Distribution and phylogenetic relationships of freshwater Thaumatomonads with a description of the new species Thaumatomonas coloniensis n. sp

The order Thaumatomonadida includes biflagellated heterotrophic flagellates that form filopodia and typically possess siliceous surface scales. We found thaumatomonads to contribute on average about 5%-10% to flagellate abundance in different benthic habitats. A new species of thaumatomonads, Thaumatomonas coloniensis n. sp., is described on the basis of morphological and molecular biological features. This new species was isolated both from groundwater at Appeldorn near Rees (Germany) and from the Rhine River at Cologne (Germany). We have sequenced the small subunit rRNA (ssu rRNA) gene and a fragment of the large subunit rRNA (lsu rRNA) gene (D3-D5 region) from the isolates of the new species, including the first sequence of a representative of the thaumatomonad genus Gyromitus. In agreement with previous studies, the differences in ribosomal genes of different thaumatomonad species are very small. For understanding the phylogenetic relationships of Thaumatomonadida and to explore their sister group relationships, we have created three sequence data sets (ssu rRNA, partial lsu rRNA, concatenated alignment of both) with the same composition of isolates (from Thaumatomonadida, Euglyphida, Cercomonadidae, and Heteromitidae). According to a Kishino-Hasegawa test, Thaumatomonadida evolved within the Cercozoa as a sister taxon to the Heteromitidae. A possibly close relationship to the Euglyphida, recently grouped together with the Thaumatomonadida in the class Imbricatea/Silicofilosea based on the rRNA data sets was not supported by our analyses.     

Evolution of the Diatoms (Bacillariophyta): IV. A Reconstruction of Their Age from Small Subunit rRNA Coding Regions and the Fossil Record

Small subunit ribosomal RNA (ssu rRNA) coding regions from 30 diatoms, 3 oomycetes, and 6 pelagophytes were used to construct linearized trees, maximum-likelihood trees, and neighbor-joining trees inferred from both unweighted and weighted distances. Stochastic accumulation of sequence substitutions among the diatoms was assessed with relative rate tests. Pennate diatoms evolved relatively slowly but within the limits set by a stochastic model; centric diatoms exceeded those limits. A rate distribution test was devised to identify those taxa showing an aberrant distribution of base substitutions within the ssu rRNA coding region. First appearance dates of diatom taxa from the fossil record were regressed against their corresponding branch lengths to infer the average and earliest possible age for the origin of the diatoms, the pennate diatoms, and the centric diatom order Thalassiosirales. Our most lenient age estimate (based on the median-evolving diatom taxon in the maximum-likelihood tree or on the average branch length in a linearized tree) suggests that their average age is approximately 164–166 Ma, which is close to their earliest fossil record. Both calculations suggest that it is unlikely that diatoms existed prior to 238–266 Ma. Rate variation among the diatoms' ssu rRNA coding regions and uncertainties associated with the origin of extant taxa in the fossil record contribute significantly to the variation in age estimates obtained. Different evolutionary models and the exclusion of fast or slow evolving taxa did not significantly affect age estimates; however, the inclusion of aberrantly fast evolving taxa did. Our molecular clock calibrations indicate that the rRNA coding regions in the diatoms are evolving at approximately 1% per 18 to 26 Ma, which is the fastest substitution rate reported in any pro- or eukaryotic group of organisms to date.     

Mikrocytos roughleyi taxonomic affiliation leads to the genus Bonamia (Haplosporidia)

Microcell-type parasites of oysters are associated with a complex of diseases in different oyster species around the world. The etiological agents are protists of very small size that are very difficult to characterize taxonomically. Associated lesions may vary according to the host species, and their occurrence may be related to variations in tissue structure. Lesion morphology cannot be used to distinguish the different agents involved. Ultrastructural observations on Mikrocytos roughleyi revealed similarities with Bonamia spp., particularly in regard to the presence of electron-dense haplosporosomes and mitochondria, whose absence from M. mackini also indicate that M. roughleyi and M. mackini are not congeneric. A partial small subunit (ssu) rRNA gene sequence of M. roughleyi was determined. This partial sequence, 951 nucleotides in length, has 95.2 and 98.4% sequence similarities with B. ostreae and B. exitiosus ssu rDNA sequences, respectively. Polymorphisms among the ssu rDNA sequences of B. ostreae, B. exitiosus and M. roughleyi allowed identification of restriction enzyme digestion patterns diagnostic for each species. Phylogenetic analysis based on the ssu rDNA data suggested that M. roughleyi belongs in the phylum Haplosporidia and that it is closely related to Bonamia spp. On the basis of ultrastructural and molecular considerations, M. roughleyi should be considered a putative member of the genus Bonamia.     

A molecular phylogenetic investigation of zoothamnium (ciliophora, peritrichia, sessilida)

The gene coding for 18S small subunit ribosomal RNA (ssu rRNA) was sequenced in seven free-living, marine species of the sessiline peritrich genus Zoothamnium. These were Zoothamnium niveum, Zoothamnium alternans, Zoothamnium pelagicum, and four unidentified species. The ssu rRNA gene also was sequenced in Vorticella convallaria, Vorticella microstoma, and in an unidentified, freshwater species of Vorticella. Phylogenetic trees were constructed using these new sequences to test a previously published phylogenetic association between Zoothamnium arbuscula, currently in the family Zoothamniidae, and peritrichs in the family Vorticellidae. Trees constructed by means of neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods all had similar topologies. The seven new sequences of Zoothamnium species grouped into three well-supported clades, each of which contained a diversity of morphological types. The three clades formed a poorly supported, larger clade that was deeply divergent from Z. arbuscula, which remained more closely associated with vorticellid peritrichs. It is apparent that Zoothamnium is a richly diverse genus and that a much more intensive investigation, involving both morphological and molecular data and a wider selection of species, will be necessary to resolve its phylogeny. A greater amount of molecular diversity than is predicted by morphological data exists within all major clades of sessiline peritrichs that have been included in molecular phylogenies, indicating that characteristics of stalk and peristomial structure traditionally used to differentiate taxa at the generic level and above may not be uniformly reliable.     

Determinants of rate variation in mammalian DNA sequence evolution

Attempts to analyze variation in the rates of molecular evolution among mammalian lineages have been hampered by paucity of data and by nonindependent comparisons. Using phylogenetically independent comparisons, we test three explanations for rate variation which predict correlations between rate variation and generation time, metabolic rate, and body size. Mitochondrial and nuclear genes, protein coding, rRNA, and nontranslated sequences from 61 mammal species representing 14 orders are used to compare the relative rates of sequence evolution. Correlation analyses performed on differences in genetic distance since common origin of each pair against differences in body mass, generation time, and metabolic rate reveal that substitution rate at fourfold degenerate sites in two out of three protein sequences is negatively correlated with generation time. In addition, there is a relationship between the rate of molecular evolution and body size for two nuclear-encoded sequences. No evidence is found for an effect of metabolic rate on rate of sequence evolution. Possible causes of variation in substitution rate between species are discussed.     

Discrimination of Porphyra species based on small subunit ribosomal RNA gene sequence

The complete nucleotide sequences of ssu rRNA genes were determined for nine species of Porphyra. Ssu rRNA gene structure was classified into four types by the presence and absence of intron(s). Gene structure even differed within the same species. Exon nucleotide sequences were identical within the same species, but differed among species. Seventeen species of Porphyra were discriminated by comparing the sequences of these diversified regions, using the results of this study and previous studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Diversity and host specificity of the Verminephrobacter-earthworm symbiosis

Symbiotic bacteria of the genus Verminephrobacter (Betaproteobacteria) were detected in the nephridia of 19 out of 23 investigated earthworm species (Oligochaeta: Lumbricidae) by 16S rRNA gene sequence analysis and fluorescence in situ hybridization (FISH). While all four Lumbricus species and three out of five Aporrectodea species were densely colonized by a mono-species culture of Verminephrobacter, other earthworm species contained mixed bacterial populations with varying proportions of Verminephrobacter; four species did not contain Verminephrobacter at all. The Verminephrobacter symbionts could be grouped into earthworm species-specific sequence clusters based on their 16S rRNA and RNA polymerase subunit B (rpoB) genes. Closely related host species harboured more closely related symbionts than did distantly related hosts. Co-diversification of the symbiotic partners could not be demonstrated unambiguously due to the poor resolution of the host phylogeny [based on histone H3 and cytochrome c oxidase subunit I (COI) gene sequence analyses]. However, there was a pattern of symbiont diversification within four groups of closely related hosts. The mean rate of symbiont 16S rRNA gene evolution was determined using a relaxed clock model, and the rate was calibrated with paleogeographical estimates of the time of origin of Lumbricid earthworms. The calibrated rates of symbiont 16S rRNA gene evolution are 0.012-0.026 substitutions per site per 50 million years and thus similar to rates reported from other symbiotic bacteria.     

A molecular phylogenetic investigation of Opisthonecta and related genera (Ciliophora, Peritrichia, Sessilida)

The gene encoding 18S small subunit ribosomal RNA (ssu rRNA) was sequenced in the sessiline peritrichs Opisthonecta minima and Opisthonecta matiensis, whose free-swimming, paedomorphic trophonts resemble telotrochs. Using these new sequences, phylogenetic trees were constructed with four different methods to test a previously published association between Opisthonecta henneguyi and members of the families Vorticellidae and Astylozoidae. All trees had similar topologies, with O. minima, O. henneguyi, Vorticella microstoma, and Astylozoon enriquesi forming a well-supported, certainly monophyletic clade. On the basis of genetic evidence, genera of the families Opisthonectidae and Astylozoidae are assigned to the family Vorticellidae, which already includes some species with free-swimming morphotypes. The ssu rRNA sequence of O. matiensis places it in the family Epistylididae; its taxonomic revision will be left to another group of authors. A close association of Ophrydium versatile with members of the family Vorticellidae was confirmed, casting doubt on the validity of the family Ophrydiidae. Epistylis galea, Campanella umbellaria, and Opercularia microdiscum are confirmed as comprising an extremely distinct, monophyletic, but morphologically heterogeneous clade that is basal to other clades of sessiline peritrichs.     

Classical plant taxonomic ambiguities extend to the molecular level

Summary The molecular evolution of cytochrome c from angiosperms is compared to that from vertebrates. On the basis of a cladistic analysis from 26 plant species, compared to that from 27 vertebrate species, we find that although the vertebrate sequences yield reasonably well-defined minimal trees that are congruent with the biological tree, the plant sequences yield multiple minimal trees that are not only highly incongruent with each other, but none of which is congruent with any reasonably biological tree. That is, the plant sequence set is much more homoplastic than that of the animal. However, as judged by the relative rate test, the extent of divergence, and degree of functional constraint, cytochrome c evolution in plants does not appear to differ from that of vertebrates.     

Biogeography in a continental island: population structure of the relict endemic centipede Craterostigmus tasmanianus (Chilopoda, Craterostigmomorpha) in Tasmania using 16S rRNA and COI

We used 16S ribosomal RNA (rRNA) and cytochrome c oxidase subunit I (COI) sequence data to investigate the population structure in the centipede Craterostigmus tasmanianus Pocock, 1902 (Chilopoda: Craterostigmomorpha: Craterostigmidae) and to look for possible barriers to gene flow on the island of Tasmania, where C. tasmanianus is a widespread endemic. We first confirmed a molecular diagnostic character in 28S rRNA separating Tasmanian Craterostigmus from its sister species Craterostigmus crabilli (Edgecombe and Giribet 2008) in New Zealand and found no shared polymorphism in this marker for the 2 species. In Tasmania, analysis of molecular variance analysis showed little variation at the 16S rRNA and COI loci within populations (6% and 13%, respectively), but substantial variation (56% and 48%, respectively) among populations divided geographically into groups. We found no clear evidence of isolation by distance using a Mantel test. Bayesian clustering and gene network analysis both group the C. tasmanianus populations in patterns which are broadly concordant with previously known biogeographical divisions within Tasmania, but we did not find that genetic distance varied in a simple way across cluster boundaries. The coarse-scale geographical sampling on which this study was based should be followed in the future by sampling at a finer spatial scale and to investigate genetic structure within clusters and across cluster boundaries.     

Detection of Cryptosporidium sp. in two new seal species, Phoca vitulina and Cystophora cristata, and a novel Cryptosporidium genotype in a third seal species, Pagophilus groenlandicus, from the Gulf of Maine

Data on the geographic distribution and host specificity of Cryptosporidium spp. are critical for developing an understanding of likely transmission patterns in nature. During a molecular-based survey of fecal samples from 293 terrestrial and aquatic animals in Maine, USA, we detected Cryptosporidium sp. in 11 harbor seals (Phoca vitulina), 1 hooded seal (Cystophora cristata), and 1 harp seal (Pagophilus groenlandicus). None of the terrestrial or freshwater mammal fecal samples or bird samples tested positive for Cryptosporidium sp. However, the sequencing results of the small subunit (ssu) rRNA gene indicate that the seals were infected with an undescribed species of Cryptosporidium , previously isolated only from ringed seals (Phoca hispida) in northern Quebec, Canada. In addition, the Cryptosporidium sp. detected in the harp seal is significantly different from the previously observed Cryptosporidium sp. in other seals. We confirmed the genetic distinctiveness of this Cryptosporidium genotype and the identity of the other Cryptosporidium sp. seal ssu rRNA sequences by using data from the 70-kDa heat shock protein gene. Based on phylogenetic reconstructions of both genes, it seems that either Cryptosporidium canis or C. felis are sister species to the seal associated Cryptosporidium spp. Our findings extend the range of " Cryptosporidium sp. seal" well south of the 55th parallel, add other species to the list of seals affected by Cryptosporidium sp., and highlight the presence of unrecognized population and potentially species level variation in Cryptosporidium.     

Phylogenetic relationships and phylogeography of the Killifish species of the subgenus Chromaphyosemion (Radda, 1971) in West Africa, inferred from mitochondrial DNA sequences

We have analyzed the phylogenetic relationships of 160 specimens from 88 samples representing all defined species of the African Aplocheiloid subgenus Chromaphyosemion in order to examine the monophyly of this group, the species interrelationships, and to reveal trends in chromosomal evolution and formulate hypotheses about their evolutionary history. The data set comprised 1153 total nucleotides from the mitochondrial 12S rRNA, cytochrome oxidase I, and D-loop. The molecular-based topologies were analyzed by maximum parsimony, maximum likelihood, distance method and Bayesian inference support the monophyly of the subgenus Chromaphyosemion. All populations with ambiguous taxonomic status were assigned to an already described species except A. sp. Rio Muni which corresponds to a still undescribed species. Aphyosemion alpha and A. lugens were in basal position in the different trees that indicate a possible origin of the subgenus Chromaphyosemion in the South Cameroon-North Gabon region. Furthermore, the South Cameroon region (between 2 degrees and 3 degrees of North latitude) that accommodates half of the Chromaphyosemion species is considered to have been a refuge zone during the late quaternary dry events that Africa experienced. Phylogenetic relationships among the subgenus also revealed that chromosomal evolution is complex and should be studied at the intraspecific level.     

海洋喇叭虫rRNA基因18S-ITS1序列及其系统发育分析

海洋喇叭虫Maristentor dinoferus 1996年在关岛(Guam)的珊瑚暗礁上被发现,至今尚未阐明其确切的系统发育地位.克隆到的海洋喇叭虫的18S-ITS1-5.8S rDNA序列包括222 bp的18S序列,77 bp的ITS1序列和22 bp的5.8S序列.比较分析了纤毛虫主要类群的ITS1序列后得出:短的ITS1序列可能是异毛类纤毛虫的特征.根据18S序列,利用邻接法构建,最大简约法和最大似然法构建系统发育树.其拓扑结构显示海洋喇叭虫属于异毛纲纤毛虫,但并不隶属喇叭虫科,应予以新的分类地位.     

Utility of nuclear DNA intron markers at lower taxonomic levels: phylogenetic resolution among nine Tragelaphus spp

Phylogenetic relationships among the nine spiral-horn antelope species of the African bovid tribe Tragelaphini are controversial. In particular, mitochondrial DNA sequencing studies are not congruent with previous morphological investigations. To test the utility of nuclear DNA intron markers at lower taxonomic levels and to provide additional data pertinent to tragelaphid evolution, we sequenced four nuclear DNA segments (MGF, PRKCI, SPTBN, and THY) and combined these data with mitochondrial DNA sequences from three genes (cytochrome b, 12S rRNA, and 16S rRNA). Our molecular supermatrix comprised 4682 characters which were analyzed independently and in combination. Parsimony and model based phylogenetic analyses of the combined nuclear DNA data are congruent with those derived from the analysis of mitochondrial gene sequences. The corroboration between nuclear and mtDNA gene trees reject the possibility that genetic processes such as lineage sorting, gene duplication/deletion and hybrid speciation account for the conflict evident in the previously published phylogenies. It suggests rather that the morphological characters used to delimit the Tragelaphid species are subject to convergent evolution. Divergence times among species, calculated using a relaxed Bayesian molecular clock, are consistent with hypotheses proposing that climatic oscillations and their impact on habitats were the major forces driving speciation in the tribe Tragelaphini.     

Phylogenetic relationships of African killifishes in the genera Aphyosemion and Fundulopanchax inferred from mitochondrial DNA sequences

We have analyzed the phylogenetic relationships of 52 species representing all defined species groups (J. J. Scheel, 1990, Atlas of Killifishes of the Old World, 448 pp.) of the African aplocheiloid fish genera Aphyosemion and Fundulopanchax in order to examine their interrelationships and to reveal trends of karyotypic evolution. The data set comprised 785 total nucleotides from the mitochondrial 12S rRNA and cytochrome b genes. The molecular-based topologies analyzed by both maximum parsimony and neighbor-joining support the monophyly of most previously defined species groups within these two killifish genera. The genus Aphyosemion is monophyletic except for the nested position of Fundulopanchax kunzi (batesi group; subgenus Raddaella) within this clade, suggesting that this taxon was improperly assigned to Fundulopanchax. The remaining Fundulopanchax species sampled were supported as being monophyletic in most analyses. Relationships among the species groups in both genera were not as strongly supported, suggesting that further data will be required to resolve these relationships. Additional sampling from the 16S rRNA gene allowed further resolution of relationships within Fundulopanchax, more specifically identifying the nonannual scheeli group as the basal lineage of this otherwise annual genus. Chromosomal evolution within Aphyosemion has been episodic, with the evolution of a reduced n = 9-10 metacentric complement having occurred in multiple, independent lineages. Polarity of chromosomal reductions within the elegans species group appears to support previous hypotheses concerning mechanisms of karyotypic change within the genus Aphyosemion.     

Implications of Fluctuations in Substitution Rates: Impact on the Uncertainty of Branch Lengths and on Relative-Rate Tests

Many tests of the lineage dependence of substitution rates, computations of the error of evolutionary distances, and simulations of molecular evolution assume that the rate of evolution is constant in time within each lineage descended from a common ancestor. However, estimates of the index of dispersion of numbers of mammalian substitutions suggest that the rate has time-dependent variations consistent with a fractal-Gaussian-rate Poisson process, which assumes common descent without assuming rate constancy. While this model does not affect certain relative-rate tests, it substantially increases the uncertainty of branch lengths. Thus, fluctuations in the rate of substitution cannot be neglected in calculations that rely on evolutionary distances, such as the confidence intervals of divergence times and certain phylogenetic reconstructions. The fractal-Gaussian-rate Poisson process is compared and contrasted with previous models of molecular evolution, including other Poisson processes, the fractal renewal process, a Lévy-stable process, a fractional-difference process, and a log-Brownian process. The fractal models are more compatible with mammalian data than the nonfractal models considered, and they may also be better supported by Darwinian theory. Although the fractal-Gaussian-rate Poisson process has not been proven to have better agreement with data or theory than the other fractal models, its Gaussian nature simplifies the exploration of its impact on evolutionary distance errors and relative-rate tests. Received: 29 September 1999 / Accepted: 20 January 2000     

Phylogeny of the Neotropical killifish family Rivulidae (Cyprinodontiformes, Aplocheiloidei) inferred from mitochondrial DNA sequences

Phylogenetic relationships of 70 taxa representing 68 species of the Neotropical killifish family Rivulidae were derived from analysis of 1516 nucleotides sampled from four different segments of the mitochondrial genome: 12S rRNA, 16S rRNA, cytochrome oxidase I, and cytochrome b. The basal bifurcation of Cynolebiatinae and Rivulinae (Costa, 1990a,b) is supported; however, Terranatos, Maratecoara, and Plesiolebias are rivulins, not cynolebiatins. These three genera, along with the other recognized annual rivulin genera, form a monophyletic clade. Austrofundulus, Rachovia, Renova, Terranatos, and 3 species of the genus Pterolebias, all from northeastern South America, form a monophyletic clade excluding other species of Pterolebias. Pterolebias as presently understood is clearly polyphyletic. Trigonectes and Moema are supported as sister groups but do not form a monophyletic group with the genera Neofundulus and Renova as previously proposed. The suite of adaptations necessary for an annual life history has clearly been lost several times in the course of rivulid evolution. Also revealed is a considerable increase in substitution rate in most annual lineages relative to the nonannual Rivulus species. The widespread and speciose genus Rivulus is paraphyletic, representing both basal and terminal clades within the Rivulidae. Previous hypotheses regarding the vicariant origin of Greater Antillean Rivulus species are supported. Most rivulid clades show considerable endemism; thus, detailed analysis of rivulid phylogeny and distribution will contribute robust hypotheses to the clarification of Neotropical biogeography.     

A phylogeny of freshwater eels inferred from mitochondrial genes

The genus Anguilla Shaw of Family Anguillidae consists entirely of freshwater eels, including 15 species and 2 subspecies. Conventionally, variegated markings and the length of the dorsal fin are the major morphological features used for reconstruction of phylogenetic relationships. The evolutionary history of these species remains unclear, especially for the Atlantic eels, whose habitats are far from the Metropolis in the Indo-Pacific region. This study reexamined the phylogenetic relationships of 12 Anguilla species by sequencing of the cytochrome b and 12S rRNA genes. In our analysis, species bearing similar coloration patterns or dorsal fin morphology are not necessarily clustered in the same clade, indicating that these morphological features might be unstable or might have occurred independently in different lineages during evolution. Combining our molecular data and geographical evidence, we speculate that (1) Anguilla first radiated about 20 million years ago, (2) the ancestors of Atlantic eels did not migrate by drifting through the Tethys Seaway at the leptocephali stage but instead trekked across the Central American Isthmus to the Sargasso Sea for spawning at the adult stage, and (3) multiple radiation events had occurred at the Metropolis during Anguilla evolution.     

Phylogenetic and systematic study of Korean <Emphasis Type="Italic">Orius</Emphasis> species (Heteroptera: Anthocoridae) on the basis of molecular and morphological data

A phylogenetic and systematic study of Orius species (Heteroptera: Anthocoridae) from Korea has been conducted using both morphological and molecular characters. Thirty morphological character states were coded for 10 strains of 9 species. Five molecular markers, partial cytochrome c oxidase I (COI), cytochrome b (CytB), 16S rRNA (16S), 18S rRNA (18S), and 28S rRNA (28S), from mitochondrial and nuclear genes, were tested. Phylogenetic analyses based on molecular data were conducted by minimum evolution, maximum parsimony, maximum likelihood, and Bayesian phylogenetic (BP) analyses. Analysis of morphological data was performed using the parsimony programs NONA, and the combined dataset of morphological and molecular data was analyzed using BP analyses. The results of this study indicate that use of COI and CytB enabled relatively effective identification of species, whereas the sequences of 16S, 18S and 28S did not enable identification of closely related species such as Orius minutus and O. strigicollis. We discuss the usefulness of the five molecular markers for determining phylogenetic relationships and identifying the species.