Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences, with emphasis on the Hypermastigea

Small subunit rRNA gene sequences were identified without cultivation from parabasalid symbionts of termites belonging to the hypermastigid orders Trichonymphida (the genera Hoplonympha, Staurojoenina, Teranympha, and Eucomonympha) and Spirotrichonymphida (Spirotrichonymphella), and from four yet-unidentified parabasalid symbionts of the termite Incisitermes minor. All these new sequences were analyzed by Bayesian, likelihood, and parsimony methods in a broad phylogeny including all identified parabasalid sequences available in databases and some as yet unidentified sequences probably derived from hypermastigids. A salient point of our study focused on hypermastigids was the polyphyly of this class. We also noted a clear dichotomy between Trichonymphida and the other parabasalid taxa. However, this hypermastigid order was apparently polyphyletic, probably reflecting its morphological diversity. Among Trichonymphida, Teranympha (Teranymphidae) grouped together with the members of the family Eucomonymphidae, suggesting that its family status is ambiguous. The monophyletic lineage composed by Spirotrichonymphida exhibited a narrower branching pattern than Trichonymphida. The root of parabasalids was examined but could not be discerned accurately.     

Molecular phylogeny of parabasalids based on small subunit rRNA sequences, with emphasis on the Trichomonadinae subfamily

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. The Trichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas faetus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.     

Joint Bayesian estimation of alignment and phylogeny

We describe a novel model and algorithm for simultaneously estimating multiple molecular sequence alignments and the phylogenetic trees that relate the sequences. Unlike current techniques that base phylogeny estimates on a single estimate of the alignment, we take alignment uncertainty into account by considering all possible alignments. Furthermore, because the alignment and phylogeny are constructed simultaneously, a guide tree is not needed. This sidesteps the problem in which alignments created by progressive alignment are biased toward the guide tree used to generate them. Joint estimation also allows us to model rate variation between sites when estimating the alignment and to use the evidence in shared insertion/deletions (indels) to group sister taxa in the phylogeny. Our indel model makes use of affine gap penalties and considers indels of multiple letters. We make the simplifying assumption that the indel process is identical on all branches. As a result, the probability of a gap is independent of branch length. We use a Markov chain Monte Carlo (MCMC) method to sample from the posterior of the joint model, estimating the most probable alignment and tree and their support simultaneously. We describe a new MCMC transition kernel that improves our algorithm's mixing efficiency, allowing the MCMC chains to converge even when started from arbitrary alignments. Our software implementation can estimate alignment uncertainty and we describe a method for summarizing this uncertainty in a single plot.     

Bayesian coestimation of phylogeny and sequence alignment

Background  

Two central problems in computational biology are the determination of the alignment and phylogeny of a set of biological sequences. The traditional approach to this problem is to first build a multiple alignment of these sequences, followed by a phylogenetic reconstruction step based on this multiple alignment. However, alignment and phylogenetic inference are fundamentally interdependent, and ignoring this fact leads to biased and overconfident estimations. Whether the main interest be in sequence alignment or phylogeny, a major goal of computational biology is the co-estimation of both.     

Simultaneous statistical multiple alignment and phylogeny reconstruction

Although the reconstruction of phylogenetic trees and the computation of multiple sequence alignments are highly interdependent, these two areas of research lead quite separate lives, the former often making use of stochastic modeling, whereas the latter normally does not. Despite the fact that reasonable insertion and deletion models for sequence pairs were already introduced more than 10 years ago, they have only recently been applied to multiple alignment and only in their simplest version. In this paper we present and discuss a strategy based on simulated annealing, which makes use of these models to infer a phylogenetic tree for a set of DNA or protein sequences together with the sequences'indel history, i.e., their multiple alignment augmented with information about the positioning of insertion and deletion events in the tree. Our method is also the first application of the TKF2 model in the context of multiple sequence alignment. We validate the method via simulations and illustrate it using a data set of primate mtDNA.     

An automated phylogenetic tree-based small subunit rRNA taxonomy and alignment pipeline (STAP)

Comparative analysis of small-subunit ribosomal RNA (ss-rRNA) gene sequences forms the basis for much of what we know about the phylogenetic diversity of both cultured and uncultured microorganisms. As sequencing costs continue to decline and throughput increases, sequences of ss-rRNA genes are being obtained at an ever-increasing rate. This increasing flow of data has opened many new windows into microbial diversity and evolution, and at the same time has created significant methodological challenges. Those processes which commonly require time-consuming human intervention, such as the preparation of multiple sequence alignments, simply cannot keep up with the flood of incoming data. Fully automated methods of analysis are needed. Notably, existing automated methods avoid one or more steps that, though computationally costly or difficult, we consider to be important. In particular, we regard both the building of multiple sequence alignments and the performance of high quality phylogenetic analysis to be necessary. We describe here our fully-automated ss-rRNA taxonomy and alignment pipeline (STAP). It generates both high-quality multiple sequence alignments and phylogenetic trees, and thus can be used for multiple purposes including phylogenetically-based taxonomic assignments and analysis of species diversity in environmental samples. The pipeline combines publicly-available packages (PHYML, BLASTN and CLUSTALW) with our automatic alignment, masking, and tree-parsing programs. Most importantly, this automated process yields results comparable to those achievable by manual analysis, yet offers speed and capacity that are unattainable by manual efforts.     

The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis

The phylogeny of the Myxosporea was studied using the small-subunit ribosomal RNA gene sequences. Maximum parsimony and Bayesian inference were used to determine myxosporean phylogenetic relationships. The analysis included 120 myxosporean sequences retrieved from GenBank and 21 newly obtained sequences of myxosporeans representing nine genera. Members of the genera Palliatus and Auerbachia were sequenced for the first time. The phylogenetic analysis supported a split of myxosporeans into two main lineages separating most of freshwater species from marine ones as described by previous authors. In addition to the two main lineages, a third lineage consisting of three species was found (Sphaerospora truttae, Sphaerospora elegans and Leptotheca ranae) and additional exceptions to the marine/freshwater myxosporean split were recognised (Sphaeromyxa hellandi, Sphaeromyxa longa and Myxidium coryphaenoideum). All three myxosporean lineages were characterised by specific lengths of SSU rDNA sequences. The lineage of marine myxosporeans split into five well-defined clades. They consisted of species with a similar site of infection and spore morphology and were referred as the Parvicapsula clade, the Enteromyxum clade, the Ceratomyxa clade, the marine Myxidium clade and the Kudoa clade, respectively. The inner topology of the freshwater clade was more complex but the trend to branch according to site of infection was observed in this clade as well. Due to the number of sequences available, a histozoic (Myxobolus clade) predominated. Interestingly, five morphologically different species infecting urinary bladder clustered within the histozoic (Myxobolus) clade. The phylogenetic trees derived from this study differ in a number of respects from the current taxonomy of the myxosporeans, which suggests that several currently utilised characters may be homoplasious or that reliance on a single gene tree may not adequately reflect the phylogeny of the group.     

A molecular phylogeny of Heterodonta (Bivalvia) based on small ribosomal subunit RNA sequences

Within Heterodonta, phylogenesis has so far been studied almost exclusively on the basis of morphological data. Results have often been discordant, and an exhaustive molecular approach has not yet been attempted. The present study was undertaken to clarify the phylogenetic relationships obtaining among Heterodonta families through the analysis of 18S rRNA gene. To do this, the whole sequence of this gene was analyzed in 29 species of eight superfamilies of the order of Veneroida (Arcticoidea, Cardioidea, Galeommatoidea, Mactroidea, Solenoidea, Tellinoidea, Tridacnoidea, and Veneroidea) and in two superfamilies of Myoida (Pholaloidea and Myoidea). The study was extended by constructing phylogenetic trees using partial sequences. This strategy made it possible to include 11 additional species by introducing three further superfamilies: Chamoidea, Corbiculoidea, and Hiatellinoidea. At variance with the conclusions reached on the basis of morphological features, the molecular data clearly show that the Myoida species included in this study belong to Veneroida, thus undermining the legitimacy of the division of Heterodonta into two orders, and that considerable differences in the phylogenetic relationships obtain among superfamilies.     

Molecular phylogeny of brachiopods and phoronids based on nuclear-encoded small subunit ribosomal RNA gene sequences

Brachiopod and phoronid phylogeny is inferred from SSU rDNA sequences of 28 articulate and nine inarticulate brachiopods, three phoronids, two ectoprocts and various outgroups, using gene trees reconstructed by weighted parsimony, distance and maximum likelihood methods. Of these sequences, 33 from brachiopods, two from phoronids and one each from an ectoproct and a priapulan are newly determined. The brachiopod sequences belong to 31 different genera and thus survey about 10% of extant genus-level diversity. Sequences determined in different laboratories and those from closely related taxa agree well, but evidence is presented suggesting that one published phoronid sequence (GenBank accession UO12648) is a brachiopod-phoronid chimaera, and this sequence is excluded from the analyses. The chiton, Acanthopleura, is identified as the phenetically proximal outgroup; other selected outgroups were chosen to allow comparison with recent, non-molecular analyses of brachiopod phylogeny. The different outgroups and methods of phylogenetic reconstruction lead to similar results, with differences mainly in the resolution of weakly supported ancient and recent nodes, including the divergence of inarticulate brachiopod sub-phyla, the position of the rhynchonellids in relation to long- and short-looped articulate brachiopod clades and the relationships of some articulate brachiopod genera and species. Attention is drawn to the problem presented by nodes that are strongly supported by non-molecular evidence but receive only low bootstrap resampling support. Overall, the gene trees agree with morphology-based brachiopod taxonomy, but novel relationships are tentatively suggested for thecideidine and megathyrid brachiopods. Articulate brachiopods are found to be monophyletic in all reconstructions, but monophyly of inarticulate brachiopods and the possible inclusion of phoronids in the inarticulate brachiopod clade are less strongly established. Phoronids are clearly excluded from a sister-group relationship with articulate brachiopods, this proposed relationship being due to the rejected, chimaeric sequence (GenBank UO12648). Lineage relative rate tests show no heterogeneity of evolutionary rate among articulate brachiopod sequences, but indicate that inarticulate brachiopod plus phoronid sequences evolve somewhat more slowly. Both brachiopods and phoronids evolve slowly by comparison with other invertebrates. A number of palaeontologically dated times of earliest appearance are used to make upper and lower estimates of the global rate of brachiopod SSU rDNA evolution, and these estimates are used to infer the likely divergence times of other nodes in the gene tree. There is reasonable agreement between most inferred molecular and palaeontological ages. The estimated rates of SSU rDNA sequence evolution suggest that the last common ancestor of brachiopods, chitons and other protostome invertebrates (Lophotrochozoa and Ecdysozoa) lived deep in Precambrian time. Results of this first DNA-based, taxonomically representative analysis of brachiopod phylogeny are in broad agreement with current morphology-based classification and systematics and are largely consistent with the hypothesis that brachiopod shell ontogeny and morphology are a good guide to phylogeny.     

The current status of the small subunit rRNA phylogeny of the coccidia (Sporozoa)

There is no current comprehensive assessment of the molecular phylogeny of the coccidia, as all recently published papers either deal with subsets of the taxa or sequence data, or provide non-robust analyses. Here, we present a comprehensive and consistent phylogenetic analysis of the available data for the small-subunit ribosomal RNA gene sequence, including a number of taxa not previously studied, based on a Bayesian tree-building analysis and the covariotide model of evolution. The assumptions of the analysis have been rigorously tested, and the benefits and limitations highlighted. Our results provide support for a number of prior conclusions, including the monophyly of the families Sarcocystidae (cyst-forming coccidia) and Eimeriidae (oocyst-forming coccidia), but with bird-host Isospora species in the Eimeriidae and mammal-host species in the Sarcocystidae. However, it is clear that a number of previously reported relationships are dependent on the evolutionary model chosen, such as the placements of Goussia janae, Lankesterella minimia and Caryospora bigenetica. Our results also confirm the monophyly of the subfamilies Toxoplasmatinae and Sarcocystinae, but only some of the previously reported groups within these subfamilies are supported by our analysis. Similarly, only some of the previously reported groups within the Eimeriidae are supported by our analysis, and the genus Eimeria is clearly paraphyletic. There are unambiguous patterns of host-parasite relationship within the coccidia, as most of the well-supported groups have a consistent and restricted range of hosts, with the exception of the Toxoplasmatinae. Furthermore, the previously reported groups for which we found no support all have a diverse range of unrelated hosts, confirming that these are unlikely to be natural groups. The most interesting unaddressed questions may relate to Isospora, which has the fewest available sequences and host-parasite relationships apparently not as straightforward as elsewhere within the suborder.     

Molecular phylogeny of onygenalean fungi based on small subunit ribosomal DNA (SSU rDNA) sequences

Phylogenetic analysis of nucleotide data from small subunit ribosomal DNA (SSU rDNA) sequences (ca. 1685 bp.) was performed on 19 taxa of the Onygenales and three related mitosporic fungi. Phylogenetic trees were constructed by the neighbor-joining method with the sequence data of related taxa obtained from DNA databases. The species in the Onygenales form two clusters and seven subclusters, and the tree topology reflects the traditional classification by Currah (1985) with some exceptions. The Myxotrichaceae is placed in the different lineage, separate from other plectomycetous taxa and among the Leotiales and the Erysiphales. Furthermore, two separate lineages in the Myxotrichaceae were found. Tree topology suggested the Onygenaceae is polyphyletic and composed of three subgroups; 1) most members of Onygenaceae, 2)Spiromastix warcupii, and 3) pathogenic dimorphic fungi classified inAjellomyces.     

Molecular phylogeny of Stentor (Ciliophora: Heterotrichea) based on small subunit ribosomal RNA sequences

To determine the phylogenetic position of Stentor within the Class Heterotrichea, the complete small subunit rRNA genes of three Stentor species, namely Stentor polymorphus, Stentor coeruleus, and Stentor roeseli, were sequenced and used to construct phylogenetic trees using the maximum parsimony, neighbor joining, and Bayesian analysis. With all phylogenetic methods, the genus Stentor was monophyletic, with S. roeseli branching basally.     

Nematode communities of small farmland ponds

The nematofauna of 14 farmland ponds, selected according to a gradient of surrounding agricultural land-use intensity, from five regions in North-West of Belgium were studied. The total nematode density (9–411 ind./10 cm2 per pond), and especially the number of species (4–12 species per pond) was especially low in these ponds. In total, 17 genera of free-living benthic nematodes, belonging to 15 families, are identified. Tobrilus gracilis and Eumonhystera filiformis were the most common species and were found in 13 and 12 of the 14 sampled ponds, respectively. The genera Tobrilus and Eumonhystera jointly comprise 77% of the total nematofauna. Consequently, the investigated water bodies were dominated by deposit feeding Monhysteridae and/or by chewing Tobrilidae. Diplogasteridae and Rhabditidae, normally related with eutrophic habitats, were almost absent. In order to explain the variation of total density, diversity, feeding-types composition and the individual density of the six most important species within ponds as well, sets of environmental variables were statistically selected. It was demonstrated that morphologically very similar species can show highly different ecological properties. The presence of a substantial mud layer and of an overall high level of eutrophication as well as the presence of possibly associated anaerobic conditions are put forward as the main factors explaining the observed low density and diversity. Total phosphate concentration and sediment characteristics seem to be the most important variables to explain the nematode community structure. However, a clear pattern of environmental variables, agricultural land use and nematode assemblages was not observed. Handling editor: K. Martens     

Effects of sequence alignment procedures on estimates of phylogeny

Previous debate about statistical variation in inferred phylogenies has focused on procedures for the estimation of evolutionary relationships from aligned sequences. Morrison and Ellis¹ have recently drawn attention to additional variation attributable to the alignment procedure used and have suggested that this may be highly significant. This raises doubts about our ability to infer reliable phylogenies. Although concerns may not be as serious as their analyses at first imply, Morrison and Ellis¹ have performed a useful service in reminding us that accurate sequence alignment is a crucial part of molecular phylogenetics. BioEssays 20 :287-290, 1998.© 1998 John Wiley & Sons, Inc.     

BAli-Phy: simultaneous Bayesian inference of alignment and phylogeny

SUMMARY: BAli-Phy is a Bayesian posterior sampler that employs Markov chain Monte Carlo to explore the joint space of alignment and phylogeny given molecular sequence data. Simultaneous estimation eliminates bias toward inaccurate alignment guide-trees, employs more sophisticated substitution models during alignment and automatically utilizes information in shared insertion/deletions to help infer phylogenies. AVAILABILITY: Software is available for download at http://www.biomath.ucla.edu/msuchard/bali-phy.     

Molecular phylogeny of the Heterotrichea (Ciliophora, Postciliodesmatophora) based on small subunit rRNA gene sequences

A comprehensive molecular analysis of the phylogenetic relationships within the Heterotrichea including all described families is still lacking. For this reason, the complete nuclear small subunit (SSU) rDNA was sequenced from further representatives of the Blepharismidae and the Stentoridae. In addition, the SSU rDNA of a new, undescribed species of the genus Condylostomides (Condylostomatidae) was sequenced. The detailed phylogenetic analyses revealed a consistent branching pattern: while the terminal branches are generally well resolved, the basal relationships remain unsolved. Moreover, the data allow some conclusions about the macronuclear evolution within the genera Blepharisma, Stentor, and Spirostomum suggesting that a single, compact macronucleus represents the ancestral state.     

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences,with emphasis on the Devescovinidae and Calonymphidae (Trichomonadea)

Small subunit rRNA sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the polymastigont Calonymphidae, including Snyderella tabogae, Calonympha grassii, and Metacoronympha senta. The yet-unidentified sequence Nk9 previously obtained from the termite Neotermes koshunensis, has also been shown to derive from the Devescovinidae Devescovina sp. by in situ hybridization. These new sequences were analyzed by distance, parsimony, and likelihood methods in a broad phylogeny including all identified parabasalid sequences available in databases. All analyses revealed the emergence of a very well supported Devescovinidae/Calonymphidae group but showed an unexpected dichotomy of the Calonymphidae represented by the "Coronympha" and "Calonympha" groups. It strongly suggests that the polymastigont state observed in the Calonymphidae might be explained by at least two independent evolutionary events. In a second phylogenetic analysis, some yet-unidentified parabasalid sequences likely deriving from the Devescovinidae/Calonymphidae taxa, were added to our data set. This analysis confirmed the polyphyly of the Calonymphidae. A tentative identification is proposed for each of these sequences, and hypotheses on the origin of the Devescovinidae and Calonymphidae are discussed. Tritrichomonas foetus or a close relative might be the best candidate for the ancestor of the Devescovinidae, fairly consistent with morphology-based hypotheses. Regarding the Calonymphidae, the origin of the "Coronympha" group might be found within the Devescovinidae, related to Foaina, whereas the "Calonympha" group may directly descend from Tritrichomonas or related species.     

Diversity and phylogeny of insect trypanosomatids based on small subunit rRNA genes: polyphyly of Leptomonas and Blastocrithidia

With the aim of further investigating phylogenetic relationships in insect trypanosomatids, we have determined the sequences of small subunit rRNA genes from ten isolates, which were originally classified as Leptomonas, Blastocrithidia, and Wallaceina based on their morphology in the hosts. The inferred maximum likelihood, parsimony, and distance trees indicate that the Leptomonas and Blastocrithidia are polyphyletic, and confirm the polyphyly of Herpetomonas and Crithidia. Blastocrithidia triatoma and Leptomonas collosoma were among the earliest branching lineages among the insect trypanosomatids, while most other isolates were found within a closely related terminal clade, which also included Crithidia fasciculata. This analysis has clearly demonstrated that the morphological classification system of insect trypanosomatids does not always reflect their genetic affinities warranting its revision in the future.