Phylogenetic comparison of local length plasticity of the small subunit of nuclear rDNAs among all Hexapoda orders and the impact of hyper-length-variation on alignment

The SSU nrDNA (18S), is one of the most frequently sequenced molecular markers in phylogenetic studies. However, the length-hyper-variation at multiple positions of this gene can affect the accuracy of alignment greatly and this length variation makes alignment across arthropod orders a serious problem. The analyses of Hexapoda phylogeny is such a case. A more clear recognition of the distribution of the length-variable-regions is needed. In this study, the secondary structure of some length-variable-regions in the SSU nrRNA of Arthropoda was adjusted by the principle of co-variation. It is found that the extent of plasticity of some length-variable-region can extraordinarily be higher than 600 bases in hexapods. And the numbers of hyper length-variable-regions are largest in Strepsiptera and Sternorrhyncha (Hemiptera). Our study shows that some length-variable-regions can serve as synapomorphies for some groups. The phylogenetic comparison also suggested that the expansion of a lateral bulge could be the origin of a helix.     

Diatom phylogenetics inferred based on direct optimization of nuclear-encoded SSU rRNA sequences

Direct optimization (DO) of 126 nuclear‐encoded SSU rRNA diatom sequences was conducted. The optimal phylogeny indicated several unique relationships with respect to those recovered from a maximum likelihood (ML) analysis of an alignment based on maximizing primary and secondary structural similarity between 126 nuclear‐encoded SSU rRNA diatom sequences ( Medlin and Kaczmarska, 2004 ). Dividing diatoms into the subdivisions Coscinodiscophytina and Bacillariophytina was not supported by the DO phylogeny, due to the paraphyly of the former. The same pertains to Coscinodiscophyceae, Mediophyceae, Thalassiosira, Fragilaria and Amphora. The ordinal‐level classification of the diatoms proposed by Round et al. (1990 ) was for the most part found to be unsupported. The DO phylogeny represented a more rigorous hypothesis than the ML tree because DO maximized character congruence during the homology testing (i.e., alignment/tree search) process whereas the non‐phylogenetic similarity‐based alignment used in the ML analysis did not. The above statement is supported by “controlled” parsimony analyses of 35 sequences, which strongly suggested that dissimilarities in the DO and ML tree structure were due to the specific homology testing approach used. It could not be precluded that differences in taxon sampling and the use of a dissimilar optimality criteria contributed to discrepancies in the structure of the optimal ML and DO trees.     

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.     

Evolution of the RNA polymerase B' subunit gene (rpoB') in Halobacteriales: a complementary molecular marker to the SSU rRNA gene

Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.     

Alpha‐Tubulin and Small Subunit rRNA Phylogenies of Peritrichs Are Congruent and Do Not Support the Clustering of Mobilids and Sessilids (Ciliophora,Oligohymenophorea)

ABSTRACT. Peritrich ciliates have been traditionally subdivided into two orders, Sessilida and Mobilida within the subclass Peritrichia. However, all the existing small subunit (SSU) rRNA phylogenetic trees showed that the sessilids and mobilids did not branch together. To shed some light on this disagreement, we tested whether or not the classic Peritrichia is a monophyletic group by assessing the reliability of the SSU rRNA phylogeny in terms of congruency with α‐tubulin phylogeny. For this purpose, we obtained 10 partial α‐tubulin sequences from peritrichs and built phylogenetic trees based on α‐tubulin nucleotide and amino acid data. A phylogenetic tree from the α‐tubulin and SSU rRNA genes in combination was also constructed and compared with that from the SSU rRNA gene using a similar species sampling. Our results show that the mobilids and sessilids are consistently separated in all trees, which reinforces the idea that the peritrichs do not constitute a monophyletic group. However, in all α‐tubulin gene trees, the urceolariids and trichodiniids do not group together, suggested mobilids may not be a monophyletic group.     

Molecular phylogenies of Parabasalia inferred from four protein genes and comparison with rRNA trees

The molecular phylogeny of parabasalids has mainly been inferred from small subunit (SSU) rRNA sequences and has conflicted substantially with systematics based on morphological and ultrastructural characters. This raises the important question, how congruent are protein and SSU rRNA trees? New sequences from seven diverse parabasalids (six trichomonads and one hypermastigid) were added to data sets of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, alpha-tubulin and beta-tubulin and used to construct phylogenetic trees. The GAPDH tree was well resolved and identical in topology to the SSU rRNA tree. This both validates the rRNA tree and suggests that GAPDH should be a valuable tool in further phylogenetic studies of parabasalids. In particular, the GAPDH tree confirmed the polyphyly of Monocercomonadidae and Trichomonadidae and the basal position of Trichonympha agilis among parabasalids. Moreover, GAPDH strengthened the hypothesis of secondary loss of cytoskeletal structures in Monocercomonadidae such as Monocercomonas and Hypotrichomonas. In contrast to GAPDH, the enolase and both tubulin trees are poorly resolved and rather uninformative about parabasalian phylogeny, although two of these trees also identify T. agilis as representing the basal-most lineage of parabasalids. Although all four protein genes show multiple gene duplications (for 3-6 of the seven taxa examined), most duplications appear to be relatively recent (i.e., species-specific) and not a problem for phylogeny reconstruction. Only for enolase are there more ancient duplications that may confound phylogenetic interpretation.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.     

A secondary structural model of the 28S rRNA expansion segments D2 and D3 for Chalcidoid wasps (Hymenoptera: Chalcidoidea)

We analyze the secondary structure of two expansion segments (D2, D3) of the 28S ribosomal (rRNA)-encoding gene region from 527 chalcidoid wasp taxa (Hymenoptera: Chalcidoidea) representing 18 of the 19 extant families. The sequences are compared in a multiple sequence alignment, with secondary structure inferred primarily from the evidence of compensatory base changes in conserved helices of the rRNA molecules. This covariation analysis yielded 36 helices that are composed of base pairs exhibiting positional covariation. Several additional regions are also involved in hydrogen bonding, and they form highly variable base-pairing patterns across the alignment. These are identified as regions of expansion and contraction or regions of slipped-strand compensation. Additionally, 31 single-stranded locales are characterized as regions of ambiguous alignment based on the difficulty in assigning positional homology in the presence of multiple adjacent indels. Based on comparative analysis of these sequences, the largest genetic study on any hymenopteran group to date, we report an annotated secondary structural model for the D2, D3 expansion segments that will prove useful in assigning positional nucleotide homology for phylogeny reconstruction in these and closely related apocritan taxa.     

Trypanosomes are monophyletic: evidence from genes for glyceraldehyde phosphate dehydrogenase and small subunit ribosomal RNA

The genomes of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major have been sequenced, but the phylogenetic relationships of these three protozoa remain uncertain. We have constructed trypanosomatid phylogenies based on genes for glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) and small subunit ribosomal RNA (SSU rRNA). Trees based on gGAPDH nucleotide and amino acid sequences (51 taxa) robustly support monophyly of genus Trypanosoma, which is revealed to be a relatively late-evolving lineage of the family Trypanosomatidae. Other trypanosomatids, including genus Leishmania, branch paraphyletically at the base of the trypanosome clade. On the other hand, analysis of the SSU rRNA gene data produced equivocal results, as trees either robustly support or reject monophyly depending on the range of taxa included in the alignment. We conclude that the SSU rRNA gene is not a reliable marker for inferring deep level trypanosome phylogeny. The gGAPDH results support the hypothesis that trypanosomes evolved from an ancestral insect parasite, which adapted to a vertebrate/insect transmission cycle. This implies that the switch from terrestrial insect to aquatic leech vectors for fish and some amphibian trypanosomes was secondary. We conclude that the three sequenced pathogens, T. brucei, T. cruzi and L. major, are only distantly related and have distinct evolutionary histories.     

Investigation of molluscan phylogeny using large-subunit and small-subunit nuclear rRNA sequences

The Mollusca represent one of the most morphologically diverse animal phyla, prompting a variety of hypotheses on relationships between the major lineages within the phylum based upon morphological, developmental, and paleontological data. Analyses of small-ribosomal RNA (SSU rRNA) gene sequence have provided limited resolution of higher-level relationships within the Mollusca. Recent analyses suggest large-subunit (LSU) rRNA gene sequences are useful in resolving deep-level metazoan relationships, particularly when combined with SSU sequence. To this end, LSU (approximately 3.5 kb in length) and SSU (approximately 2 kb) sequences were collected for 33 taxa representing the major lineages within the Mollusca to improve resolution of intraphyletic relationships. Although the LSU and combined LSU+SSU datasets appear to hold potential for resolving branching order within the recognized molluscan classes, low bootstrap support was found for relationships between the major lineages within the Mollusca. LSU+SSU sequences also showed significant levels of rate heterogeneity between molluscan lineages. The Polyplacophora, Gastropoda, and Cephalopoda were each recovered as monophyletic clades with the LSU+SSU dataset. While the Bivalvia were not recovered as monophyletic clade in analyses of the SSU, LSU, or LSU+SSU, the Shimodaira-Hasegawa test showed that likelihood scores for these results did not differ significantly from topologies where the Bivalvia were monophyletic. Analyses of LSU sequences strongly contradict the widely accepted Diasoma hypotheses that bivalves and scaphopods are closely related to one another. The data are consistent with recent morphological and SSU analyses suggesting scaphopods are more closely related to gastropods and cephalopods than to bivalves. The dataset also presents the first published DNA sequences from a neomeniomorph aplacophoran, a group considered critical to our understanding of the origin and early radiation of the Mollusca.     

Nearly complete rRNA genes from 371 Animalia: updated structure-based alignment and detailed phylogenetic analysis

This study presents a manually constructed alignment of nearly complete rRNA genes from most animal clades (371 taxa from ~33 of the ~36 metazoan phyla), expanded from the 197 sequences in a previous study. This thorough, taxon-rich alignment, available at http://www.wsu.edu/~jmallatt/research/rRNAalignment.html and in the Dryad Repository (doi: http://dx.doi.org/10.5061/dryad.1v62kr3q), is based rigidly on the secondary structure of the SSU and LSU rRNA molecules, and is annotated in detail, including labeling of the erroneous sequences (contaminants). The alignment can be used for future studies of the molecular evolution of rRNA. Here, we use it to explore if the larger number of sequences produces an improved phylogenetic tree of animal relationships. Disappointingly, the resolution did not improve, neither when the standard maximum-likelihood method was used, nor with more sophisticated methods that partitioned the rRNA into paired and unpaired sites (stem, loop, bulge, junction), or accounted for the evolution of the paired sites. For example, no doublet model of paired-site substitutions (16-state, 16A and 16B, 7A-F, or 6A-C models) corrected the placement of any rogue taxa or increased resolution. The following findings are from the simplest, standard, ML analysis. The 371-taxon tree only imperfectly supported the bilaterian clades of Lophotrochozoa and Ecdysozoa, and this problem remained after 17 taxa with unstably positioned sequences were omitted from the analysis. The problem seems to stem from base-compositional heterogeneity across taxa and from an overrepresentation of highly divergent sequences among the newly added taxa (e.g., sequences from Cephalopoda, Rotifera, Acoela, and Myxozoa). The rogue taxa continue to concentrate in two locations in the rRNA tree: near the base of Arthropoda and of Bilateria. The approximately uncertain (AU) test refuted the monophyly of Mollusca and of Chordata, probably due to long-branch attraction of the highly divergent cephalopod and urochordate sequences out of those clades. Unlikely to be correct, these refutations show for the first time that rRNA phylogeny can support some 'wrong' clades. Along with its weaknesses, the rRNA tree has strengths: It recovers many clades that are supported by independent evidence (e.g., Metazoa, Bilateria, Hexapoda, Nonoculata, Ambulacraria, Syndermata, and Thecostraca with Malacostraca) and shows good resolution within certain groups (e.g., in Platyhelminthes, Insecta, Cnidaria). As another strength, the newly added rRNA sequences yielded the first rRNA-based support for Carnivora and Cetartiodactyla (dolphin+llama) in Mammalia, for basic subdivisions of Bryozoa ('Gymnolaemata+Stenolaemata' versus Phylactolaemata), and for Oligostraca (ostracods+branchiurans+pentastomids+mystacocarids). Future improvement could come from better sequence-evolution models that account for base-compositional heterogeneity, and from combining rRNA with protein-coding genes in phylogenetic reconstruction.     

A New Species of Scutellospora with a Coiled Germination Shield

During a survey of mycorrhizal fungi on the upper part of theCisadane River, on the slopes of Mount Pangrango in Gede PangrangoNational Park, West Java, an undescribed species of Scutellospora(Glomales)was discovered. This species has metallic golden to yellow toyellowish-brown spores that possess columnar protuberances.It is described and named Scutellospora projecturata sp. nov.The sequence of the nearly complete SSU rRNA gene was analysedand phylogenetic trees constructed. Copyright 2000 Annals ofBotany Company Scutellospora projecturata, Glomales, new species, 18s SSU rRNA, West Java, phylogenetic tree, phylogeny, arbuscular mycorrhizal fungi, AMF     

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 reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level

Although the molecular phylogeny, evolution and biodiversity of arbuscular mycorrhizal fungi (AMF) are becoming clearer, phylotaxonomically reliable sequence data are still limited. To fill this gap, a data set allowing resolution and environmental tracing across all taxonomic levels is provided. Two overlapping nuclear DNA regions, totalling c. 3 kb, were analysed: the small subunit (SSU) rRNA gene (up to 1800 bp) and a fragment spanning c. 250 bp of the SSU rDNA, the internal transcribed spacer (ITS) region (c. 475-520 bp) and c. 800 bp of the large subunit (LSU) rRNA gene. Both DNA regions together could be analysed for 35 described species, the SSU rDNA for c. 76 named and 18 as yet undefined species, and the ITS region or LSU rDNA, or a combination of both, for c. 91 named and 16 as yet undefined species. Present phylogenetic analyses, based on the three rDNA markers, provide reliable and robust resolution from phylum to species level. Altogether, 109 named species and 27 cultures representing as yet undefined species were analysed. This study provides a reference data set for molecular systematics and environmental community analyses of AMF, including analyses based on deep sequencing.     

The RDP-II (Ribosomal Database Project)

The Ribosomal Database Project (RDP-II), previously described by Maidak et al. [Nucleic Acids Res. (2000), 28, 173-174], continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 8.0 (June 1, 2000) consisted of 16 277 aligned prokaryotic small subunit (SSU) rRNA sequences while the number of eukaryotic and mitochondrial SSU rRNA sequences in aligned form remained at 2055 and 1503, respectively. The number of prokaryotic SSU rRNA sequences more than doubled from the previous release 14 months earlier, and approximately 75% are longer than 899 bp. An RDP-II mirror site in Japan is now available (http://wdcm.nig.ac.jp/RDP/html/index.h tml). RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment polymorphism experiments. The RDP-II email address for questions and comments has been changed from curator@cme.msu.edu to rdpstaff@msu.edu.     

The impact of length-variable data and alignment criterion on the phylogeny of Decapodiformes (Mollusca: Cephalopoda)

In molecular phylogenetics, mode of analysis is typically the primary reason cited for obtaining different topologies. However, sequence alignment is as important in determining topology as analytical method or optimality criterion, particularly for length-variable sequences. We investigate the effect of alignment strategy and parameters in an analysis of length-variable 18S rDNA sequences from 41 oceanic squids (Decapodiformes), focusing particularly on the impact of including variable regions on the resulting topology. Decapodiformes are an enigmatic group of molluscs whose evolutionary relationships remain unclear because standard molecular analyses have continued to produce poorly supported or poorly resolved trees, possibly due to elevated levels of molecular evolution. We find that alignment strategy (Clustal X versus POY) has a greater affect on topology than does parameter choice. Some groups were robust to shifts in methodology, including cuttlefishes (Sepiidae), bobtail squids (Sepiolidae) and pygmy squids (Idiosepiidae). No alignment strategy supported the monophyly of Oegopsida, although Myopsida did consistently form a monophyletic group. The variable regions consistently provided more well-supported and well-resolved trees than the conserved regions alone, indicating the phylogenetic importance of including length-variable data. © The Willi Hennig Society 2007.     

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.