A comprehensive molecular phylogeny of tardigrades—adding genes and taxa to a poorly resolved phylum‐level phylogeny

Tardigrades constitute a phylum of miniaturized metazoans with ca. 1030 living species, a fossil record that probably dates back to the Cambrian, and physiological properties that allow them to live in almost any environment known to host life on Earth—they can also survive in space. Despite broad consensus regarding their membership of the superclade Ecdysozoa, the exact position of the phylum remains contested (some analyses suggest onychophorans and arthropods as their closest relatives, while others suggest a relationship to nematodes and nematomorphs) and the internal relationships of the phylum are still poorly understood. In the present study, we present a hypothesis of tardigrade relationships by examining more taxa and more markers than any previously published phylogeny of the group. We generated novel data for three markers (18S rRNA, 28S rRNA, COI) for 42 individuals of 16 carefully identified species, comprising 12 genera and five families from the classes Heterotardigrada and Eutardigrada, and analysed them in conjunction with nearly all data available from GenBank. Our results show certain disagreement with current taxonomy both at higher ranks (families, orders, classes) and at low (generic) taxonomic levels. When studying the sensitivity to outgroup choice, the class Eutardigrada was monophyletic under only one combination of outgroups; all other combinations placed the eutardigrade order Apochela as sister to the class Heterotardigrada. Phylogenetic relationships within the other eutardigrade order, Parachela, were stable to outgroup choice. Eutardigrade superfamilies recently proposed by Sands and collaborators in the order Parachela were tested with the introduction of new sequences from additional genera, and the possible morphological synapomorphies supporting those superfamilies are discussed. © The Will Henning Society 2011.     

Evolutionary relationships within the protostome phylum Sipuncula: a molecular analysis of ribosomal genes and histone H3 sequence data

The phylogenetic relationships of the members of the phylum Sipuncula are investigated by means of DNA sequence data from three nuclear markers, two ribosomal genes (18S rRNA and the D3 expansion fragment of 28S rRNA), and one protein-coding gene, histone H3. Phylogenetic analysis via direct optimization of DNA sequence data using parsimony as optimality criterion is executed for 12 combinations of parameter sets accounting for different indel costs and transversion/transition cost ratios in a sensitivity analysis framework. Alternative outgroup analyses are also performed to test whether they affected rooting of the sipunculan topology. Nodal support is measured by parsimony jackknifing and Bremer support values. Results from the different partitions are highly congruent, and the combined analysis for the parameter set that minimizes overall incongruence supports monophyly of Sipuncula, but nonmonophyly of several higher taxa recognized for the phylum. Mostly responsible for this is the split of the family Sipunculidae in three main lineages, with the genus Sipunculus being the sister group to the remaining sipunculans, the genus Phascolopsis nesting within the Golfingiiformes, and the genus Siphonosoma being associated to the Phascolosomatidea. Other interesting results are the position of Phascolion within Golfingiidae and the position of Antillesoma within Aspidosiphonidae. These results are not affected by the loci selected or by the outgroup chosen. The position of Apionsoma is discussed, although more data would be needed to better ascertain its phylogenetic affinities. Monophyly of the genera with multiple representatives (Themiste, Aspidosiphon, and Phascolosoma) is well supported, but not the monophyly of the genera Nephasoma or Golfingia. Interesting phylogeographic questions arise from analysis of multiple representatives of a few species.     

MicroRNAs resolve an apparent conflict between annelid systematics and their fossil record

Both the monophyly and inter-relationships of the major annelid groups have remained uncertain, despite intensive research on both morphology and molecular sequences. Morphological cladistic analyses indicate that Annelida is monophyletic and consists of two monophyletic groups, the clitellates and polychaetes, whereas molecular phylogenetic analyses suggest that polychaetes are paraphyletic and that sipunculans are crown-group annelids. Both the monophyly of polychaetes and the placement of sipunculans within annelids are in conflict with the annelid fossil record—the former because Cambrian stem taxa are similar to modern polychaetes in possessing biramous parapodia, suggesting that clitellates are derived from polychaetes; the latter because although fossil sipunculans are known from the Early Cambrian, crown-group annelids do not appear until the latest Cambrian. Here we apply a different data source, the presence versus absence of specific microRNAs—genes that encode approximately 22 nucleotide non-coding regulatory RNAs—to the problem of annelid phylogenetics. We show that annelids are monophyletic with respect to sipunculans, and polychaetes are paraphyletic with respect to the clitellate Lumbricus, conclusions that are consistent with the fossil record. Further, sipunculans resolve as the sister group of the annelids, rooting the annelid tree, and revealing the polarity of the morphological change within this diverse lineage of animals.     

The complete mitochondrial genome sequence of Whitmania pigra (Annelida, Hirudinea): the first representative from the class Hirudinea

The mitochondrial genome is a significant tool for investigating the evolutionary history of metazoan animals. The currently available mitochondrial genome data in GenBank is limited to understand the detail evolutionary relationship among the metazoan animals, especially in the phylum Annelida. Here we present the mitochondrial gene organization, gene order and codon usage of the leech Whitmania pigra (Annelida), which is the first representative from the class Hirudinea. It is a circular molecule of 14,426bp, and encodes 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. All 37 genes of W. pigra mitochondrial genome are transcribed from the same strand, which is identical to studied annelids, two echiurans, two sipunculans and many other lophotrochozoans. Five conserved gene clusters can be found in mitochondrial genomes of nine studied annelids, including (1) cox1-N-cox2; (2) cox3-Q-nad6-cob-W-atp6; (3) H-nad5-F-E-P-T-nad4L-nad4; (4) srRNA-V-lrRNA; and (5) nad3-S(1)-nad2. Compared with that of other studied annelids, translocations of transfer RNAs were found in the gene arrangement of W. pigra mitochondrial genome. Phylogenetic analysis strongly support that the species from Hirudinina and Oligochaeta form a monophyletic group Clitellata (BPM=100, BPP=100), which is consistent with previous research based on morphological and other molecular data. Both gene order data and amino acid sequences reveal that echiurans are derived annelids and sipunculans should be clustered with annelids and echiurans.     

Using Regression Models to Analyze Randomized Trials: Asymptotically Valid Hypothesis Tests Despite Incorrectly Specified Models

Summary .  Regression models are often used to test for cause-effect relationships from data collected in randomized trials or experiments. This practice has deservedly come under heavy scrutiny, because commonly used models such as linear and logistic regression will often not capture the actual relationships between variables, and incorrectly specified models potentially lead to incorrect conclusions. In this article, we focus on hypothesis tests of whether the treatment given in a randomized trial has any effect on the mean of the primary outcome, within strata of baseline variables such as age, sex, and health status. Our primary concern is ensuring that such hypothesis tests have correct type I error for large samples. Our main result is that for a surprisingly large class of commonly used regression models, standard regression-based hypothesis tests (but using robust variance estimators) are guaranteed to have correct type I error for large samples, even when the models are incorrectly specified. To the best of our knowledge, this robustness of such model-based hypothesis tests to incorrectly specified models was previously unknown for Poisson regression models and for other commonly used models we consider. Our results have practical implications for understanding the reliability of commonly used, model-based tests for analyzing randomized trials.     

A modern approach to rotiferan phylogeny: combining morphological and molecular data

The phylogeny of selected members of the phylum Rotifera is examined based on analyses under parsimony direct optimization and Bayesian inference of phylogeny. Species of the higher metazoan lineages Acanthocephala, Micrognathozoa, Cycliophora, and potential outgroups are included to test rotiferan monophyly. The data include 74 morphological characters combined with DNA sequence data from four molecular loci, including the nuclear 18S rRNA, 28S rRNA, histone H3, and the mitochondrial cytochrome c oxidase subunit I. The combined molecular and total evidence analyses support the inclusion of Acanthocephala as a rotiferan ingroup, but do not support the inclusion of Micrognathozoa and Cycliophora. Within Rotifera, the monophyletic Monogononta is sister group to a clade consisting of Acanthocephala, Seisonidea, and Bdelloidea-for which we propose the name Hemirotifera. We also formally propose the inclusion of Acanthocephala within Rotifera, but maintaining the name Rotifera for the new expanded phylum. Within Monogononta, Gnesiotrocha and Ploima are also supported by the data. The relationships within Ploima remain unstable to parameter variation or to the method of phylogeny reconstruction and poorly supported, and the analyses showed that monophyly was questionable for the families Dicranophoridae, Notommatidae, and Brachionidae, and for the genus Proales. Otherwise, monophyly was generally supported for the represented ploimid families and genera.     

Molecular signatures for the phylum Synergistetes and some of its subclades

Species belonging to the phylum Synergistetes are poorly characterized. Though the known species display Gram-negative characteristics and the ability to ferment amino acids, no single characteristic is known which can define this group. For eight Synergistetes species, complete genome sequences or draft genomes have become available. We have used these genomes to construct detailed phylogenetic trees for the Synergistetes species and carried out comprehensive analysis to identify molecular markers consisting of conserved signature indels (CSIs) in protein sequences that are specific for either all Synergistetes or some of their sub-groups. We report here identification of 32 CSIs in widely distributed proteins such as RpoB, RpoC, UvrD, GyrA, PolA, PolC, MraW, NadD, PyrE, RpsA, RpsH, FtsA, RadA, etc., including a large >300 aa insert within the RpoC protein, that are present in various Synergistetes species, but except for isolated bacteria, these CSIs are not found in the protein homologues from any other organisms. These CSIs provide novel molecular markers that distinguish the species of the phylum Synergistetes from all other bacteria. The large numbers of other CSIs discovered in this work provide valuable information that supports and consolidates evolutionary relationships amongst the sequenced Synergistetes species. Of these CSIs, seven are specifically present in Jonquetella, Pyramidobacter and Dethiosulfovibrio species indicating a cladal relationship among them, which is also strongly supported by phylogenetic trees. A further 15 CSIs that are only present in Jonquetella and Pyramidobacter indicate a close association between these two species. Additionally, a previously described phylogenetic relationship between the Aminomonas and Thermanaerovibrio species was also supported by 9 CSIs. The strong relationships indicated by the indel analysis provide incentives for the grouping of species from these clades into higher taxonomic groups such as families or orders. The identified molecular markers, due to their specificity for Synergistetes and presence in highly conserved regions of important proteins suggest novel targets for evolutionary, genetic and biochemical studies on these bacteria as well as for the identification of additional species belonging to this phylum in different environments.     

A molecular approach to arthrotardigrade phylogeny (Heterotardigrada,Tardigrada)

The marine order Arthrotardigrada (class Heterotardigrada, phylum Tardigrada) is known for its conspicuously high morphological diversity and has been traditionally recognized as the most ancestral group within the phylum. Despite its potential importance in understanding the evolution of the phylum, the phylogenetic relationships of Arthrotardigrada have not been clarified. This study conducted molecular phylogenetic analyses of the order encompassing all families except Neoarctidae using nuclear 18S and 28S rRNA fragments. Data from two rare families, Coronarctidae and Renaudarctidae, were included for the first time. The analyses confirmed the monophyly of Heterotardigrada and inferred Coronarctidae as the sister group to all other heterotardigrade taxa. Furthermore, the results support a monophyletic Renaudarctidae + Stygarctidae clade, which has been previously suggested on morphology. Our data indicated that two subfamilies currently placed in Halechiniscidae are only distantly related to this family. We propose that these taxa are each elevated to family level (Styraconyxidae (new rank) and Tanarctidae (new rank)). The morphology of tardigrades is discussed in the context of the inferred phylogeny.     

Distribution and diversity of sipunculan fauna in high Arctic fjords (west Svalbard)

Sipuncula is a relatively species poor and generally rarely investigated phylum; nonetheless, it may play a considerable role in the ecosystem. During this study sipunculan species distribution patterns in four fjords of west Spitsbergen (Kongsfjorden, Hornsund, Isfjorden and van Mijenfjorden) were examined. Material was collected during ten cruises undertaken from 1997 to 2006. A total of 381 samples were taken at 132 stations located in the four fjords and, a total number of 920 sipunculans specimens were found in 114 of those samples. The highest sipunculan species richness was observed in Hornsund (six species), followed by Kongsfjorden and Isfjorden (five species in each fjord). Sipunculan fauna in all fjords was strongly dominated by Golfingia vulgaris (80% of all sipunculan individuals in Kongsfjorden), and Golfingia margaritacea (84% in van Mijenfjorden and 40% in Hornsund) or Nephasoma diaphanes (54% in Isfjorden). Locally, sipunculans were found in high densities (max. 62 ind. 0.1 m⁻² and up to 11% of macrobenthic densities) and biomass (max. 110.87 g 0.1 m⁻² and up to 80% of total fauna biomass). At such sites, sipunculans may play an important role in bioturbation of sediments and as a food source for higher trophic levels. Sipunculans did not occur within close proximity of the glacier where they might be eliminated due to high sedimentation rate and low amounts of organic matter. Because of their importance in benthic systems, a need to include sipunculans in routine macrobenthic surveys is emphasized.     

Novelty in phylogeny of gastrotricha: evidence from 18S rRNA gene

Gastrotricha form a phylum which is crucial for defining the origin of pseudocoelomates, in that they share a number of characters with Rotifera and Nematoda but also with acoelomates, and even the evolutionary relationships within the phylum are anything but defined. For this reason the first extensive molecular data on Gastrotricha from the 18S rRNA sequences of both orders have been obtained and analyzed. Sequence analyses show that the phylum Gastrotricha is strictly monophyletic along an evolutionary line quite distinct from that of both Rotifera and Nematoda. A new view of the evolutionary history of the phylum Gastrotricha is put forward, in which Chaetonotida, and not Macrodasyida, are the most primitive forms of the group, contrary to the commonly held view. A polyphyletic origin of aschelminthes is supported, and the misleading term pseudocoelomates should be discarded.     

Molecular evidence for Acanthocephala as a subtaxon of Rotifera

Rotifers are free-living animals usually smaller than 1 mm that possess a characteristic wheel organ. Acanthocephalans (thorny-headed worms) are larger endoparasitic animals that use vertebrates and arthropods to complete their life cycle. The taxa Acanthocephala and Rotifera are considered separate phyla, often within the taxon Aschelminthes. We have reexamined the relationship between Rotifera and Acanthocephala using 18S rRNA gene sequences. Our results conclusively show that Acanthocephala is the sister group of the rotifer class Bdelloidea. Rotifera was nonmonophyletic in all molecular analyses, which supports the hypothesis that the Acanthocephala represent a taxon within the phylum Rotifera and not a separate phylum. These results agree with a previous cladistic study of morphological characters. Correspondence to: J.R. Garey     

A molecular phylogenetic study of the subtribe Glycininae (Leguminosae) derived from the chloroplast DNA rps16 intron sequences

Phylogenetic relationships among 13 genera of the subtribe Glycininae, two genera of the allied subtribe Diocleinae that were included within Glycininae by Polhill, and two genera of the subtribe Erythrininae as outgroups were inferred from chloroplast DNA rps16 intron sequence variation. Pairwise sequence divergence values ranged from identity between Teramnus mollis and T. micans and between T. flexilis and T. labialis to 7.89% between Pueraria wallichii and Pseudeminia comosa across all accessions. Phylogenies estimated using parsimony and neighbor-joining methods revealed that (1) Glycininae is monophyletic if Pachyrhizus and Calopogonium (both Diocleinae) are included within Glycininae; (2) the genus Teramnus is closely related to Glycine, and Amphicarpaea showed a sister relationship to the clade comprising Teramnus and Glycine; (3) the expanded Glycininae including two genera of Diocleinae is divided into three branches, temporarily named I (comprising the rest of the examined taxa), II (Pueraria wallichii), and III (Mastersia), but their relationships are equivocal; and (4) the genus Pueraria, regarded as a closely related genus to Glycine, is not monophyletic and should be divided into at least four genera (a hypothesis supported previously by Lackey).     

A simplified estimator of two and four gene relationship coefficients

Molecular marker data provide a means of circumventing the problem of not knowing the population structure of a natural population, as observed similarities between a pair's genotypes provide information on their genetic relationship. Numerous method-of-moment (MOM) estimators have been developed for estimating relationship coefficients using this information. Here, I present a simplified form of Wang's 2002 relationship estimator that is not dependent upon a previously required weighting scheme, thus improving the efficiency of the estimator when used with genuinely related pairs. The new estimator is compared against other estimators under a range of conditions, including situations where the parameter estimates are truncated to lie within the legitimate parameter space. The advantages of the new estimator are most notable for the two-gene coefficient of relatedness. Truncating the MOM estimators results in parameter estimates whose properties are similar to maximum likelihood estimates, with them having generally lower sampling variances, but being biased.     

Reconstructing ordinal relationships in the Demospongiae using mitochondrial genomic data

Class Demospongiae (phylum Porifera) encompasses most of sponges' morphological and species diversity. It also represents one of the most challenging and understudied groups in animal phylogenetics, with many higher-level relationships still being unresolved. Among the unanswered questions are the most fundamental, including those about the monophyly of the Demospongiae and the relationships among the 14 recognized orders within the class. The lack of resolved phylogeny hampers progress in studies of demosponge biology, evolution and biodiversity and may interfere with the efficient conservation and economic use of this group. We addressed the question of demosponge relationships using mitochondrial genomic data. We assembled a mitochondrial genomic dataset comprising all orders of demosponges that includes 17 new and five previously published complete demosponge mitochondrial genomes. To test for the congruence between mtDNA-based and nuclear rRNA-based phylogenies, we also determined and analyzed 18S rRNA sequences for the same set of species. Our results provide strong support for five major clades within the Demospongiae: Homoscleromorpha=G0 (order Homosclerophorida), Keratosa=G1 (orders Dendroceratida, Dictyoceratida, and Verticillitida), Myxospongiae=G2 (orders Chondrosida, Halisarcida, and Verongida), marine Haplosclerida=G3 and the rest of demosponges=G4 (orders Agelasida, Astrophorida, Hadromerida, Halichondrida, Poecilosclerida, Spirophorida, and freshwater Haploscerida), and for the (G0((G1+G2)(G3+G4)) relationships among these clades. Conversely, mitochondrial genomic data do not support the monophylies of traditional subclasses Ceractinomorpha and Tetractinomorpha as well as several currently recognized orders of demosponges. Furthermore, we demonstrate that mitochondrial gene arrangements can also be informative for the inference of order-level demosponge relationships and propose a modified method for the analysis of gene order data that works well when translocation of tRNA genes are more frequent than other rearrangements.     

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 phylogenetic convergence within Elasmobranchii revealed by cytochrome oxidase subunits

The phylogenetic relationships within the subclass of Elasmobranchii are under question within the academic community and their systematic classification based on morphological, or physiological characteristics has not yet been fully justified. Modern cladistic studies suggested that batoids are derived sharks, a taxonomic status known as the Hypnosqualean hypothesis. The main purpose of this study was to address this issue using a data set of aligned, directly sequenced, mitochondrial cytochrome oxidase subunits I and II. Maximum Likelihood, Maximum Parsimony, Minimum Evolution and Bayesian inference were implemented for tree reconstructions. The results provided evidence that supported the rejection of the above hypothesis, in accordance with other recent molecular phylogenetic studies. More specifically Rajiformes species were presented as separate lineages from sharks. Prionace species on the other hand was grouped within Carcharhinoformes, which was clustered as sister group to Lamniformes. COI and COII regions supported, monophylies of Squaliformes and paraphylies of Carchariniformes.     

Early Cambrian sipunculan worms from southwest China

We report the discovery of sipunculan worms from the Lower Cambrian Maotianshan Shale, near Kunming (southwest China). Their sipunculan identity is evidenced by the general morphology of the animals (sausage-shaped body with a slender retractable introvert and a wider trunk) and by other features, both external (e.g. perioral crown of tentacles, and hooks, papillae and wrinkle rings on the body surface) and internal (U-shaped gut, and the anus opening near the introvert-trunk junction). The three fossil forms (Archaeogolfingia caudata gen. et sp. nov., Cambrosipunculus tentaculatus gen. et sp. nov. and Cambrosipunculus sp.) have striking similarities to modern sipunculans, especially the Golfingiidae to which their evolutionary relationships are discussed. This study suggests that most typical features of extant sipunculans have undergone only limited changes since the Early Cambrian, thus indicating a possible evolutionary stasis over the past 520 Myr.     

An ecological law and its macroecological consequences as revealed by studies of relationships between host densities and parasite prevalence

Epidemiological models predict a positive relationship between host population density and abundance of macroparasites. Here I lest these by a comparative study. I used data on communities of four groups of parasites inhabiting the gastrointestinal tract of mammals, nematodes of the orders Oxyurida. Ascarida. Enoplida and Spirurida. respectively. The data came from 44 mammalian species and represent examination of 16886 individual hosts. I studied average prevalence of all nematodes within an order in a host species, a measure of community level abundance, and considered the potential confounding effects of host body weight, fecundity, age at maturity and diet. Host population density was positively correlated with parasite prevalence within the order Oxyurida, where all species have direct life cycles. Considering the effects of other variables did not change this. This supports the assumption that parasite transmission rate generally is a positive function of host population density_: It also strengthens the hypothesis that host densities generally act as important determinants of species richness among directly transmitted parasites and suggests that negative influence of such parasites on host population growth rate increase with increasing host population density among host species. Within the other three nematode orders, where a substantial number of the species have indirect life cycles, no relationships between prevalence and host population density were seen, Again. considering the effects of other variables did not affect this conclusion. This suggests that host population density is a poor predictor of species richness of indirectly transmitted parasites and that effects of such parasites on host population dynamics do not scale with host densities among species of hosts.