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.     

Divergent paralogues of ribosomal DNA in eucalypts (Myrtaceae)

The presence of divergent paralogues of nuclear ribosomal DNA, from the 18S-5.8S-26S cistron, is reported in members of Eucalyptus subg. Eucalyptus. These paralogues, which include non-functional pseudogenes, probably diverged prior to the differentiation of species groups in subg. Eucalyptus. When compared with presumably functional sequences, the pseudogenes show greater sequence variation between species, particularly in the 5.8S gene. They are also characterised by reduced GC content, associated with a reduced number of CpG and CpNpG methylation sites, and an increase in the inferred number of methylation-induced substitutions. Some pseudogenes also lack motifs that are usually conserved in plants, both in ITS1 and the 5.8S gene. Two main lineages of pseudogenes are identified, one isolated from a group of western Australian species, one from a group of eastern Australian species. It is not clear whether these two lineages of pseudogenes are orthologous, or represent independent divergences from functional sequence types. The presence of divergent rDNA paralogues highlights the need for caution when interpreting eucalypt phylogenies based on ITS sequences.     

A cladistic study of Arillastrum, Angophora and Eucalyptus (Myrtaceae)

A cladistic study of Anllastrum, Angophora and Eucalyptus (Myrtaceae). Transformed cladistic; character compatibility; branch and bound, and Farris-Wagner methods gave similar solutions in a cladistic study of Arillastrum, Angophora and Eucalyptus. These analyses, based on morphological characters, indicate that Eucalyptus is a monophyletic group and that its sister taxon is Angophora.
Within Eucalyptus , subgenera Blakella and Corymbia are sister taxa to all other groups; subgenera Monocalyptus, Idiogenes and Gaubaea form a monophyletic group with subgenus Monocalyptus sister to subgenera Idiogenes and Gaubaea ; subgenera Symphyomyrtus and Telocalyptus together also form a monophyletic group and, with Eucalyptus similis (subgenus Eudesmia group 4), are sister to the Monocalyptus group. Eucalyptus subgenus Telocalyptus (4 species), Eucalyptus subgenus Idiogenes (1 species) and Eucalyptus subgenus Gaubaea (2 species) should not be recognized as subgenera and some individual species need further examination. Eucalyptus subgenus Eudesmia is a paraphyletic group.
Some characters are identified as parallelisms, e.g. axillary inflorescences, sepaline operculum, bristle glands, and clustered anthers. A more congruent interpretation of the single operculum of Eucalyptus subgenus Monocalyptus as at least partly petaline rather than solely sepaline in origin is suggested.
The area relationships for the taxa are concordant with those derived from geological and climatological information. New Caledonia is sister area to Australia, and within Australia southwestern Australia is sister area to south-eastern and north-eastern Australia.     

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.     

Pseudogenes and DNA-based diet analyses: a cautionary tale from a relatively well sampled predator-prey system

Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1-5 pseudogene haplotypes per sample representing 5-100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA recovered per sample were highly variable and appear to be related to PCR cycling characteristics. This is a well-sampled system where we can reliably identify the putative pseudogenes and separate them from their mitochondrial paralogues using a number of recommended means. In many other cases, it would be virtually impossible to determine whether a putative prey sequence is actually a pseudogene derived from either the predator or prey DNA. The implications of this for DNA-based dietary studies, in general, are discussed.     

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.     

New species of Mycosphaerella from Myrtaceae in plantations and native forests in eastern Australia

The majority of Mycosphaerella species from eucalypts (Eucalyptus, Corymbia and Angophora) in Australia have been recorded only from trees growing in plantations. This illustrates a bias in research in the past two decades toward commercial enterprise, and it emphasises a lack of understanding of the occurrence of these important fungi under natural conditions. Surveys of foliar fungi in native forests in eastern Australia, as well as adjacent plantations, thus have been initiated in recent years. In this study we describe four new species of Mycosphaerella from Eucalyptus spp. as well as other Myrtaceae. Mycosphaerella tumulosa sp. nov. (anamorph: Pseudocercospora sp.) was found on more than seven species of Eucalyptus and Corymbia in native forests and plantations in northeastern New South Wales and southeastern Queensland and appears to be relatively common, although not damaging to these trees. Mycosphaerella multiseptata sp. nov. was recorded from several locations on species of Angophora in native forests and amenity plantings. Mycosphaerella pseudovespa sp. nov. was found in one location in native forest on E. biturbinata. The first species of Mycosphaerella to be described from Syncarpia, M. syncarpiae sp. nov., was found in native forests in numerous locations from Sydney through to northeastern New South Wales and appears to be relatively common.     

Flow cytometry and cytogenetic tools in eucalypts: genome size variation × karyotype stability

The eucalypts comprise a group of woody plants used in commercial forest plantations owing to their high growth rates, adaptability to various ecological conditions and multiple applications. Despite the enormous amount of molecular data available for eucalypts, a basic understanding of the nature of its genome still requires information regarding the DNA amount in the genus. In this work, we estimated the genome size and base composition of 25 eucalypt species. With a comparative karyotype approach, we aimed to identify possible chromosomal alterations correlated with the genome size variation. Classical cytogenetic and genomic in situ hybridization experiments were conducted for this purpose. The studied species showed genome size ranging from 2C = 0.91 (Corymbia intermedia) to 2C = 1.37 pg (Eucalyptus paniculata) and AT/CG ratios varying from AT = 61.3 (Eucalyptus urophylla) to AT = 62.85% (C. intermedia). Comparative karyotype analysis revealed no remarkable differences in chromosome number (2n = 22) or morphology among eucalypt species despite considerable differences in nuclear DNA content. The genome in situ hybridization method did not distinguish non-homologous chromosomal regions of Eucalyptus baileyana and Corymbia citriodora, despite the difference of 0.45 pg between their genome sizes. The results found in the present work corroborate the consideration of small and dispersed DNA changes as the main cause of genome size variation in eucalypts.     

The vestigial olfactory receptor subgenome of odontocete whales: phylogenetic congruence between gene-tree reconciliation and supermatrix methods

The macroevolutionary transition of whales (cetaceans) from a terrestrial quadruped to an obligate aquatic form involved major changes in sensory abilities. Compared to terrestrial mammals, the olfactory system of baleen whales is dramatically reduced, and in toothed whales is completely absent. We sampled the olfactory receptor (OR) subgenomes of eight cetacean species from four families. A multigene tree of 115 newly characterized OR sequences from these eight species and published data for Bos taurus revealed a diverse array of class II OR paralogues in Cetacea. Evolution of the OR gene superfamily in toothed whales (Odontoceti) featured a multitude of independent pseudogenization events, supporting anatomical evidence that odontocetes have lost their olfactory sense. We explored the phylogenetic utility of OR pseudogenes in Cetacea, concentrating on delphinids (oceanic dolphins), the product of a rapid evolutionary radiation that has been difficult to resolve in previous studies of mitochondrial DNA sequences. Phylogenetic analyses of OR pseudogenes using both gene-tree reconciliation and supermatrix methods yielded fully resolved, consistently supported relationships among members of four delphinid subfamilies. Alternative minimizations of gene duplications, gene duplications plus gene losses, deep coalescence events, and nucleotide substitutions plus indels returned highly congruent phylogenetic hypotheses. Novel DNA sequence data for six single-copy nuclear loci and three mitochondrial genes (> 5000 aligned nucleotides) provided an independent test of the OR trees. Nucleotide substitutions and indels in OR pseudogenes showed a very low degree of homoplasy in comparison to mitochondrial DNA and, on average, provided more variation than single-copy nuclear DNA. Our results suggest that phylogenetic analysis of the large OR superfamily will be effective for resolving relationships within Cetacea whether supermatrix or gene-tree reconciliation procedures are used.     

Human U1 RNA pseudogenes may be generated by both DNA- and RNA-mediated mechanisms.

Analysis of cloned human genomic loci homologous to the small nuclear RNA U1 established that such sequences are abundant and dispersed in the human genome and that only a fraction represent bona fide genes. The majority of genomic loci bear defective gene copies, or pseudogenes, which contain scattered base mismatches and in some cases lack the sequence corresponding to the 3' end of U1 RNA. Although all of the U1 genes examined to date are flanked by essentially identical sequences and therefore appear to comprise a single multigene family, we present evidence for the existence of at least three structurally distinct classes of U1 pseudogenes. Class I pseudogenes had considerable flanking sequence homology with the U1 gene family and were probably derived from it by a DNA-mediated event such as gene duplication. In contrast, the U1 sequence in class II and III U1 pseudogenes was flanked by single-copy genomic sequences completely unrelated to those flanking the U1 gene family; in addition, short direct repeats flanked the class III but not the class II pseudogenes. We therefore propose that both class II and III U1 pseudogenes were generated by an RNA-mediated mechanism involving the insertion of U1 sequence information into a new chromosomal locus. We also noted that two other types of repetitive DNA sequences in eucaryotes, the Alu family in vertebrates and the ribosomal DNA insertions in Drosophila, bore a striking structural resemblance to the classes of U1 pseudogenes described here and may have been created by an RNA-mediated insertion event.     

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).     

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。     

Quercitol links the physiology, taxonomy and evolution of 279 eucalypt species

Aim  Increasing aridity over geological time-scales has driven a high degree of speciation within the Eucalyptus group in Australia. Isolation of gene pools by climatic and edaphic conditions and high rates of out-crossing have given rise to a large diversity of adaptive traits. Among these traits, adaptations of cellular biochemistry are likely to be significant in preserving cellular function during arid conditions. The aim of this study was to determine the quantitative and qualitative distribution of soluble carbohydrates and polyols in Eucalyptus .
Location  Australia.
Methods  We sampled 279 of the 700+ documented eucalypts (in the three genera comprising the eucalypts: Angophora Cav., Corymbia Hill & Johnson and Eucalyptus L'Hér.) and analysed leaf tissues for the occurrence of low-molecular-weight carbohydrates and polyols.
Results  We have uncovered a discrete pattern in concentration of quercitol (a cyclitol) that correlates strongly with the current taxonomic classification based on both morphology and DNA sequencing. We also uncovered a further and stronger correlation between the presence of quercitol in leaf tissues and a reduced growth (mallee) form.
Main conclusions  These findings, together with the chemical properties of quercitol, suggest that we have uncovered a chemical marker of structural adaptations to arid conditions, thus providing a putative, broad-scale functional link to adaptation to aridity.     

Amplified ribosomal spacer sequence: structure and evolutionary origin

A novel class of repeated sequences consisting of tandem arrays of ribosomal spacer sequence has been discovered in a mouse genome. Comparison to normal ribosomal DNA reveals that one repeat unit consists of two separate parts of spacer sequence. This amplified spacer sequence has a pseudogene-like structure but is distinct from the previously reported pseudogenes and orphons in regions lacking coding sequences. So far the amplified spacer sequence has been found only in the BALB/c mouse genome but not in ten other laboratory strains and several wild-type mouse stocks. Surprisingly, a part of the amplified spacer sequence unit had a higher homology to the corresponding part of the ribosomal DNA sequence of Mus musculus molossinus, a Japanese wild-type mouse, than to the corresponding part of the rDNA of the BALB/c mouse. These findings suggest that the amplified spacer sequence of the BALB/c mouse might have partly originated in M. m. molossinus or in a related subspecies.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

Inferring the history of the polyploid Silene aegaea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.     

Detection of recombination in DNA multiple alignments with hidden Markov models.

Conventional phylogenetic tree estimation methods assume that all sites in a DNA multiple alignment have the same evolutionary history. This assumption is violated in data sets from certain bacteria and viruses due to recombination, a process that leads to the creation of mosaic sequences from different strains and, if undetected, causes systematic errors in phylogenetic tree estimation. In the current work, a hidden Markov model (HMM) is employed to detect recombination events in multiple alignments of DNA sequences. The emission probabilities in a given state are determined by the branching order (topology) and the branch lengths of the respective phylogenetic tree, while the transition probabilities depend on the global recombination probability. The present study improves on an earlier heuristic parameter optimization scheme and shows how the branch lengths and the recombination probability can be optimized in a maximum likelihood sense by applying the expectation maximization (EM) algorithm. The novel algorithm is tested on a synthetic benchmark problem and is found to clearly outperform the earlier heuristic approach. The paper concludes with an application of this scheme to a DNA sequence alignment of the argF gene from four Neisseria strains, where a likely recombination event is clearly detected.