The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes.

We have determined the complete nucleotide sequence (4712 nucleotides) of the mouse 28S rRNA gene. Comparison with all other homologs indicates that the potential for major variations in size during the evolution has been restricted to a unique set of a few sites within a largely conserved secondary structure core. The D (divergent) domains, responsible for the large increase in size of the molecule from procaryotes to higher eukaryotes, represent half the mouse 28S rRNA length. They show a clear potential to form self-contained secondary structures. Their high GC content in vertebrates is correlated with the folding of very long stable stems. Their comparison with the two other vertebrates, xenopus and rat, reveals an history of repeated insertions and deletions. During the evolution of vertebrates, insertion or deletion of new sequence tracts preferentially takes place in the subareas of D domains where the more recently fixed insertions/deletions were located in the ancestor sequence. These D domains appear closely related to the transcribed spacers of rRNA precursor but a sizable fraction displays a much slower rate of sequence variation.     

Comparative evolution of the mitochondrial cytochrome b gene and nuclear beta-fibrinogen intron 7 in woodpeckers

Most molecular phylogenetic studies of vertebrates have been based on DNA sequences of mitochondrial-encoded genes. MtDNA evolves rapidly and is thus particularly useful for resolving relationships among recently evolved groups. However, it has the disadvantage that all of the mitochondrial genes are inherited as a single linkage group so that only one independent gene tree can be inferred regardless of the number of genes sequenced. Introns of nuclear genes are attractive candidates for independent sources of rapidly evolving DNA: they are pervasive, most of their nucleotides appear to be unconstrained by selection, and PCR primers can be designed for sequences in adjacent exons where nucleotide sequences are conserved. We sequenced intron 7 of the beta-fibrinogen gene (beta-fibint7) for a diversity of woodpeckers and compared the phylogenetic signal and nucleotide substitution properties of this DNA sequence with that of mitochondrial-encoded cytochrome b (cyt b) from a previous study. A few indels (insertions and deletions) were found in the beta-fibint7 sequences, but alignment was not difficult, and the indels were phylogentically informative. The beta-fibint7 and cyt b gene trees were nearly identical to each other but differed in significant ways from the traditional woodpecker classification. Cyt b evolves 2.8 times as fast as beta-fibint7 (14. 0 times as fast at third codon positions). Despite its relatively slow substitution rate, the phylogenetic signal in beta-fibint7 is comparable to that in cyt b for woodpeckers, because beta-fibint7 has less base composition bias and more uniform nucleotide substitution probabilities. As a consequence, compared with cyt b, beta-fibint7 nucleotide sites are expected to enter more distinct character states over the course of evolution and have fewer multiple substitutions and lower levels of homoplasy. Moreover, in contrast to cyt b, in which nearly two thirds of nucleotide sites rarely vary among closely related taxa, virtually all beta-fibint7 nucleotide sites appear free of selective constraints, which increases informative sites per unit sequenced. However, the estimated gamma distribution used to model rate variation among sites suggests constraints on some beta-fibint7 sites. This study suggests that introns will be useful for phylogenetic studies of recently evolved groups.     

Molecular systematics of sponges (Porifera)

The first application of molecular systematics to sponges was in the 1980s, using allozyme divergence to dis-criminate between conspecific and congeneric sponge populations. Since this time, a fairly large database has been accumulated and, although the first findings seemed to indicate that sponge species were genetically more divergent than those of other marine invertebrates, a recent review of the available dataset indicates that levels of interspecific gene identities in most sponges fall within the normal range found between species of other invertebrates. Nevertheless, some sponge genera have species that are extremely divergent from each other, suggesting a possible polyphyly of these genera. In the 1990s, molecular studies comparing sequences of ribosomal RNA have been used to reappraise the phylogenetic relationships among sponge genera, families, orders and classes. Both the 18S small subunit and the 28S large subunit rRNA genes have been sequenced (41 complete or partial and 75 partial sequences, respectively). Sequences of 18S rRNA show good support for Porifera being true Metazoa, but they are not informative for resolving relationships among genera, families or orders. 28S rRNA domains D1 and D2 appear to be more informative for the terminal nodes and provide resolution for internal topologies in sufficiently closely related species, but the deep nodes between orders or classes cannot be resolved using this molecule. Recently, a more conserved gene, Hsp70, has been used to try to resolve the relationships in the deep nodes. Metazoan monophyly is very well supported. Nevertheless, the divergence between the three classes of Porifera, as well as the divergence between Porifera, Cnidaria and Ctenophora, is not resolved. Research is in progress using other genes such as those of the homeodomain, the tyrosine kinase domain, and those coding for the aggregation factor. For the moment the dataset for these genes is too restricted to resolve the phylogenetic relationships of these phyla. However, whichever the genes, the phylogenies obtained suggest that Porifera could be paraphyletic and that the phylogenetic relationships of most of the families and orders of the Demospongiae have to be reassessed. The Calcarea and Hexactinellida are still to be studied at the molecular level.     

Analysis of the Secondary Structure of ITS1 in Pectinidae: Implications for Phylogenetic Reconstruction and Structural Evolution

It is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, we have developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into four structural domains. Compensatory changes were found in all four structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

The secondary structure of large-subunit rRNA divergent domains, a marker for protist evolution

The secondary structure of the large-subunit ribosomal RNA (24-26S rRNA) has been studied with emphasis on comparative analysis of the folding patterns of the divergent domains in the available protist sequences, that is Prorocentrum micans (dinoflagellate), Saccharomyces carlsbergensis (yeast), Tetrahymena thermophila (ciliate), Physarum polycephalum and Dictyostelium discoideum (slime moulds), Crithidia fasciculata and Giardia lamblia (parasitic flagellates). The folding for the D3, D7a and D10 divergent domains has been refined and a consensus model for the protist 24-26S rRNA structure is proposed. Two hundred seventy-seven nucleotide-long aligned sequences representing all or part of the D3, H32-33, D8, D9 and D10 divergent domains are used for the construction of unrooted phylogenetic trees either calculated from a nucleotide difference matrix, or determined with the PAUP programme based on the parsimony method. Both phylogenies suggest three major branchings, the first leading to the dinoflagellate (which branches off first), ciliate and yeast, the second to the slime moulds, and the last to the parasitic flagellates.     

Sequences of three closely related variants of a complex satellite DNA diverge at specific domains

Major differences among the sequences of the repeat units of a very complex satellite DNA are located in domains which are sensitive to S1 nuclease under torsional stress, indicating that the domains assume unusual secondary or tertiary structures. Repeat units of the satellite, which accounts for 3% of the DNA of a land crab, have been inserted into pBR322 and the primary sequences of three cloned variants determined. The variants selected for sequencing include 1) RU (2089 base pairs (bp) ), representative of the average size of repeat units of cellular satellite; 2) TRU (1674 bp), truncated at an extra EcoRI site; and 3) EXT (2639 bp), extended by a 5-fold amplification of a 142-bp segment, one copy of which is present in RU and TRU (Bonnewell, V., Fowler, R.F., and Skinner, D.M. (1983) Science 221, 862-865). It appears that every copy of the satellite may be different and that the variants do not arise from cloning accidents. Extensive domains, as long as approximately 560 bp, are greater than 95% homologous among RU, TRU, and EXT; these conserved domains are composed of DNA whose base composition and sequences do not have remarkable features. By contrast, the sequences that comprise the divergent domains are unusually rich in 1) tracts of (dG X dC) 13-23 and arrangements of similar but not identical repetitive oligonucleotides or 2) alternating purines and pyrimidines (pu/py).     

Informative characteristics of 12 divergent domains in complete large subunit rDNA sequences from the harmful dinoflagellate genus, Alexandrium (Dinophyceae)

The genus Alexandrium includes organisms of interest, both for the study of dinoflagellate evolution and for their impacts as toxic algae affecting human health and fisheries. Only partial large subunit (LSU) rDNA sequences of Alexandrium and other dinoflagellates are available, although they contain much genetic information. Here, we report complete LSU rDNA sequences from 11 strains of Alexandrium, including Alexandrium affine, Alexandrium catenella, Alexandrium fundyense, Alexandrium minutum, and Alexandrium tamarense, and discuss their segmented domains and structure. Putative LSU rRNA coding regions were recorded to be around 3,400 bp long. Their GC content (about 43.7%) is among the lowest when compared with other organisms. Furthermore, no AT-rich regions were found in Alexandrium LSU rDNA, although a low GC content was recorded within the LSU rDNA. No intron-like sequences were found. The secondary structure of the LSU rDNA and parsimony analyses showed that most variation in LSU rDNA is found in the divergent (D) domains with the D2 region being the most informative. This high D domain variability can allow members of the diverse Alexandrium genus to be categorized at the species level. In addition, phylogenetic analysis of the alveolate group using the complete LSU sequences strongly supported previous findings that the dinoflagellates and apicomplexans form a clade.     

Complete chloroplast genome sequences of Solanum bulbocastanum, Solanum lycopersicum and comparative analyses with other Solanaceae genomes

Despite the agricultural importance of both potato and tomato, very little is known about their chloroplast genomes. Analysis of the complete sequences of tomato, potato, tobacco, and Atropa chloroplast genomes reveals significant insertions and deletions within certain coding regions or regulatory sequences (e.g., deletion of repeated sequences within 16S rRNA, ycf2 or ribosomal binding sites in ycf2). RNA, photosynthesis, and atp synthase genes are the least divergent and the most divergent genes are clpP, cemA, ccsA, and matK. Repeat analyses identified 33–45 direct and inverted repeats ≥30 bp with a sequence identity of at least 90%; all but five of the repeats shared by all four Solanaceae genomes are located in the same genes or intergenic regions, suggesting a functional role. A comprehensive genome-wide analysis of all coding sequences and intergenic spacer regions was done for the first time in chloroplast genomes. Only four spacer regions are fully conserved (100% sequence identity) among all genomes; deletions or insertions within some intergenic spacer regions result in less than 25% sequence identity, underscoring the importance of choosing appropriate intergenic spacers for plastid transformation and providing valuable new information for phylogenetic utility of the chloroplast intergenic spacer regions. Comparison of coding sequences with expressed sequence tags showed considerable amount of variation, resulting in amino acid changes; none of the C-to-U conversions observed in potato and tomato were conserved in tobacco and Atropa. It is possible that there has been a loss of conserved editing sites in potato and tomato.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Secondary structure prediction for complete rDNA sequences (18S, 5.8S,and 28S rDNA) of Demodex folliculorum, and comparison of divergent domains structures across Acari

According to base pairing, the rRNA folds into corresponding secondary structures, which contain additional phylogenetic information. On the basis of sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2 and 28S rDNA) of Demodex, we predicted the secondary structure of the complete rDNA sequence (18S, 5.8S, and 28S rDNA) of Demodex folliculorum, which was in concordance with that of the main arthropod lineages in past studies. And together with the sequence data from GenBank, we also predicted the secondary structures of divergent domains in SSU rRNA of 51 species and in LSU rRNA of 43 species from four superfamilies in Acari (Cheyletoidea, Tetranychoidea, Analgoidea and Ixodoidea). The multiple alignment among the four superfamilies in Acari showed that, insertions from Tetranychoidea SSU rRNA formed two newly proposed helixes, and helix c3-2b of LSU rRNA was absent in Demodex (Cheyletoidea) taxa. Generally speaking, LSU rRNA presented more remarkable differences than SSU rRNA did, mainly in D2, D3, D5, D7a, D7b, D8 and D10.     

Comparison of primary and secondary 26S rRNA structures in twoTetrahymena species: Evidence for a strong evolutionary and structural constraint in expansion segments

Summary We have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for twoTetrahymena species,T. thermophila andT. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the twoTetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes.The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to presesrve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Evolutionary rates of insertion and deletion in noncoding nucleotide sequences of primates

Insertions and deletions are responsible for gaps in aligned nucleotide sequences, but they have been usually ignored when the number of nucleotide substitutions was estimated. We compared six sets of nuclear and mitochondrial noncoding DNA sequences of primates and obtained the estimates of the evolutionary rate of insertion and deletion. The maximum-parsimony principle was applied to locate insertions and deletions on a given phylogenetic tree. Deletions were about twice as frequent as insertions for nuclear DNA, and single-nucleotide insertions and deletions were the most frequent in all events. The rate of insertion and deletion was found to be rather constant among branches of the phylogenetic tree, and the rate (approximately 2.0/kb/Myr) for mitochondrial DNA was found to be much higher than that (approximately 0.2/kb/Myr) for nuclear DNA. The rates of nucleotide substitution were about 10 times higher than the rate of insertion and deletion for both nuclear and mitochondrial DNA.      

Comparisons of large subunit rRNAs reveal some eukaryote-specific elements of secondary structure

All large rRNAs possess a common core of secondary structure. However, large variations in the size of the molecule have arisen during evolution, which are accommodated over a dozen rapidly evolving domains. Most of the enlargement of the eukaryotic molecules (as compared to prokaryotes) is in fact restricted over only two of these divergent domains, which are dramatically expanded in vertebrates. We have derived secondary structure models for these two domains through a systematic comparison of all the pro- and eukaryotic sequences published so far. Within each of these domains, a subset of secondary structure elements which are specific to eukaryotes is detected. Archaebacterial-specific secondary structures can also be identified which appear to be maintained through a strong selective constraint. The relative preservation of such group-specific structures raises the issue of their potential involvement in some diversification of ribosomal functions among the three fundamental phylogenetic groups, eubacteria, archaebacteria and eukaryotes. We also show that eukaryotic ribosomal RNAs are subjected, over their entire length, to a unique type of compositional constraint which may largely differ among the major eukaryotic taxa.     

Species interactions mediate phylogenetic community structure in a hyperdiverse lizard assemblage from arid Australia

Evolutionary history can exert a profound influence on ecological communities, but few generalities have emerged concerning the relationships among phylogeny, community membership, and niche evolution. We compared phylogenetic community structure and niche evolution in three lizard clades (Ctenotus skinks, agamids, and diplodactyline geckos) from arid Australia. We surveyed lizard communities at 32 sites in the northwestern Great Victoria Desert and generated complete species-level molecular phylogenies for regional representatives of the three clades. We document a striking pattern of phylogenetic evenness within local communities for all groups: pairwise correlations in species abundance across sites are negatively related to phylogenetic similarity. By modeling site suitability on the basis of species' habitat preferences, we demonstrate that phylogenetic evenness generally persists even after controlling for habitat filtering among species. This phylogenetic evenness is coupled with evolutionary lability of habitat-associated traits, to the extent that closely related species are more divergent in habitat use than distantly related species. In contrast, lizard diets are phylogenetically conserved, and pairwise dietary overlap between species is negatively related to phylogenetic distance in two of the three clades. Our results suggest that contemporary and historical species interactions have led to similar patterns of community structure across multiple clades in one of the world's most diverse lizard communities.     

Phylogenetic signal and its decay in mitochondrial SSU and LSU rRNA gene fragments of Anisoptera

The phylogeny of Anisoptera, dragonflies in the strict sense, has proven to be notoriously difficult to resolve. Based on morphological characters, several recent publications dealing with the phylogeny of dragonflies proposed contradicting inter- and intrafamily relationships. We explored phylogenetic information content of mitochondrial large-subunit (LSU) and small-subunit (SSU) ribosomal gene fragments for these systematic problems. Starting at published universal primers, we developed primer sets suitable for amplifying large parts of the LSU and SSU rRNA genes within dragonflies. These fragments turned out to harbor sufficient phylogenetic information to satisfyingly resolve intrafamily relationships, but they contain insufficient phylogenetic structure to permit reliable conclusions about several interfamily relationships. We demonstrate that decay of phylogenetic signal progresses from intrafamily to interfamily to outgroup relationships and is correlated with an increase of genetic distances. As expected, signal decay is most pronounced in fast-changing sites. Additionally, base composition among fast-changing sites significantly deviates from the expected homogeneity. Homogeneity of base composition among all included taxa was restored only after removing fast-changing sites from the data set. The molecular data tentatively support interfamily relationships proposed by the most recent publication based on morphological characters of fossil and extant dragonflies.     

Multiple Lineages of the Mitochondrial Gene NADH Dehydrogenase Subunit 1 (ND1) in Parasitic Helminths: Implications for Molecular Evolutionary Studies of Facultatively Anaerobic Eukaryotes

The task of using partial ND1 sequences to infer a phylogeny for species of the genus Paragonimus (Trematoda: Digenea) was complicated by the discovery of at least two ND1 lineages within individual worms. The divergence of the ND1 lineages is shown by phylogenetic analysis not only to predate the divergence of the three Paragonimus species or species groups investigated but also the divergence of some trematode families. Some sequences are clearly pseudogenes as they contain single base deletions and/or premature termination codons. The presence of both pseudogenes and/or mitochondrial heteroplasmy are invoked to explain the presence of multiple and divergent ND1 lineages in these trematodes, which have two distinct cytochemical types of mitochondria. The implications for phylogenetic studies generally and of parasitic helminths specifically, using ND1 sequence data, are discussed. The ability of these organisms to adapt their metabolic processes to the variable availability of oxygen as an electron acceptor are proposed to explain some of the molecular diversity observed in parasitic helminths and possibly also in other anaerobically adapted eukaryotes. Received: 18 October 1999 / Accepted: 23 June 2000     

苔藓动物主要类群28S rDNA多变区的分子形态分析及其系统学意义

测定了苔藓动物主要代表类群3种苔虫的28S rDNA全序列(3 459～3 613 bp),结合已公布的相关类群的28S rDNA全序列数据,分析了其一级结构特征,并分别绘制了D2、D3和D8区段的二级结构.比较分析结果显示:各类群的全序列总长均超过3 300 bp,GC含量为41.8%～57.7%;D3和D8区段的二级结构保守性较高,D2区差异显著;各区段的长度变化较大,但是,大多都具有相似的主干结构.基于二级结构多变区分子形态学特征的比较基本和基于一级结构的分子系统学结论相吻合,提示它们同样包含着重要的系统发育信息.