Molecular phylogenetic position of the genera Stephanonympha and Caduceia (Parabasalia) inferred from nuclear small subunit rRNA gene sequences

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.     

Phylogenetic relationships within the class Spirotrichea (Ciliophora) inferred from small subunit rRNA gene sequences

The small subunit rDNAs of five species belonging to the Euplotidae and eight species of the Oxytrichidae were sequenced to obtain a more detailed picture of the phylogenetic relationships within the Spirotrichea (Ciliophora). Various tree reconstruction algorhythms yielded nearly identical topologies. All Euplotidae were separated from the other Spirotrichea by a deep split. Further, a large genetic distance between the marine genus Moneuplotes and the freshwater species of Euplotoides was found. Differences between the methods used occurred only within the Oxytrichidae. Whereas the monophyly of the Stylonychinae was supported in all trees, the monophyly of the Oxytrichinae was not. However, the molecular data support the morphological and ontogenetic evidence that the pattern of 18 frontal-ventral-transversal cirri evolved in the stemline of the Oxytrichidae and was modified several times independently. Our results are also in agreement with taxonomic revisions: the separation of both Sterkiella nova from Oxytricha and Tetmemena pustulata from Stylonychia.     

Affinities among anostracan (Crustacea: Branchiopoda) families inferred from phylogenetic analyses of multiple gene sequences

To gain insights into the relationships among anostracan families, molecular phylogenetic analyses were performed on nuclear (28S D1-D3 ribosomal DNA) and mitochondrial (16S rDNA, COI) gene regions for representatives of seven families and an outgroup. Data matrices used in the analyses included 951 base pairs (bp) of aligned sequences for 28S, 465 bp for 16S, and 658 bp (219 amino acids) for COI. Maximum-parsimony and maximum-likelihood methods were used to construct phylogenetic trees, enabling the evaluation of both previous hypotheses of taxonomic relationships among families based on morphology, and of the relative merits of independent versus simultaneous analyses of multiple data sets for phylogeny construction. Data from various combinations of the gene regions produced relatively congruent patterns of phylogenetic affinity. In most analyses, two monophyletic groups were resolved: one cluster included the families Polyartemiidae, Chirocephalidae, Branchinectidae, Streptocephalidae, and Thamnocephalidae, while the other contained the Artemiidae and Branchipodidae. Comparative analyses showed that combining gene regions in a single matrix generally resulted in increased resolution and support for each cluster relative to those obtained from single-gene analyses. Statistical tests demonstrated that morphology-based hypotheses of relationships among families had poorer support than those determined from molecular data, reflecting the homoplasy in characters used to differentiate families.     

The phylogenetic position of Allocreadiidae (Trematoda: Digenea) from partial sequences of the 18S and 28S ribosomal RNA genes

Species of Allocreadiidae are an important component of the parasite fauna of freshwater vertebrates, particularly fishes, and yet their systematic relationships with other trematodes have not been clarified. Partial sequences of the 18S and 28S ribosomal RNA genes from 3 representative species of Allocreadiidae, i.e., Crepidostomum cooperi, Bunodera mediovitellata, and Polylekithum ictaluri, and from 79 other taxa representing 78 families of trematodes obtained from GenBank, were used in a phylogenetic analysis to address the relationships of Allocreadiidae with other plagiorchiiforms/plagiorchiidans. Maximum parsimony and Bayesian analyses of combined 18S and 28S rRNA gene sequence data place 2 of the allocreadiids, Crepidostomum cooperi and Bunodera mediovitellata, in a clade with species of Callodistomidae and Gorgoderidae, which, in turn is sister to a clade containing Polylekithum ictaluri and representatives of Encyclometridae, Dicrocoelidae, and Orchipedidae, a grouping supported by high bootstrap values. These results suggest that Polylekithum ictaluri is not an allocreadiid, a conclusion that is supported by reported differences between its cercaria and that of other allocreadiids. Although details of the life cycle of callodistomids, the sister taxon to Allocreadiidae, remain unknown, the relationship of Allocreadiidae and Gorgoderidae is consistent with their larval development in bivalve, rather than gastropod, molluscs, and with their host relationships (predominantly freshwater vertebrates). The results also indicate that, whereas Allocreadiidae is not a basal taxon, it is not included within the suborder Plagiorchiata. No support was found for a direct relationship between allocreadiids and opecoelids either.     

Phylogeny of six oligohymenophoreans (Protozoa, Ciliophora) inferred from small subunit rRNA gene sequences

The small subunit rRNA (SSrRNA) genes of six marine oligohymenophoreans, namely Uronemella filificum , Schizocalyptra sp.-WYG07060701, Schizocalyptra aeschtae , Pleuronema sinica , P. czapikae and Paratetrahymena sp., were sequenced. Phylogenetic trees were constructed with four different methods to assess the inter- and intrageneric relationships among the scuticociliates and the phylogenetic assignment of the order Loxocephalida. The SSrRNA phylogeny indicates that: (i) Paratetrahymena is most closely related to Cardiostomatella ; (ii) the order Loxocephalida and the family Uronematidae both appear to be polyphyletic; (iii) the order Philasterida is a well-defined taxon; (iv) Cyclidium porcatum falls outside the order Pleuronematida in all analyses; (v) the validity of the genus Uronemella is confirmed; (vi) Schizocalyptra is a member of the family Pleuronematidae. Furthermore, the predicted secondary structures of the variable region 4 of the SSrRNA gene sequences show that the size of the terminal bulge in Helix E23–7 is probably different for the orders Philasterida and Pleuronematida. Also, compared to Uronema and Homalogastra , Uronemella has distinct patterns in Helices E23–1, E23–7, E23–8 and E23–9.     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.      

Diagnosis of candidosis by amplification of small subunit of 18S rRNA gene

A PCR assay for the diagnosis of infection produced by Candida sp. was developed. The primers, designated 520 and 522, were selected from highly-conserved areas of the small subunit (ssu) 18S rRNA gene of Candida spp. To check the value of the results a Candida albicans oligonucleotide probe, digoxigenin-labeled, and a general Candida probe were used in hybridization experiments with the amplified products. We were able to detect a Candida- specific fragment of 1800 bp from different clinical samples. The procedure described could provide an interesting complement to present diagnostic methods of detecting Candida sp in clinical samples.     

Phylogeny of protostome moulting animals (Ecdysozoa) inferred from 18 and 28S rRNA gene sequences

Reliability of reconstruction of phylogenetic relationships within a group of protostome moulting animals was evaluated by means of comparison of 18 and 28S rRNA gene sequences sets both taken separately and combined. Reliability of reconstructions was evaluated by values of the bootstrap support of major phylogenetic tree nodes and by degree of congruence of phylogenetic trees inferred by various methods. By both criteria, phylogenetic trees reconstructed from the combined 18 and 28S rRNA gene sequences were better than those inferred from 18 and 28S sequences taken separately. Results obtained are consistent with phylogenetic hypothesis separating protostome animals into two major clades, moulting Ecdysozoa (Priapulida + Kinorhyncha, Nematoda + Nematomorpha, Onychophora + Tardigrada, Myriapoda + Chelicerata, Crustacea + Hexapoda) and unmoulting Lophotrochozoa (Plathelminthes, Nemertini, Annelida, Mollusca, Echiura, Sipuncula). Clade Cephalorhyncha does not include nematomorphs (Nematomorpha). Conclusion was taken that it is necessary to use combined 18 and 28S data in phylogenetic studies.     

Molecular systematics of Volvocales (Chlorophyceae, Chlorophyta) based on exhaustive 18S rRNA phylogenetic analyses

The taxonomy of Volvocales (Chlorophyceae, Chlorophyta) was traditionally based solely on morphological characteristics. However, because recent molecular phylogeny largely contradicts the traditional subordinal and familial classifications, no classification system has yet been established that describes the subdivision of Volvocales in a manner consistent with the phylogenetic relationships. Towards development of a natural classification system at and above the generic level, identification and sorting of hundreds of sequences based on subjective phylogenetic definitions is a significant step. We constructed an 18S rRNA gene phylogeny based on 449 volvocalean sequences collected using exhaustive BLAST searches of the GenBank database. Many chimeric sequences, which can cause fallacious phylogenetic trees, were detected and excluded during data collection. The results revealed 21 strongly supported primary clades within phylogenetically redefined Volvocales. Phylogenetic classification following PhyloCode was proposed based on the presented 18S rRNA gene phylogeny along with the results of previous combined 18S and 26S rRNA and chloroplast multigene analyses.     

Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences.

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.     

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.     

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.     

Phylogeny of the Northeast Pacific brown algal (Phaeophycean) orders as inferred from 18S rRNA gene sequences

Partial 18S rRNA gene sequences have been determined for thirteen brown algae representing nine Northeast Pacific brown algal orders: Chordariales, Desmarestiales, Dictyosiphonales, Dictyotales, Ectocarpales, Fucales, Scytosiphonales, Sphacelariales and Syringodermatales. These sequences were compared with published sequences from a kelp (Laminariales), a xanthophyte and a bacillariophyte. A preliminary phylogeny generated by the neighbor-joining phylogeny inference method indicated that the class Phaeophyceae is a monophyletic group in relation to the xanthophyte and the bacillariophyte. Further, bootstrap analysis of the phylogeny consistently grouped together all the representatives belonging to the orders Ectocarpales, Chordariales, Dictyosiphonales and Scytosiphonales and separated them from the representatives belonging to the other brown algal orders. These results offer valuable insights into the controversial brown algal orders' phylogeny and provide additional data to the phylogenetic relationship study among the chromophyte classes.     

Reevaluation of the phylogenetic relationship between mobilid and sessilid peritrichs (Ciliophora, Oligohymenophorea) based on small subunit rRNA genes sequences

Based on morphological characters, peritrich ciliates (Class Olygohymenophorea, Subclass Peritrichia) have been subdivided into the Orders Sessilida and Mobilida. Molecular phylogenetic studies on peritrichs have been restricted to members of the Order Sessilida. In order to shed more light into the evolutionary relationships within peritrichs, the complete small subunit rRNA (SSU rRNA) sequences of four mobilid species, Trichodina nobilis, Trichodina heterodentata, Trichodina reticulata, and Trichodinella myakkae were used to construct phylogenetic trees using maximum parsimony, neighbor joining, and Bayesian analyses. Whatever phylogenetic method used, the peritrichs did not constitute a monophyletic group: mobilid and sessilid species did not cluster together. Similarity in morphology but difference in molecular data led us to suggest that the oral structures of peritrichs are the result of evolutionary convergence. In addition, Trichodina reticulata, a Trichodina species with granules in the center of the adhesive disc, branched separately from its congeners, Trichodina nobilis and Trichodina heterodentata, trichodinids without such granules. This indicates that granules in the adhesive disc might be a phylogenetic character of high importance within the Family Trichodinidae.     

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.     

V4 region of small subunit rDNA indicates polyphyly of the Fellodistomidae (Digenea) which is supported by morphology and life-cycle data

There is no morphological synapomorphy for the disparate digeneans, the Fellodistomidae Nicoll, 1909. Although all known life-cycles of the group include bivalves as first intermediate hosts, there is no convincing morphological synapomorphy that can be used to unite the group. Sequences from the V4 region of small subunit (18S) rRNA genes were used to infer phylogenetic relationships among 13 species of Fellodistomidae from four subfamilies and eight species from seven other digenean families: Bivesiculidae; Brachylaimidae; Bucephalidae; Gorgoderidae; Gymnophallidae; Opecoelidae; and Zoogonidae. Outgroup comparison was made initially with an aspidogastrean. Various species from the other digenean families were used as outgroups in subsequent analyses. Three methods of analysis indicated polyphyly of the Fellodistomidae and at least two independent radiations of the subfamilies, such that they were more closely associated with other digeneans than to each other. The Tandanicolinae was monophyletic (100% bootstrap support) and was weakly associated with the Gymnophallidae (<50–55% bootstrap support). Monophyly of the Baccigerinae was supported with 78–87% bootstrap support, and monophyly of the Zoogonidae + Baccigerinae received 77–86% support. The remaining fellodistomid species, Fellodistomum fellis, F. agnotum and Coomera brayi (Fellodistominae) plus Proctoeces maculatus and Complexobursa sp. (Proctoecinae), formed a separate clade with 74–92% bootstrap support. On the basis of molecular, morphological and life-cycle evidence, the subfamilies Baccigerinae and Tandanicolinae are removed from the Fellodistomidae and promoted to familial status. The Baccigerinae is promoted under the senior synonym Faustulidae Poche, 1926, and the Echinobrevicecinae Dronen, Blend & McEachran, 1994 is synonymised with the Faustulidae. Consequently, species that were formerly in the Fellodistomidae are now distributed in three families: Fellodistomidae; Faustulidae (syn. Baccigerinae Yamaguti, 1954); and Tandanicolidae Johnston, 1927. We infer that the use of bivalves as intermediate hosts by this broad range of families indicates multiple host-switching events within the radiation of the Digenea.     

Molecular evolution of pteridophytes and their relationship to seed plants: Evidence from complete 18S rRNA gene sequences

Complete 18S ribosomal RNA sequence data from representatives of all extant pteridophyte lineages together with RNA sequences from different seed plants were used to infer a molecular phylogeny of vascular plants that included all major land plant lineages. The molecular data indicate that lycopsids are monophyletic and are the earliest diverging group within the vascular land plants, whereasPsilotum nudum is more closely related to the seed plants than to other pteridophyte lineages. The phylogenetic trees based on maximum likelihood, parsimony and distance analyses show substantial agreement with the evolutionary relationships of land plants as interpreted from the fossil record.     

Ecdysozoan phylogeny and Bayesian inference: first use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin

Relationships among the ecdysozoans, or molting animals, have been difficult to resolve. Here, we use nearly complete 28S+18S ribosomal RNA gene sequences to estimate the relations of 35 ecdysozoan taxa, including newly obtained 28S sequences from 25 of these. The tree-building algorithms were likelihood-based Bayesian inference and minimum-evolution analysis of LogDet-transformed distances, and hypotheses were tested wth parametric bootstrapping. Better taxonomic resolution and recovery of established taxa were obtained here, especially with Bayesian inference, than in previous parsimony-based studies that used 18S rRNA sequences (or 18S plus small parts of 28S). In our gene trees, priapulan worms represent the basal ecdysozoans, followed by nematomorphs, or nematomorphs plus nematodes, followed by Panarthropoda. Panarthropoda was monophyletic with high support, although the relationships among its three phyla (arthropods, onychophorans, tardigrades) remain uncertain. The four groups of arthropods-hexapods (insects and related forms), crustaceans, chelicerates (spiders, scorpions, horseshoe crabs), and myriapods (centipedes, millipedes, and relatives)-formed two well-supported clades: Hexapoda in a paraphyletic crustacea (Pancrustacea), and 'Chelicerata+Myriapoda' (a clade that we name 'Paradoxopoda'). Pycnogonids (sea spiders) were either chelicerates or part of the 'chelicerate+myriapod' clade, but not basal arthropods. Certain clades derived from morphological taxonomy, such as Mandibulata, Atelocerata, Schizoramia, Maxillopoda and Cycloneuralia, are inconsistent with these rRNA data. The 28S gene contained more signal than the 18S gene, and contributed to the improved phylogenetic resolution. Our findings are similar to those obtained from mitochondrial and nuclear (e.g., elongation factor, RNA polymerase, Hox) protein-encoding genes, and should revive interest in using rRNA genes to study arthropod and ecdysozoan relationships.