苏铁nrDNA ITS区的序列多态性:不完全致同进化的证据

本研究对苏铁(Cycas revoluta) nrDNA ITS进行克隆测序, 并以cDNA ITS为参照, 比较分析获得的序列的碱基变异、GC含量、5.8S二级结构的稳定性和5.8S保守基序的有无以及系统发育关系。结果发现苏铁nrDNA ITS存在较高的基因组内多样性, 同时, 这些分化的nrDNA ITS拷贝中包含有假基因的存在, 而且假基因与功能拷贝之间已经形成了较大的遗传分化, 这暗示假基因起源有较长历史。苏铁核仁组织区不仅多达16个, 而且分布在13条染色体上, 这可能是其nrDNA ITS致同进化不完全的主要原因。     

Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.     

Evolution of the 5.8S nrDNA gene and internal transcribed spacers in Carapichea ipecacuanha (Rubiaceae) within a phylogeographic context

Nuclear ribosomal DNA (nrDNA) constitutes a multicopy gene family that is used widely to test evolutionary hypotheses across a broad range of organisms. It is presumed that, as a result of concerted evolution, tandem nrDNA repeats are homogeneous within species and different between species. We sampled 77 specimens of a disjunct species (Carapichea ipecacuanha) from throughout its three geographic ranges and obtained 266 nrDNA sequences, of which 26 were obtained by direct sequencing and 240 by cloning of PCR products. Complementary sequence analyses, which included analyses of secondary structure stability, the pattern of base substitutions, GC content, and the presence of conserved motifs, were used to characterize the internal transcribed spacer (ITS) region (ITS1-5.8S nrDNA-ITS2). Our results showed that concerted evolution of the ITS region was incomplete in C. ipecacuanha, particularly in the Atlantic range. In the highly polymorphic populations of the Atlantic range, intraindividual variation was observed and involved 56 functional paralogs and 15 pseudogenes from two highly divergent ribogroups. The Amazonian range (with 12 functional paralogs) and the Central-American range (with five functional paralogs) were genetically depauperate and exhibited no pseudogenes. In the two latter ranges, almost complete homogenization of the ITS sequences had occurred. We argue that it is important to consider past evolutionary history when making inferences about the efficiency with which concerted evolution homogenizes tandem nrDNA repeats a single sequence.     

Divergent and reticulate species relationships in Leucaena (Fabaceae) inferred from multiple data sources: insights into polyploid origins and nrDNA polymorphism

Previous analyses of species relationships and polyploid origins in the mimosoid legume genus Leucaena have used chloroplast DNA (cpDNA) restriction site data and morphology. Here we present an analysis of a new DNA sequence data set for the nuclear ribosomal DNA (nrDNA) 5.8S subunit and flanking ITS 1 and ITS 2 spacers, a simultaneous analysis of the morphology, ITS and cpDNA data sets for the diploid species, and a detailed comparison of the cpDNA and ITS gene trees, which include multiple accessions of all five tetraploid species. Significant new insights into species relationships and polyploid origins, including that of the economically important tropical forage tree L. leucocephala, are discussed. Heterogeneous ITS copy types, including 26 putative pseudogene sequences, were found within individuals of four of the five tetraploid and one diploid species. Potential pseudogenes were identified using two pairwise comparison approaches as well as a tree-based method that compares observed and expected proportions of total ITS variation contributed by the 5.8S subunit optimized onto branches of one of the ITS gene trees. Inclusion of putative pseudogene sequences in the analysis provided evidence that some pseudogenes in allopolyploid L. leucocephala are not the result of post-allopolyploidization gene silencing, but were inherited from its putative diploid maternal progenitor L. pulverulenta.     

The Evolution of Ribosomal DNA: Divergent Paralogues and Phylogenetic Implications

Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serve as good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies.     

Non-concerted ITS evolution, early origin and phylogenetic utility of ITS pseudogenes in Pyrus

Molecular studies of 19 species of the genus Pyrus L. revealed different degrees of intra-individual polymorphism of the internal transcribed spacer (ITS 1, 5.8S rDNA and ITS 2) region due to the existence of putative non-functional copies (pseudogenes), putative recombinants and non-concerted evolution among functional copies. Different types of ITS pseudogenes displaying lower GC content and unstable secondary structure were preferentially amplified under normal PCR conditions. Functional ITS copies were successfully obtained in all investigated accessions under the modified PCR conditions. All pseudogenes were highly divergent from their corresponding functional copies and formed a monophyletic group in the phylogenetic tree based on all paralogs, indicating they were of relatively early origin. Functional ITS copies led to confused and poorly resolved phylogeny as a result of low sequence divergence, existence of unidentified ancient recombinants and a high degree of intra-individual functional ITS polymorphism, while certain types of pseudogenes and some relict pseudogenes offered more credible clues for the evolutionary history of Pyrus species.     

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

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

Recent origin and phylogenetic utility of divergent ITS putative pseudogenes: a case study from Naucleeae (Rubiaceae)

The internal transcribed spacer (ITS) of nuclear ribosomal DNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. Although the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make unambiguous alignment difficult. In addition, long branches often can lead to spurious relationships, particularly in parsimony analyses. We have discovered unusually high levels of ITS polymorphism (up to 30%, 40%, and 14%, respectively) in three tropical tree species of the coffee family (Rubiaceae), Adinauclea fagifolia, Haldina cordifolia, and Mitragyna rubrostipulata. Both secondary structure stability and patterns of nucleotide substitutions in a highly conserved region (5.8S gene) were used for distinguishing presumed functional sequences from putative pseudogenes. The combination of both criteria was the most powerful approach. The sequences from A. fagifolia appear to be a mix of functional genes and highly distinct putative pseudogenes, whereas those from H. cordifolia and M. rubrostipulata were identified as putative pseudogenes. We explored the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identified the same monophyletic groups and indicated that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. The resulting trees are similar to those produced by previous analyses of chloroplast genes. In contrast to results of previous studies therefore, divergent putative pseudogenes can be useful for phylogenetic analyses, especially when no sequences of their functional counterparts are available. Our studies clearly show that ITS polymorphism may not necessarily mislead phylogenetic inference. Despite using many different PCR conditions (different primers, higher denaturing temperatures, and absence or presence of DMSO and BSA-TMACl), we recovered only a few functional ITS copies from A. fagifolia and none from H. cordifolia and M. rubrostipulata, which suggests that PCR selection is occurring and/or the presumed functional alleles are located at minor loci (with few ribosomal DNA copies).     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.      

Extensive 5.8S nrDNA polymorphism in <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae) with special reference to the identification of pseudogenic internal transcribed spacer regions

The internal transcribed spacer (ITS) region (ITS1, 5.8S rDNA, ITS2) represents the most widely applied nuclear marker in eukaryotic phylogenetics. Although this region has been assumed to evolve in concert, the number of investigations revealing high degrees of intra-individual polymorphism connected with the presence of pseudogenes has risen. The 5.8S rDNA is the most important diagnostic marker for functionality of the ITS region. In Mammillaria, intra-individual 5.8S rDNA polymorphisms of up to 36% and up to nine different types have been found. Twenty-eight of 30 cloned genomic Mammillaria sequences were identified as putative pseudogenes. For the identification of pseudogenic ITS regions, in addition to formal tests based on substitution rates, we attempted to focus on functional features of the 5.8S rDNA (5.8S motif, secondary structure). The importance of functional data for the identification of pseudogenes is outlined and discussed. The identification of pseudogenes is essential, because they may cause erroneous phylogenies and taxonomic problems. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes

Many early reports of ITS region (ITS 1, 5.8S, and ITS 2) variation in flowering plants indicated that nrDNA arrays within individuals are homogeneous. However, both older and more recent studies have found intra-individual nrDNA polymorphism across a range of plant taxa including presumed non-hybrid diploids. In addition, polymorphic individuals often contain potentially non-functional nrDNA copies (pseudogenes). These findings suggest that complete concerted evolution should not be assumed when embarking on phylogenetic studies using nrDNA sequences. Here we (1). discuss paralogy in relation to species tree reconstruction and conclude that a priori determinations of orthology and paralogy of nrDNA sequences should not be made based on the functionality or lack of functionality of those sequences; (2). discuss why systematists might be particularly interested in identifying and including pseudogene sequences as a test of gene tree sampling; (3). examine the various definitions and characterizations of nrDNA pseudogenes as well as the relative merits and limitations of a subset of pseudogene detection methods and conclude that nucleotide substitution patterns are particularly appropriate for the identification of putative nrDNA pseudogenes; and (4). present and discuss the advantages of a tree-based approach to identifying pseudogenes based on comparisons of sequence substitution patterns from putatively conserved (e.g., 5.8S) and less constrained (e.g., ITS 1 and ITS 2) regions. Application of this approach, through a method employing bootstrap hypothesis testing, and the issues discussed in the paper are illustrated through reanalysis of two previously published matrices. Given the apparent robustness of the test developed and the ease of carrying out percentile bootstrap hypothesis tests, we urge researchers to employ this statistical tool. While our discussion and examples concern the literature on plant systematics, the issues addressed are relevant to studies of nrDNA and other multicopy genes in other taxa.     

A Phylogeographic Split in Buxus balearica (Buxaceae) as Evidenced by Nuclear Ribosomal Markers: When ITS Paralogues Are Welcome

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin. Intragenomic polymorphic sites were detected for western and eastern accessions of B. balearica and in two accessions of the sister species B. sempervirens. Overall, 19 different ribotypes were found in B. balearica after sequencing 48 clones, whereas 15 ribotypes were detected in 19 clones of B. sempervirens. The integrity and secondary structure stability of the ribosomal sequences suggest that they are not pseudogenes. The high number of ribotypes recovered through cloning suggested that some sequences could be chimeric or generated in vivo by partial homogenization through gene conversion or unequal crossing-over. Average sequence divergence among B. balearica clones was 0.768%, and the most divergent sequences differed by 1.62%. Available evidence does not suggest that B. balearica paralogues have been obtained from other extant Buxus species through interspecific hybridization. The presence of several ribosomal sequences in box implies that the molecular forces driving the concerted evolution of this multigene family are not fully operational in this genus. Phylogenetic analyses of cloned ITS sequences from B. balearica displayed very poor resolution and only two clades received moderate bootstrap support. Despite the marked intragenomic sequence divergence found, ribosomal data suggest a clear phylogeographic split in B. balearica between western and eastern accessions. The distinct, nonchimeric sequences that are postulated as being present in each biogeographic group suggest that box populations from Anatolia (eastern Mediterranean) are relict. [Reviewing Editor: Dr. Rafael Zardoya]     

四照花亚属的nrDNA ITS致同进化不完全

四照花亚属(Cornus subg.Syncarpea)隶属于山茱萸科山茱萸属(Cornus),我国该亚属共有5种8亚种。为探讨四照花亚属nrDNA ITS序列的致同进化不完全现象及假基因产生的可能原因,分析了该亚属4种(每种1~2个居群)共21个个体的nrDNA ITS序列。结果表明,这些类群的nrDNA ITS存在多态性,通过分析这些nrDNA ITS克隆序列的G+C含量、5.8S保守基序和二级结构最小自由能,推测其可能存在假基因。系统发育研究结果显示所有nrDNA ITS序列分成5个分支,同一个体的不同拷贝被分别置于两个甚至多个分支中,且不同分支显示了不同种间关系。四照花亚属物种个体内部存在nrDNA ITS不完全致同进化,可能归咎于不完全的世系分选(incomplete lineage sorting)、种间杂交或多倍化等进化事件,从而导致基因组内nrITS区序列出现多态性,同时也导致难以通过外部形态来划分亚属内种间界限。     

Reticulate evolution and the origins of ribosomal internal transcribed spacer diversity in apomictic Meloidogyne

Among root knot nematodes of the genus Meloidogyne, the polyploid obligate mitotic parthenogens M. arenaria, M. javanica, and M. incognita are widespread and common agricultural pests. Although these named forms are distinguishable by closely related mitochondrial DNA (mtDNA) haplotypes, detailed sequence analyses of internal transcribed spacers (ITSs) of nuclear ribosomal genes reveal extremely high diversity, even within individual nematodes. This ITS diversity is broadly structured into two very different groups that are 12%-18% divergent: one with low diversity (< 1.0%) and one with high diversity (6%-7%). In both of these groups, identical sequences can be found within individual nematodes of different mtDNA haplotypes (i.e., among species). Analysis of genetic variance indicates that more than 90% of ITS diversity can be found within an individual nematode, with small but statistically significant (5%-10%; P < 0.05) variance distributed among mtDNA lineages. The evolutionarily distinct parthenogen M. hapla shows a similar pattern of ITS diversity, with two divergent groups of ITSs within each individual. In contrast, two diploid amphimictic species have only one lineage of ITSs with low diversity (< 0.2%). The presence of divergent lineages of rDNA in the apomictic taxa is unlikely to be due to differences among pseudogenes. Instead, we suggest that the diversity of ITSs in M. arenaria, M. javanica, and M. incognita is due to hybrid origins from closely related females (as inferred from mtDNA) and combinations of more diverse paternal lineages.     

Ribosomal ITS sequences and plant phylogenetic inference

One of the most popular sequences for phylogenetic inference at the generic and infrageneric levels in plants is the internal transcribed spacer (ITS) region of the 18S-5.8S-26S nuclear ribosomal cistron. The prominence of this source of nuclear DNA sequence data is underscored by a survey of phylogenetic publications involving comparisons at the genus level or below, which reveals that of 244 papers published over the last five years, 66% included ITS sequence data. Perhaps even more striking is the fact that 34% of all published phylogenetic hypothesis have been based exclusively on ITS sequences. Notwithstanding the many important contributions of ITS sequence data to phylogenetic understanding and knowledge of genome relationships, a number of molecular genetic processes impact ITS sequences in ways that may mislead phylogenetic inference. These molecular genetic processes are reviewed here, drawing attention to both underlying mechanism and phylogenetic implications. Among the most prevalent complications for phylogenetic inference is the existence in many plant genomes of extensive sequence variation, arising from ancient or recent array duplication events, genomic harboring of pseudogenes in various states of decay, and/or incomplete intra- or inter-array homogenization. These phenomena separately and collectively create a network of paralogous sequence relationships potentially confounding accurate phylogenetic reconstruction. Homoplasy is shown to be higher in ITS than in other DNA sequence data sets, most likely because of orthology/paralogy conflation, compensatory base changes, problems in alignment due to indel accumulation, sequencing errors, or some combination of these phenomena. Despite the near-universal usage of ITS sequence data in plant phylogenetic studies, its complex and unpredictable evolutionary behavior reduce its utility for phylogenetic analysis. It is suggested that more robust insights are likely to emerge from the use of single-copy or low-copy nuclear genes.