balsam poplars. The incongruence between phylogenetic trees based on nuclear- and chloroplast-DNA sequence data suggests a reticulate evolution in the genus Populus.     

ITS regions in diploids of Aegilops (Poaceae) and their phylogenetic implications

The nucleotide sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA in nine diploid species representing six sections of Aegilops were determined by direct sequencing of PCR-amplified DNA fragments. These sequences were aligned with two ITS sequences of additional species from Genbank. Sequence divergences were estimated using Kimura two-parameter model, and the phylogenetic analyses were performed using the maximum parsimony (MP) and the neighbor-joining (NJ) methods with PAUP and PHYLIP, respectively. The sequence divergences between the diploid species varied from 0.5% to 4.68%. The resulting MP tree and NJ tree showed relatively congruent phylogenetic relationships among these species, except Ae. caudata. Particularly, Ae. speltoides was basal within the two trees. The paraphyletic relationships between Ae. speltoides and two species of Sect. Sitopsis, and between Ae. uniaristata and two species of Sect. Comopyrum were supported strongly. The ITS data suggest that currently recognized sections within Aegilops should be reconsidered.     

DNA序列在植物系统学研究中的应用

植物DNA序列由于进化速率上的差异而适用于不同分类阶元的系统发育研究,因此,针对某一特定的系统学问题选择相应合适的分子片段是分子系统学研究中最为关键的一步。在前人研究的基础上,主要讨论了目前分子系统发育和进化研究中一些常用的DNA序列的适用范围,包括nrDNA的18S基因及ITS等非编码区,cpDNA的编码基因（rbcI、matK、ndhF、atpB）及非编码区序列（rpL16、rpoC1、rps16、trnL-F和trnT-L）和应用较少的mtDNA。研究表明,18S、rbcI等编码基因及mtDNA一般适用于较高分类阶元甚至整个种子植物谱系间的系统发育的探讨,而ITS及cpDNA的非编码区序列等因其较快的进化速率多用于较低分类阶元的系统关系研究。