The phylogeny of land plants inferred from 18S rDNA sequences: pushing the limits of rDNA signal?

Previous studies of the phylogeny of land plants based on analysis of 18S ribosomal DNA (rDNA) sequences have generally found weak support for the relationships recovered and at least some obviously spurious relationships, resulting in equivocal inferences of land plant phylogeny. We hypothesized that greater sampling of both characters and taxa would improve inferences of land plant phylogeny based on 18S rDNA sequences. We therefore conducted a phylogenetic analysis of complete (or nearly complete) 18S rDNA sequences for 93 species of land plants and 7 green algal relatives. Parsimony analyses with equal weighting of characters and characters state changes and parsimony analyses weighting (1) stem bases half as much as loop bases and (2) transitions half as much as transversions did not produce substantially different topologies. Although the general structure of the shortest trees is consistent with most hypotheses of land plant phylogeny, several relationships, particularly among major groups of land plants, appear spurious. Increased character and taxon sampling did not substantially improve the performance of 18S rDNA in phylogenetic analyses of land plants, nor did analyses designed to accommodate variation in evolutionary rates among sites. The rate and pattern of 18S rDNA evolution across land plants may limit the usefulness of this gene for phylogeny reconstruction at deep levels of plant phylogeny. We conclude that the mosaic structure of 18S rDNA, consisting of highly conserved and highly variable regions, may contain historical signal at two levels. Rapidly evolving regions are informative for relatively recent divergences (e.g., within angiosperms, seed plants, and ferns), but homoplasy at these sites makes it difficult to resolve relationships among these groups. At deeper levels, changes in the highly conserved regions of small-subunit rDNAs provide signal across all of life. Because constraints imposed by the secondary structure of the rRNA may affect the phylogenetic information content of 18S rDNA, we suggest that 18S rDNA sequences be combined with other data and that methods of analysis be employed to accommodate these differences in evolutionary patterns, particularly across deep divergences in the tree of life.     

rRNA二级结构序列用于真菌系统学研究的方法初探

本文首次利用核酸二级结构特征代替核酸碱基作为探讨类群之间亲缘关系的信号,构建了基于结构特征的子囊菌部分类群的系统进化树。该方法以S(规范的碱基对),Q(不规范的碱基对),I(单链),B(侧环),M(多分枝环)和H(发卡结构)为代码将二级结构特征区分为6种不同的亚结构类型,然后将二级结构特征转换为结构序列,并进行结构序列分析。该方法使rRNA不只局限于碱基比较,拓展了其应用范围,为揭示分子的功能与进化的关系提供了线索。结果表明,结构序列分析可用于子囊菌的系统学研究;相对于核酸序列分析,结构分析的结果似乎更加清晰地体现子囊果的演化过程。     

以子囊菌部分类群的rRNA数据为例,评估不同分区策略对贝叶斯分析效果的影响(英文)

在使用rRNA基因进行系统发育分析过程中,不同位点间进化速度的差异性可能是导致系统误差的一个重要原因。以52个真菌为研究对象,利用rRNA二级结构特征构建分区策略,探讨不同分区策略对贝叶斯分析的影响。结果显示各结构分区的最优核酸替代模型及其参数与分区类型密切相关。与传统的贝叶斯方法相比,使用结构环的分区策略对结果没有显著影响,而引入臂元素的方法却导致更高的边际似然值和支持率。此外,不考虑结构特征,简单的增加子分区数量的分区策略尽管也能导致贝叶斯因素值的增加,却没有提高解决亲缘关系的能力,说明一个合理的分区策略应该基于生物学功能(或二级结构特征)而非纯数学因素。     

Phylogenetic analysis of molluscan mitochondrial LSU rDNA sequences and secondary structures

Mollusks are an extraordinarily diverse group of animals with an estimated 200,000 species, second only to the phylum Arthropoda. We conducted a comparative analysis of complete mitochondrial ribosomal large subunit sequences (LSU) of a chiton, two bivalves, six gastropods, and a cephalopod. In addition, we determined secondary structure models for each of them. Comparative analyses of nucleotide variation revealed substantial length variation among the taxa, with stylommatophoran gastropods possessing the shortest lengths. Phylogenetic analyses of the nucleotide sequence data supported the monophyly of Albinaria, Euhadra herklotsi + Cepaea nemoralis, Stylommatophora, Cerithioidea, and when only transversions are included, the Bivalvia. The phylogenetic limits of the mitochondrial LSU rRNA gene within mollusks appear to be up to 400 million years, although this estimate will have to be tested further with additional taxa. Our most novel finding was the discovery of phylogenetic signal in the secondary structure of rRNA of mollusks. The absence of entire stem/loop structures in Domains II, III, and V can be viewed as three shared derived characters uniting the stylommatophoran gastropods. The absence of the aforementioned stem/loop structure explains much of the observed length variation of the mitochondrial LSU rRNA found within mollusks. The distribution of these unique secondary structure characters within mollusks should be examined.     

How slippage-derived sequences are incorporated into rRNA variable-region secondary structure: implications for phylogeny reconstruction

We analyzed the type and frequency of mutational changes in hypervariable rRNA regions, using the highly length-variable region V4 of the small subunit rRNA locus of tiger beetles (Cicindelidae) as an example. Phylogenetic analysis of indels in closely related species showed that (1) most indels are single nucleotides (usually A or T and sometimes G) or di-nucleotides of A and T. These occur at numerous foci, and they exhibit a strong bias for duplication of 5' single and di-nucleotide motifs but not 3' motifs. (2) Insertions/deletions in stem-forming regions affected paired and unpaired bases with about equal frequency but they did not disrupt the secondary structure. (3) Recurring mutations involving short repeats of the same bases caused parallel evolution of similar sequence motifs in the rRNA of different lineages. The observed types of change are consistent with the propostion that slippage is the main mutational mechanism. Slippage-derived sequences tend to be self-complementary, and therefore the stem-loop structure could be self-organizing as a consequence of the underlying mutational mechanism. Thus, the secondary structure in the cicindelid V4 region may be conserved due to the dynamics of the mutational mechanism rather than to functional constraints. These processes may also have a tendency to produce similar primary sequences irrespective of phylogenetic associations. The findings have implications for sequence alignment in phylogenetic analysis and should caution against the use of secondary structure to improve the determination of positional homology in hypervariable regions.     

Phylogeny of sipunculan worms: A combined analysis of four gene regions and morphology

The intra-phyletic relationships of sipunculan worms were analyzed based on DNA sequence data from four gene regions and 58 morphological characters. Initially we analyzed the data under direct optimization using parsimony as optimality criterion. An implied alignment resulting from the direct optimization analysis was subsequently utilized to perform a Bayesian analysis with mixed models for the different data partitions. For this we applied a doublet model for the stem regions of the 18S rRNA. Both analyses support monophyly of Sipuncula and most of the same clades within the phylum. The analyses differ with respect to the relationships among the major groups but whereas the deep nodes in the direct optimization analysis generally show low jackknife support, they are supported by 100% posterior probability in the Bayesian analysis. Direct optimization has been useful for handling sequences of unequal length and generating conservative phylogenetic hypotheses whereas the Bayesian analysis under mixed models provided high resolution in the basal nodes of the tree.     

Consideration of RNA secondary structure significantly improves likelihood-based estimates of phylogeny: examples from the bilateria

Sequences from ribosomal RNA (rRNA) genes have made a huge contribution to our current understanding of metazoan phylogeny and indeed the phylogeny of all of life. That said, some parts of this rRNA-based phylogeny remain unresolved. One approach to increase the resolution of these trees would be to use more appropriate models of sequence evolution in phylogenetic analysis. RNAs transcribed from rRNA genes have a complex secondary structure mediated by base pairing between sometimes distant regions of the rRNA molecule. The pairing between the stem nucleotides has important consequences for their evolution which differs from that of unpaired loop nucleotides. These differences in evolution should ideally be accounted for when using rRNA sequences for phylogeny estimation. We use a novel permutation approach to demonstrate the significant superiority of models of sequence evolution that allow stem and loop regions to evolve according to separate models and, in common with previous studies, we show that 16-state models that take base pairing of stems into account are significantly better than simpler, 4-state, single-nucleotide models. One of these 16-state models has been applied to the phylogeny of the Bilateria using small subunit rRNA (SSU) sequences. Our optimal tree largely echoes previous results based on SSU in particular supporting the tripartite Bilaterian tree of deuterostomes, lophotrochozoans, and ecdysozoans. There are also a number of differences, however, perhaps most important of which is the observation of a clade consisting of the gastrotrichs plus platyheminthes that is basal to all other lophotrochozoan taxa. Use of 16-state models also appears to reduce the Bayesian support given to certain biologically improbable groups found using standard 4-state models.     

Phylogenetic relationships among the Malagasy lemuriforms (Primates: Strepsirrhini) as indicated by mitochondrial sequence data from the 12S rRNA gene

Numerous phylogenetic hypotheses have been advanced for the Malagasy lemuriform radiation, drawing on data from morphology, physiology, behaviour and molecular genetics. Almost all possible relationships have been proposed and most nodes have been contested. We present a phylogenetic analysis, using several analytical methods, of a partial sequence from the 12s rRNA mitochondrial gene. This gene codes for the small ribosomal subunit, and functional constraints require that the secondary structure of the molecule is strongly conserved, which in inturn exerts constraints on the primary sequence structure. Although previous studies have suggested a very wide range of phylogenetic applicability for this molecule, our results indicate that it is most useful in strepsirrhine primates for estimating relationships among genera within families and among relatively recently diverged families (mean sequence divergence about 11%). Relationships among families separated by larger genetic distances (>12% divergence; e.g. Cheirogaleidae, Daubentoniidae, Megaladapidae) are difficult to resolve consistently. Our data show strong support for an Indridae-Lemuridae sister group and for monophyly of the Lemuridae with Varecia as the sister to all other lemurids. They also support, albeit less strongly, sister group relationships between Lemur and Hapalemur within the Lemuridae and between PmpLthecus and Avahi in the Indridae.     

Three-dimensional model of Escherichia coli ribosomal 5 S RNA as deduced from structure probing in solution and computer modeling.

The conformation of Escherichia coli 5 S rRNA was investigated using chemical and enzymatic probes. The four bases were monitored at one of their Watson-Crick positions with dimethylsulfate (at C(N-3) and A(N-1], with a carbodiimide derivative (at G(N-1) and U(N-3] and with kethoxal (at G(N-1, N-2]. Position N-7 of purine was probed with diethylpyrocarbonate (at A(N-7] and dimethylsulfate (at G(N-7]. Double-stranded or stacked regions were tested with RNase V1 and unpaired guanine residues with RNase T1. We also used lead(II) that has a preferential affinity for interhelical and loop regions and a high sensitivity for flexible regions. Particular care was taken to use uniform conditions of salt, magnesium, pH and temperature for the different enzymatic chemical probes. Derived from these experimental data, a three dimensional model of the 5 S rRNA was built using computer modeling which integrates stereochemical constraints and phylogenetic data. The three domains of 5 S rRNA secondary structure fold into a Y-shaped structure that does not accommodate long-range tertiary interactions between domains. The three domains have distinct structural and dynamic features as revealed by the chemical reactivity and the lead(II)-induced hydrolysis: domain 2 (loop B/helix III/loop C) displays a rather weak structure and possesses dynamic properties while domain 3 (helix V/region E/helix IV/loop D) adopts a highly structured and overall helical conformation. Conserved nucleotides are not crucial for the tertiary folding but maintain an intrinsic structure in the loop regions, especially via non-canonical pairing (A.G, G.U, G.G, A.C, C.C), which can close the loops in a highly specific fashion. In particular, nucleotides in the large external loop C fold into an organized conformation leading to the formation of a five-membered loop motif. Finally, nucleotides at the hinge region of the Y-shape are involved in a precise array of hydrogen bonds based on a triple interaction between U14, G69 and G107 stabilizing the quasi-colinearity of helices II and V. The proposed tertiary model is consistent with the localization of the ribosomal protein binding sites and possesses strong analogy with the model proposed for Xenopus laevis 5 S rRNA, indicating that the Y-shape model can be generalized to all 5 S rRNAs.     

Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae)

The nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA including the 5.8S rRNA gene and the 5′ end of the 28S rRNA gene have been determined for 19 species in 10 genera of the powdery mildew fungi in order to analyze their phylogenetic relationship. These fungi were divided into two large groups based on the nucleotide length of the ITS regions, and this grouping was in line with that based on the morphological characters of the anamorphic stage rather than the teleomorphic stage. Although the variable ITS sequences were often ambiguously aligned, conserved sites were also found. Thus, a neighbor-joining tree was constructed using the nucleotide sequence data of the conserved sites of the ITS regions, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene. The phylogenetic tree displayed the presence of four groups in the powdery mildews, which were distinguished by their morphology and/or host ranges. In the ITS2 region, the presence of a common secondary structure having four hairpin domains was suggested, in spite of the highly variable nucleotide sequences of this region. The predicted secondary structure was supported by the compensatory mutations as well as compensatory conserved sequences and high G+C content in the predicted stem regions. Contribution No. 142 from the Laboratory of Plant Pathology, Mie University.     

Secondary structure of mitochondrial 12S rRNA among fish and its phylogenetic applications.

The complete 12S ribosomal RNA(rRNA) sequences from 23 gobioid species and nine diverse assortments of other fish species were employed to establish a core secondary structure model for fish 12S rRNA. Of the 43 stems recognized, 41 were supported by at least some compensatory evidence among vertebrates. The rates of nucleotide substitution were lower in stems than in loops. This may produce less phylogenetic information in stems when recently diverged taxa are compared. An analysis of compensatory substitution shows that the percentage of covariation is 68%, and the weighting factor for phylogenetic analyses to account for the dependence of mutations should be 0.66. Different stem-loop weighting schemes applied to the analyses of phylogenetic relationships of the Gobioidei indicate that down-weighting paired regions because of nonindependence could not improve the present phylogenetic analysis. A biased nucleotide composition (adenine% [A%] > thymine% [T%], cytosine% [C%] > guanine% [G%]) in the loop regions was also observed in the mammalian counterpart. The excess of A and C in the loop regions may be because of the asymmetric mechanism of mtDNA replication, which leads to the spontaneous deamination of C and A. This process may also be responsible for a transition-transversion bias and the patterns of nucleotide substitutions in both stems and loops.     

A case study to estimate the applicability of secondary structures of SSU‐rRNA gene in taxonomy and phylogenetic analyses of ciliates

Phylogenetic studies of ciliates are mainly based on the primary structure information of the nuclear genes. Some regions of the small subunit ribosomal RNA (SSU‐rRNA) gene have distinctive secondary structures, which have demonstrated value as phylogenetic/taxonomic characters. In the current work, we predict the secondary structures of four variable regions (V2, V4, V7 and V9) in the SSU‐rRNA gene of 45 urostylids. Structure comparisons indicate that the V4 region is the most effective in revealing interspecific relationships, while the V9 region appears suitable at the family level or higher. The V2 region also offers some taxonomic information, but is too conserved to reflect phylogenetic relationships at the family or lower level, at least for urostylids. The V7 region is the least informative. We constructed several phylogenetic trees, based on the primary sequence alignment and based on an improved alignment according to the secondary structures. The results suggest that including secondary structure information in phylogenetic analyses provides additional insights into phylogenetic relationships. Using urostylid ciliates as an example, we show that secondary structure information results in a better understanding of their relationships, for example generic relationships within the family Pseudokeronopsidae.     

An Empirical Analysis of mt 16S rRNA Covarion-Like Evolution in Insects: Site-Specific Rate Variation Is Clustered and Frequently Detected

The structural and functional analysis of rRNA molecules has attracted considerable scientific interest. Empirical studies have demonstrated that sequence variation is not directly translated into modifications of rRNA secondary structure. Obviously, the maintenance of secondary structure and sequence variation are in part governed by different selection regimes. The nature of those selection regimes still remains quite elusive. The analysis of individual bacterial models cannot adequately explore this topic. Therefore, we used primary sequence data and secondary structures of a mitochondrial 16S rRNA fragment of 558 insect species from 15 monophyletic groups to study patterns of sequence variation, and variation of secondary structure. Using simulation studies to establish significance levels of change, we found that despite conservation of secondary structure, the location of sequence variation within the conserved rRNA structure changes significantly between groups of insects. Despite our conservative estimation procedure we found significant site-specific rate changes at 56 sites out of 184. Additionally, site-specific rate variation is somewhat clustered in certain helices. Both results confirm what has been predicted from an application of non-stationary maximum likelihood models to rRNA sequences. Clearly, constraints on sequence variation evolve and leave footprints in the form of evolutionary plasticity in rRNA sequences. Here, we show that a better understanding of the evolution of rRNA sequences can be obtained by integrating both phylogenetic and structural information.     

Variation patterns of the mitochondrial 16S rRNA gene with secondary structure constraints and their application to phylogeny of cyprinine fishes (Teleostei: Cypriniformes)

The mitochondrial 16S ribosomal RNA (rRNA) gene sequences from 93 cyprinid fishes were examined to reconstruct the phylogenetic relationships within the diverse and economically important subfamily Cyprininae. Within the subfamily a biased nucleotide composition (A>T, C>G) was observed in the loop regions of the gene, and in stem regions apparent selective pressures of base pairing showed a bias in favor of G over C and T over A. The bias may be associated with transition-transversion bias. Rates of nucleotide substitution were lower in stems than in loops. Analysis of compensatory substitutions across these taxa demonstrates 68% covariation in the gene and a logical weighting factor to account for dependence in mutations for phylogenetic inference should be 0.66. Comparisons of varied stem-loop weighting schemes indicate that the down-weightings for stem regions could improve the phylogenetic analysis and the degree of non-independence of stem substitutions was not as important as expected. Bayesian inference under four models of nucleotide substitution indicated that likelihood-based phylogenetic analyses were more effective in improving the phylogenetic performance than was weighted parsimony analysis. In Bayesian analyses, the resolution of phylogenies under the 16-state models for paired regions, incorporating GTR + G + I models for unpaired regions was better than those under other models. The subfamily Cyprininae was resolved as a monophyletic group, as well as tribe Labein and several genera. However, the monophyly of the currently recognized tribes, such as Schizothoracin, Barbin, Cyprinion + Onychostoma lineages, and some genera was rejected. Furthermore, comparisons of the parsimony and Bayesian analyses and results of variable length bootstrap analysis indicates that the mitochondrial 16S rRNA gene should contain important character variation to recover well-supported phylogeny of cyprinid taxa whose divergences occurred within the recent 8 MY, but could not provide resolution power for deep phylogenies spanning 10-19 MYA.     

Molecular modelling of the 3-D structure of RNA tetraloops with different nucleotide sequences.

One surprisingly common element of RNA secondary structure consists of a hairpin capped by a four-base loop (or the tetraloop). Recently the 3-D structures of two RNA-tetraloops have been determined by NMR-studies. Both structures have a similar architecture: the first and the last bases of the loop form a hydrogen bonded pair which is stacked on the stem base pair. We have analysed the ability of tetraloops, with the other combinations of the first and the fourth bases, to adopt such a 'diloop' conformation using computer modelling. The analysis has shown that the 'diloop' conformation has many covalent and steric constraints which give a possibility for reliable structural predictions. As a result, a set of the tetraloop 3-D structures in which hydrogen bonded pairing of the first and the last bases does not cause covalent and steric hindrances has been selected. In most cases several predicted 3-D structures corresponded to one tetraloop sequence. Taking into consideration the folding pathway of RNA hairpins we have resolved this ambiguity and predicted the most probable 3-D structure for every possible nucleotide sequence of the tetraloop. On the basis of these results a conclusion has been drawn on the possible reasons of the tetraloop phylogenetic preference.     

Systematics of New World Monkeys (Platyrrhini, Primates) Based on 16S Mitochondrial DNA Sequences: A Comparative Analysis of Different Weighting Methods in Cladistic Analysis

In order to investigate the effects of different weighting methods on a phylogeny reconstruction based on DNA sequences and to evaluate the phylogenetic information content of various secondary structures, a fragment of the large ribosomal mitochondrial gene (16S) was sequenced from 13 species of New World monkeys, three species of catarrhines, and Tarsius. The data were analyzed cladistically without weighting characters or changes, and with different weighting methods: a priori differential weights for transitions and transversions, two variants of dynamic weighting for each kind and direction of change, and successive approximations, using both the character consistency index (CI) and the rescaled consistency index (RC). The results were compared with published trees constructed from nuclear sequences of ε-globins and morphological characters by different authors. The result of the analysis of the mtDNA data set with successive approximations, using the RC as weighting function, was the closest to the topology on which all molecular and morphological trees concur. Other relationships were unique to this tree. "Loops" were the type of secondary structure that showed maximum variation in sequence length and sites with the lowest character CI and RC. A large number of sites within loops showed high values for these indices, however, which suggests that uniform downweighting of these regions represents a large loss of phylogenetic information. Successive weighting, which assigns a weight for each particular character, seems to be a desirable alternative to this practice. We propose a new variant of dynamic weighting, which we call homoplasy-correcting dynamic weighting, that like dynamic weighting, is applicable to any kind of sequence, coding or noncoding.     

A Phylogeny for the African Treefrog Family Hyperoliidae Based on Mitochondrial rDNA

Mitochondrial 12S rRNA sequences were used to construct a phylogeny for the African treefrog family, Hyperoliidae. The sequences were aligned using the well-established mt 12S rRNA secondary structure as a map to designate stem and loop positions. Conservation of secondary structure permits a more accurate basis for assessing homologies than does primary sequence alone. The molecular trees showed many similarities to the morphological trees constructed previously. Species within a genus always grouped together. The generaHyperolius, Heterixalus, Afrixalus,andKassinawere resolved, but not with statistical significance.Leptopeliswas the basal group. The analysis shows clearly thatPhlyctimantisgroups withKassinaas it did in Drewes' morphological tree and thatTachycnemisgroups closely withHeterixalus,a relationship not suggested by the morphological data. Character weighting (including compensatory base changes), mitochondrial trees vs gene trees, and the biogeography of the group are discussed.     

Application of the secondary structure model of rRNA for phylogeny: D2-D3 expansion segments of the LSU gene of plant-parasitic nematodes from the family Hoplolaimidae Filipjev, 1934

Knowledge of rRNA structure is increasingly important to assist phylogenetic analysis through reconstructing optimal alignment, utilizing molecule features as an additional source of data and refining appropriate models of evolution of the molecule. We describe a procedure of optimization for alignment and a new coding method for nucleotide sequence data using secondary structure models of the D2 and D3 expansion fragments of the LSU-rRNA gene reconstructed for fifteen nematode species of the agriculturally important and diverse family Hoplolaimidae, order Tylenchida. Using secondary structure information we converted the original sequence data into twenty-eight symbol codes and submitted the transformed data to maximum parsimony analysis. We also applied the original sequence data set for Bayesian inference. This used the doublet model with sixteen states of nucleotide doublets for the stem region and the standard model of DNA substitution with four nucleotide states for loops and bulges. By this approach, we demonstrate that using structural information for phylogenetic analyses led to trees with lower resolved relationships between clades and likely eliminated some artefactual support for misinterpreted relationships, such as paraphyly of Helicotylenchus or Rotylenchus. This study as well as future phylogenetic analyses is herein supported by the development of an on-line database, NEMrRNA, for rRNA molecules in a structural format for nematodes. We also have developed a new computer program, RNAstat, for calculation of nucleotide statistics designed and proposed for phylogenetic studies.