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.     

樟属植物ITS序列多态性分析

对樟科樟属(Cinnamomum Schaeffer) 17个代表样本的核糖体DNA内转录间隔区(nrDNA ITS)进行克隆测序。对获得的87条不同ITS序列的长度变异、GC含量、5.8S区二级结构的稳定性、遗传距离、进化模式以及系统发育关系进行了相关分析。研究结果显示, ITS序列在樟属植物内存在明显的多态性, 87条序列中的22条序列被鉴定为假基因序列, 其余65条序列为功能基因序列; 假基因序列采用中性进化模式, 变异明显大于功能序列。ITS序列在樟属植物中出现一致性进化不完全和假基因现象也可能发生在樟科其它类群中, 这可能是导致樟科植物ITS序列直接测序方式成功率低的重要原因。     

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).     

Nuclear Ribosomal ITS Functional Paralogs Resolve the Phylogenetic Relationships of a Late-Miocene Radiation Cycad Cycas (Cycadaceae)

Cycas is the most widespread and diverse genus among the ancient cycads, but the extant species could be the product of late Miocene rapid radiations. Taxonomic treatments to date for this genus are quite controversial, which makes it difficult to elucidate its evolutionary history. We cloned 161 genomic ITS sequences from 31 species representing all sections of Cycas. The divergent ITS paralogs were examined within each species and identified as putative pseudogenes, recombinants and functional paralogs. Functional paralogs were used to reconstruct phylogenetic relationships with pseudogene sequences as molecular outgroups, since an unambiguous ITS sequence alignment with their closest relatives, the Zamiaceae, is unachievable. A fully resolved and highly supported tree topology was obtained at the section level, with two major clades including six minor clades. The results fully supported the classification scheme proposed by Hill (2004) at the section level, with the minor clades representing his six sections. The two major clades could be recognised as two subgenera. The obtained pattern of phylogenetic relationships, combined with the different seed dispersal capabilities and paleogeography, allowed us to propose a late Miocene rapid radiation of Cycas that might have been promoted by vicariant events associated with the complex topography and orogeny of South China and adjacent regions. In contrast, transoceanic dispersals might have played an important role in the rapid diversification of sect. Cycas, whose members have evolved a spongy layer in their seeds aiding water dispersals.     

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.     

云南山茶(Camellia reticulata) nrDNA ITS序列多态性分析

对云南山茶 (Camellia reticulata) 8个品种的核核糖体DNA内转录间隔区(nrDNA ITS)进行克隆测序,将获得的ITS序列进行GC含量、 5.8S区二级结构的稳定性、替代模式、核苷酸多样性及系统发育关系的相关分析.实验结果显示,云南山茶8个品种的ITS序列存在丰富的基因组内多态性,同时包含中性进化的假基因,表明其ITS序列逃离了一致性进化.云南山茶ITS序列多态性的原因可能来自广泛的种间杂交,以及rDNA位点在基因组中有不确定的物理位置.ITS假基因为品种的物种形成研究提供了更全面的遗传证据,同时也提示了利用ITS假基因进行系统发育分析可能会对其真实的系统关系造成影响.     

ITS secondary structure derived from comparative analysis: implications for sequence alignment and phylogeny of the Asteraceae

An RNA secondary structure model is presented for the nuclear ribosomal internal transcribed spacers (ITS) based on comparative analysis of 340 sequences from the angiosperm family Asteraceae. The model based on covariation analysis agrees with structural features proposed in previous studies using mainly thermodynamic criteria and provides evidence for additional structural motifs within ITS1 and ITS2. The minimum structure model suggests that at least 20% of ITS1 and 38% of ITS2 nucleotide positions are involved in base pairing to form helices. The sequence alignment enabled by conserved structural features provides a framework for broadscale molecular evolutionary studies and the first family-level phylogeny of the Asteraceae based on nuclear DNA data. The phylogeny based on ITS sequence data is very well resolved and shows considerable congruence with relationships among major lineages of the family suggested by chloroplast DNA studies, including a monophyletic subfamily Asteroideae and a paraphyletic subfamily Cichorioideae. Combined analyses of ndhF and ITS sequences provide additional resolution and support for relationships in the family.     

ITS sequence data resolve higher level relationships among the eucalypts.

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA were obtained for 35 species of Eucalyptus s.s. and seven taxa representing five outgroup genera (Allosyncarpia, Angophora, Arillastrum, Corymbia, and Stockwellia). The sequences were analyzed cladistically. The data distinguished clearly between the two major subgenera of Eucalyptus s.s. (Symphyomyrtus and Monocalyptus) but indicated that subgenus Eudesmia may be paraphyletic. ITS sequence data demonstrated the potential to resolve relationships between sections within subgenus Symphyomyrtus. Within sections, however, taxa were poorly differentiated. At the generic level, Corymbia appeared to be paraphyletic due to the exclusion of Angophora. The positions of Allosyncarpia and Arillastrum relative to the ingroup remain unresolved. ITS sequence data may prove valuable for resolving other phylogenetic relationships at higher taxonomic levels within Eucalyptus.     

Non-concerted ITS evolution in Mammillaria (Cactaceae)

Molecular studies of 21 species of the large Cactaceae genus Mammillaria representing a variety of intrageneric taxonomic levels revealed a high degree of intra-individual polymorphism of the internal transcribed spacer region (ITS1, 5.8S rDNA, ITS2). Only a few of these ITS copies belong to apparently functional genes, whereas most are probably non-functional (pseudogenes). As a multiple gene family, the ITS region is subjected to concerted evolution. However, the high degree of intra-individual polymorphism of up to 36% in ITS1 and up to 35% in ITS2 suggests a non-concerted evolution of these loci in Mammillaria. Conserved angiosperm motifs of ITS1 and ITS2 were compared between genomic and cDNA ITS clones of Mammillaria. Some of these motifs (e.g., ITS1 motif 1, 'TGGT' within ITS2) in combination with the determination of GC-content, length comparisons of the spacers and ITS2 secondary structure (helices II and III) are helpful in the identification of pseudogene rDNA regions.     

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.     

Molecular phylogeny of North American Branchiobdellida (Annelida: Clitellata)

Branchiobdellidans, or crayfish worms, are ectosymbiotic clitellate annelids associated primarily with freshwater crayfishes. The main objectives of our study were to infer a molecular phylogeny for the North American Branchiobdellida, examine its congruence with morphology-based hypotheses of relationships at the subfamily and genus level, and use our dataset to assess consistency of GenBank-archived branchiobdellidan sequences. We used nucleotide sequence data from two mtDNA genes (COI and 16S rDNA) and three nuclear genes (28S rDNA, 18S rDNA, and ITS1) to estimate phylogenetic relationships among 47 described and one undescribed species of Branchiobdellida. We recovered a monophyletic branchiobdellidan clade with generally short branch lengths, suggesting that a large portion of the taxon has likely undergone a recent and rapid radiation in North America. Results from our phylogenetic analyses indicate that current taxonomic groupings are largely unsupported by the molecular data. All four subfamilies are either paraphyletic or polyphyletic, and only three of seven sampled non-monotypic genera were monophyletic. We found a high rate (49%) of inconsistency in GenBank-archived sequences, over 70% of which can be attributed to field- or laboratory-based error.     

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.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Phylogeny and cryptic diversity in geckos (Phyllopezus; Phyllodactylidae; Gekkota) from South America's open biomes

The gecko genus Phyllopezus occurs across South America's open biomes: Cerrado, Seasonally Dry Tropical Forests (SDTF, including Caatinga), and Chaco. We generated a multi-gene dataset and estimated phylogenetic relationships among described Phyllopezus taxa and related species. We included exemplars from both described Phyllopezus pollicaris subspecies, P. p. pollicaris and P. p.przewalskii. Phylogenies from the concatenated data as well as species trees constructed from individual gene trees were largely congruent. All phylogeny reconstruction methods showed Bogertia lutzae as the sister species of Phyllopezus maranjonensis, rendering Phyllopezus paraphyletic. We synonymized the monotypic genus Bogertia with Phyllopezus to maintain a taxonomy that is isomorphic with phylogenetic history. We recovered multiple, deeply divergent, cryptic lineages within P. pollicaris. These cryptic lineages possessed mtDNA distances equivalent to distances among other gekkotan sister taxa. Described P. pollicaris subspecies are not reciprocally monophyletic and current subspecific taxonomy does not accurately reflect evolutionary relationships among cryptic lineages. We highlight the conservation significance of these results in light of the ongoing habitat loss in South America's open biomes.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

A phylogeny of all species of Arceuthobium (Viscaceae) using nuclear and chloroplast DNA sequences

The genus Arceuthobium (dwarf mistletoes, Viscaceae) comprises 42 species that parasitize hosts in Pinaceae and Cupressaceae in the Old and New Worlds. Maximum parsimony analyses were conducted on two data partitions (separately and combined): nuclear ribosomal internal transcribed spacer (ITS) sequences for all 42 currently recognized species and chloroplast trnT-L-F sequences for 34 New World species. The Old and New World species were phylogenetically distinct using ITS, thus making subgenus Arceuthobium paraphyletic. Arceuthobium pendens and A. guatemalense comprise the basalmost clade of subgenus Vaginata, characterized by the presence of flabellate secondary branching. The trnT-L-F sequences, which vary widely in length depending upon taxon, contain three times less phylogenetic signal than ITS, although homoplasy for this partition is lower. Several of the clades obtained from analysis of nuclear ITS sequences are also recovered using trnT-L-F sequences such as A. guatemalense and A. pendens, the A. rubrum group, the A. vaginatum group, and the A. campylopodum group. The ITS + trnT-L-F tree is well resolved except for four internal nodes. A revised classification of the genus is discussed that recognizes only monophyletic species that are well differentiated by molecular data.     

Phylogeny and host-plant association in the leaf beetle genus Trirhabda LeConte (Coleoptera: Chrysomelidae)

The leaf beetle genus Trirhabda contains 26 described species from the United States and Canada, feeding on host plants from the families Asteraceae and Hydrophyllaceae. In this study, we present a phylogeny for the genus that was reconstructed from mitochondrial COI and 12S rRNA fragments, nuclear ITS2 rRNA, and morphological characters. Both parsimony and mixed-model Bayesian likelihood analyses were performed. Under both methods, the mitochondrial and nuclear partitions support the same backbone phylogeny, as do the combined data. The utility of the molecular data is contrasted with the low phylogenetic signal among morphological characters. The phylogeny was used to trace the evolution of the host-plant association in Trirhabda. The recovered phylogeny shows that although the host-plant association is phylogenetically conservative, Trirhabda experienced one shift to a distantly related host-plant family, 6 shifts between host-plant tribes, and 6 between genera within tribes. The phylogeny reveals that Trirhabda were plesiomorphically adapted to tolerate complex secondary compounds of its host plants and this adaptation is retained in Trirhabda species, as evidenced by multiple shifts from chemically simpler host plants back to the more complex host plants.     

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。