Conditionally Neutral Phylogenetic Markers of Major Taxa: A New Aspect of the Evolution of Macromolecules

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda.     

Conditionally neutral phylogenetic markers of major taxa: a new aspect of the evolution of macromolecules

The current phase of molecular phylogenetics can be named the 18S rRNA gene era, which is now approaching the end. To date, almost all phyla of metazoans and many taxa of protists are represented in databases of 18S rRNA gene sequences. The elements of the phylogenetic tree of Metazoa inferred from 18S rRNA genes are characterized by unequal validity: some of them seem to be well grounded; others are not adequately supported, and probably will be revised later. The validity of phylogenetic reconstruction is influenced by two main factors: (1) erroneous grouping of long branches that occur because of abnormally high evolution rate; (2) deficit of phylogenetically informative characters. A method for overcoming these difficulties is suggested in addition to known tools: using phylogenetic markers that are stable within individual taxa and evolve by punctuated equilibrium. These markers are least influenced by the convergence caused by a high evolution rate of the entire gene. The nature of these markers of ancient taxa, paradoxical from the perspective of neutral evolution, is discussed, as well as their importance for establishing monophyly of both new large-scale taxonomic groups of invertebrates (Bilateria + Rhombozoa + Orthonectida + Myxozoa + Cnidaria + Placozoa and Echinodermata + Hemichordata) and some major taxa of Nematoda.     

Molecular phylogeny of the hyperdiverse genus Sarcophaga (Diptera: Sarcophagidae), and comparison between algorithms for identification of rogue taxa

The hyperdiverse genus Sarcophaga Meigen, with about 890 valid species arranged within 169 subgenera, accounts for almost half of the diversity of the subfamily Sarcophaginae. Current phylogenetic hypotheses for this genus are poorly supported or based on small taxon sets, or both. Here, we use molecular data from the genes COI and 28S to reconstruct the phylogeny of Sarcophaga based on the most comprehensive sampling for the group to date: 144 species from 47 subgenera, including representatives from all regional faunas for the first time. Of the total sequences of Sarcophaga used in the present study, 94.7% were newly generated. The secondary structure of the D1–D3 expansion segments of 28S is presented for the first time for the family Sarcophagidae, and is used in a multiple sequence alignment. Branch support and tree resolution increased remarkably through rogue taxa identification and exclusion. Rogue behaviour was explained mostly as a missing data problem. The RogueNaRok web service and the algorithms chkmoves, IterPCR and prunmajor implemented in the computer program TNT were equally good at identifying critical rogue species, but chkmoves and IterPCR also identified rogue clades. Pruning rogues increased the number of monophyletic subgenera in consensus trees from one to six out of 19 subgenera with more than one representative species. Bayesian inference, maximum‐likelihood and parsimony analyses recovered more monophyletic subgenera after the removal of rogue taxa, with parsimony showing the largest improvements in branch support and resolution. Although with low support, Nearctic taxa were found to be the earliest diverging lineages, followed by a subsequent diversification of Old World faunas, which is in agreement with currently available evidence of a New World origin and early diversification of Sarcophaga.     

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.     

The phylogenetic position of the comb jellies (Ctenophora) and the importance of taxonomic sampling

The transition to a vermiform body shape is one of the most important events in animal evolution, having led to the impressive radiation of Bilateria. However, the sister group of Bilateria has remained obscure. Cladistic analyses of morphology indicate that Ctenophora is the sister group of Bilateria. Previous analyses of SSU rRNA sequences have yielded conflicting results; in many studies Ctenophora forms the sister group of Cnidaria + Bilateria, but in others the ctenophores group with poriferans. Here we re‐examine the SSU sequence by analyzing a dataset with 528 metazoan + outgroup sequences, including almost 120 poriferan and diploblast sequences. We use parsimony ratchet and jackknife methods, as well as Bayesian methods, to analyze the data. The results indicate strong phylogenetic signals for a cnidarian + bilaterian group and for the comb jellies to have branched off early within a group uniting all epithelial animals [(Ct,(Cn,Bi))]. We demonstrate the importance of inclusive taxonomic coverage of ribosomal sequences for resolving this problematic part of the metazoan tree: topological stability increases dramatically with the addition of taxa, and the jackknife frequencies of the internal nodes uniting the lineages [(Cn,Bi) and ((Ct,(Cn,Bi))] also increase. We consider the reconstructed topology to represent the current best hypothesis of the interrelationships of these old lineages. Some morphological features supporting alternative hypotheses are discussed in the light of this result. © The Willi Hennig Society 2004.     

Sensitivity of Ribosomal RNA Character Sampling in the Phylogeny of Rhabditida

Near-full-length 18S and 28S rRNA gene sequences were obtained for 33 nematode species. Datasets were constructed based on secondary structure and progressive multiple alignments, and clades were compared for phylogenies inferred by Bayesian and maximum likelihood methods. Clade comparisons were also made following removal of ambiguously aligned sites as determined using the program ProAlign. Different alignments of these data produced tree topologies that differed, sometimes markedly, when analyzed by the same inference method. With one exception, the same alignment produced an identical tree topology when analyzed by different methods. Removal of ambiguously aligned sites altered the tree topology and also reduced resolution. Nematode clades were sensitive to differences in multiple alignments, and more than doubling the amount of sequence data by addition of 28S rRNA did not fully mitigate this result. Although some individual clades showed substantially higher support when 28S data were combined with 18S data, the combined analysis yielded no statistically significant increases in the number of clades receiving higher support when compared to the 18S data alone. Secondary structure alignment increased accuracy in positional homology assignment and, when used in combination with paired-site substitution models, these structural hypotheses of characters and improved models of character state change yielded high levels of phylogenetic resolution. Phylogenetic results included strong support for inclusion of Daubaylia potomaca within Cephalobidae, whereas the position of Fescia grossa within Tylenchina varied depending on the alignment, and the relationships among Rhabditidae, Diplogastridae, and Bunonematidae were not resolved.     

Consideration of RNA secondary structure significantly improves likelihood-based estimates of phylogeny: examples from the bilateria

Sequences from ribosomal RNA (rRNA) genes have made a huge contribution to our current understanding of metazoan phylogeny and indeed the phylogeny of all of life. That said, some parts of this rRNA-based phylogeny remain unresolved. One approach to increase the resolution of these trees would be to use more appropriate models of sequence evolution in phylogenetic analysis. RNAs transcribed from rRNA genes have a complex secondary structure mediated by base pairing between sometimes distant regions of the rRNA molecule. The pairing between the stem nucleotides has important consequences for their evolution which differs from that of unpaired loop nucleotides. These differences in evolution should ideally be accounted for when using rRNA sequences for phylogeny estimation. We use a novel permutation approach to demonstrate the significant superiority of models of sequence evolution that allow stem and loop regions to evolve according to separate models and, in common with previous studies, we show that 16-state models that take base pairing of stems into account are significantly better than simpler, 4-state, single-nucleotide models. One of these 16-state models has been applied to the phylogeny of the Bilateria using small subunit rRNA (SSU) sequences. Our optimal tree largely echoes previous results based on SSU in particular supporting the tripartite Bilaterian tree of deuterostomes, lophotrochozoans, and ecdysozoans. There are also a number of differences, however, perhaps most important of which is the observation of a clade consisting of the gastrotrichs plus platyheminthes that is basal to all other lophotrochozoan taxa. Use of 16-state models also appears to reduce the Bayesian support given to certain biologically improbable groups found using standard 4-state models.     

Molecular phylogeny of the Raphidiidae (Raphidioptera)*

We present a molecular phylogeny of the family Raphidiidae including representatives of 21 of the 26 genera. Sequences from the nuclear gene for the large subunit ribosomal RNA (28S rRNA) and the mitochondrial cytochrome c oxidase subunit 3 gene (cox3) were used. For the phylogenetic reconstructions we applied automated and manual approaches for sequence alignment and different evolutionary models and tree building algorithms. The trees based on the two alignment approaches were rather similar in their overall topology. A combination of both marker sequences increased the resolution of the trees. The six clades within the raphidiid family that emerged represent either single genera or groups of genera, namely: (i) the Nearctic genus Agulla Navás, (ii) the Nearctic/Central American genus Alena Navás, (iii) the Central Asiatic and Eastern Palaearctic genus Mongoloraphidia H. Aspöck & U. Aspöck, (iv) the Palaearctic Puncha clade, (v) the western Mediterranean Ohmella clade, and (vi) the Palaearctic Phaeostigma clade. The New World taxa Agulla and Alena are placed as successive out‐groups to a monophyletic Palaearctic clade. The Mongoloraphidia clade is distributed in the eastern Palearctic while the remaining three clades are exclusively (Ohmella clade) or mainly distributed in the western Palaearctic. The early radiation of extant Raphidiidae is interpreted based on the phylogenetic tree obtained in the present study, and the geological and palaeobiological processes around the K–T boundary.     

基于细胞核rDNA ITS片段的水青冈属的分子系统发育

对山毛榉科水青冈属6种、1亚种、1栽培变种的ITS区片段进行了测序和分析,并对其中2个具有ITS序列多态性的分类群进行了ITS区克隆。水青冈属ITS系统发育树聚成两支,位于基部的是分布于北美的大叶水青冈,另一分支则包括了欧洲和东亚的类群。在欧洲和东亚分支中,又包括两支,其中日本北部的波叶水青冈位于基部,台湾水青冈和欧亚大陆的水青冈形成另外一支。ITS区分析与现行的水青冈属基于形态学性状的属下分类系统有一定差异,而与本属现存物种的地理分布格局较为一致。各类群间TIS区序列差异较小,显示属内现存物种的分化时间不是太长。     

The distribution of ascus types and photobiontal selection in Lecanoromycetes (Ascomycota) against the background of a revised SSU nrDNA phylogeny

Within the last decade, molecular methods have revealed the relationships in many groups of lichenized Ascomycetes. However, the published phylogenies were often contradictory with respect to higher taxonomic levels. To achieve a more convincing overall picture of phylogenetic relationships of and within the Lecanoromycetes, we set up an alignment of all publicly available SSU nrDNA sequences of the Pezizomycotina, discarded those of insufficient quality, and added 21 newly obtained sequences representing groups that were not or under-represented so far. The alignment of 635 taxa and a subset of 166 Lecanoromycetes were analysed with different phylogenetic algorithms. The best phylogenetic trees, with respect to length, resolution, and support, were obtained with the Parsimony Ratchet method and DNAPARS (Phylip). According to these results, the Lecanoromycetes were subdivided into eight monophyletic clades representing the following orders: Acarosporales ad. int., Agyriales, Baeomycetales, Gyalectales, Icmadophilales ad. int., Lecanorales, Ostropales, and Pertusariales. These clades are distributed in two monophyletic groups corresponding to the two different types of ascomatal ontogeny. The taxa of the Lecanorales-clade differ from the other orders by the absence of a primary cyanobiont and of phycobionts of the genera Coccomyxa and Trentepohlia, in combination with the presence of an amyloid reaction of the ascus tips (lacking amyloid flanks). The Lecanorales-clade is subdivided into nine groups which are treated here at the level of suborders: Lecideineae, Teloschistineae, Physciineae, Bacidiineae ad int., Sphaerophorineae ad int., Rhizocarpineae ad int., Psorineae ad int., Cladoniineae, and Lecanorineae. The amyloid reaction pattern of the ascus apex correlates with this grouping at suborder rank. Peltigerales were excluded from the Lecanorales but included in the Lecanoromycetes based on morpho-anatomical features. The Lichinales were excluded from the Lecanoromycetes, while the position of the Umbilicariaceae remains unclear.     

Incorporation of gap characters and lineage-specific regions into phylogenetic analyses of gene families from divergent clades: an example from the kinesin superfamily across eukaryotes

The kinesin superfamily across eukaryotes was used to examine how incorporation of gap characters scored from conserved regions shared by all members of a gene family and incorporation of amino acid and gap characters scored from lineage‐specific regions affect gene‐tree inference of the gene family as a whole. We addressed these two questions in the context of two different densities of sequence sampling, four alignment programs, and two methods of tree construction. Taken together, our findings suggest the following. First, gap characters should be incorporated into gene‐tree inference, even for divergent sequences. Second, gene regions that are not conserved among all or most sequences sampled should not be automatically discarded without evaluation of potential phylogenetic signal that may be contained in gap and/or sequence characters. Third, among the four alignment programs evaluated using their default alignment parameters, Clustal may be expected to output alignments that result in the greatest gene‐tree resolution and support. Yet, this high resolution and support should be regarded as optimistic, rather than conservative, estimates. Fourth, this same conclusion regarding resolution and support holds for Bayesian gene‐tree analyses relative to parsimony‐jackknife gene‐tree analyses. We suggest that a more conservative approach, such as aligning the sequences using DIALIGN‐T or MAFFT, analyzing the appropriate characters using parsimony, and assessing branch support using the jackknife, is more appropriate for inferring gene trees of divergent gene families. © The Willi Hennig Society 2007.     

Efficiently resolving the basal clades of a phylogenetic tree using Bayesian and parsimony approaches: a case study using mitogenomic data from 100 higher teleost fishes

Many phylogenetic analyses that include numerous terminals but few genes show high resolution and branch support for relatively recently diverged clades, but lack of resolution and/or support for "basal" clades of the tree. The various benefits of increased taxon and character sampling have been widely discussed in the literature, albeit primarily based on simulations rather than empirical data. In this study, we used a well-sampled gene-tree analysis (based on 100 mitochondrial genomes of higher teleost fishes) to test empirically the efficiency of different methods of data sampling and phylogenetic inference to "correctly" resolve the basal clades of a tree (based on congruence with the reference tree constructed using all 100 taxa and 7990 characters). By itself, increased character sampling was an inefficient method by which to decrease the likelihood of "incorrect" resolution (i.e., incongruence with the reference tree) for parsimony analyses. Although increased taxon sampling was a powerful approach to alleviate "incorrect" resolution for parsimony analyses, it had the general effect of increasing the number of, and support for, "incorrectly" resolved clades in the Bayesian analyses. For both the parsimony and Bayesian analyses, increased taxon sampling, by itself, was insufficient to help resolve the basal clades, making this sampling strategy ineffective for that purpose. For this empirical study, the most efficient of the six approaches considered to resolve the basal clades when adding nucleotides to a dataset that consists of a single gene sampled for a small, but representative, number of taxa, is to increase character sampling and analyze the characters using the Bayesian method.     

AliGROOVE – visualization of heterogeneous sequence divergence within multiple sequence alignments and detection of inflated branch support

Background

Masking of multiple sequence alignment blocks has become a powerful method to enhance the tree-likeness of the underlying data. However, existing masking approaches are insensitive to heterogeneous sequence divergence which can mislead tree reconstructions. We present AliGROOVE, a new method based on a sliding window and a Monte Carlo resampling approach, that visualizes heterogeneous sequence divergence or alignment ambiguity related to single taxa or subsets of taxa within a multiple sequence alignment and tags suspicious branches on a given tree.

Results

We used simulated multiple sequence alignments to show that the extent of alignment ambiguity in pairwise sequence comparison is correlated with the frequency of misplaced taxa in tree reconstructions. The approach implemented in AliGROOVE allows to detect nodes within a tree that are supported despite the absence of phylogenetic signal in the underlying multiple sequence alignment. We show that AliGROOVE equally well detects heterogeneous sequence divergence in a case study based on an empirical data set of mitochondrial DNA sequences of chelicerates.

Conclusions

The AliGROOVE approach has the potential to identify single taxa or subsets of taxa which show predominantly randomized sequence similarity in comparison with other taxa in a multiple sequence alignment. It further allows to evaluate the reliability of node support in a novel way.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-294) contains supplementary material, which is available to authorized users.     

新疆红井子盐碱土壤非培养放线菌多样性

【目的】研究新疆红井子盐碱土壤中的放线菌物种多样性。【方法】应用基于16S rRNA基因序列系统发育分析的免培养方法进行放线菌物种多样性分析。利用放线菌特异性引物,以土壤样品总DNA为模板,扩增16S rRNA基因,构建16S rRNA基因克隆文库,并对文库中的插入序列进行RFLP分析。【结果】随机挑选的246个阳性克隆通过酶切筛选出61个不同图谱的重组克隆并测序。分析结果显示这61个克隆序列分属于42个OTUs,分布于放线菌纲(Actinobacteria)的放线菌亚纲(Actinobacteridae)、酸微菌亚纲(Acidimicrobidae)和红色杆菌亚纲(Rubrobacteridae);该环境中有71.4%的序列与已有效发表菌株的序列相似性小于97%,代表着放线菌新类群,其中部分序列形成了几个独立的进化分支,可能代表更高级的新分类单元。【结论】红井子土壤中的放线菌组成具有丰富的多样性,并有新放线菌分类单位的潜在资源,值得进一步进行开发研究。     

Evolutionary Relationships Among Old World Fruitbats (Megachiroptera: Pteropodidae) Based on 12S rRNA, tRNA Valine, and 16S rRNA Gene Sequences

Old World fruitbats were divided into the cynopterine, epomophorine, rousettine, eonycterine, and notopterine sections by Knud Andersen (1912). Among these, the eonycterine and notopterine sections together comprise the subfamily Macroglossinae, which includes forms with specializations for nectarivory. Single-copy DNA hybridization data argue against the monophyly of four of Andersen's sections and further suggest paraphyly or polyphyly of the Macroglossinae. DNA hybridization data provide support for an endemic African clade that includes Megaloglossus (an eonycterine), Epomophorus (an epomophorine), and Lissonycteris (a rousettine). Analyses of mitochondrial 12S rRNA-tRNA valine gene sequences corroborate the African clade but provide less resolution than hybridization data for most branches on the pteropodid tree. Here, we report 11 new 16S rRNA sequences and analyze a mitochondrial data set that includes 12S rRNA, tRNA valine, and 16S rRNA for 18 pteropodid genera. Parsimony, minimum evolution, and maximum likelihood were all employed in phylogenetic analyses. The addition of 16S rRNA sequences to the mitochondrial data set resulted in increased support for several clades, including Macroglossus + Syconycteris, Cynopterus + Thoopterus, Rousettus + the endemic African clade, and Eonycteris + Rousettus + the endemic African clade. Statistical tests suggest that another endemic African genus, Eidolon, is dissociated from the African clade and represents an independent invasion into Africa. We constructed a molecular phylogenetic framework that incorporated clades that were strongly supported by both single-copy DNA hybridization and 12S rRNA-tRNA valine-16S rRNA sequences. Using this framework as a backbone phylogenetic constraint, we then analyzed a morphological data matrix for 34 pteropodid genera with parsimony. Results of this analysis suggest that other epomophorines and Myonycteris (a cynopterine) are also part of the endemic African clade.     

Aligned 18S and insect phylogeny

The nuclear small subunit rRNA (18S) has played a dominant role in the estimation of relationships among insect orders from molecular data. In previous studies, 18S sequences have been aligned by unadjusted automated approaches (computer alignments that are not manually readjusted), most recently with direct optimization (simultaneous alignment and tree building using a program called "POY"). Parsimony has been the principal optimality criterion. Given the problems associated with the alignment of rRNA, and the recent availability of the doublet model for the analysis of covarying sites using Bayesian MCMC analysis, a different approach is called for in the analysis of these data. In this paper, nucleotide sequence data from the 18S small subunit rRNA gene of insects are aligned manually with reference to secondary structure, and analyzed under Bayesian phylogenetic methods with both GTR+I+G and doublet models in MrBayes. A credible phylogeny of Insecta is recovered that is independent of the morphological data and (unlike many other analyses of 18S in insects) not contradictory to traditional ideas of insect ordinal relationships based on morphology. Hexapoda, including Collembola, are monophyletic. Paraneoptera are the sister taxon to a monophyletic Holometabola but weakly supported. Ephemeroptera are supported as the sister taxon of Neoptera, and this result is interpreted with respect to the evolution of direct sperm transfer and the evolution of flight. Many other relationships are well-supported but several taxa remain problematic, e.g., there is virtually no support for relationships among orthopteroid orders. A website is made available that provides aligned 18S data in formats that include structural symbols and Nexus formats.     

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.     

First combined cladistic analysis of marsupial mammal interrelationships

We combine osteological, dental, and soft tissue data with sequences from three nuclear and five mitochondrial genes, sampling all major living clades of marsupials plus several extinct taxa, to perform a simultaneous analysis of marsupial interrelationships. These data were analyzed using direct optimization and sensitivity analysis on a parallel supercomputing cluster, and compared with trees produced with conventional parsimony and likelihood algorithms using a static alignment. A major issue in marsupial phylogeny is the relationships among australidelphians. Optimal analyses using direct optimization and those based on the static alignment support the basal positions of peramelians (bandicoots) and Dromiciops ('monito del monte') within Australidelphia, and in all but one case these analyses support a monophyletic Eometatheria, a group consisting of all australidelphians excluding peramelians. Dromiciops is basal within Eometatheria in analyses that maximize congruence across partitions, including the equally weighted parameter set. The topologies resulting from direct optimization under all parameter sets show some differences, but all show a high degree of resolution. Direct optimization supports high-level clades supported by analyses of partitioned molecular (e.g., Notoryctes as sister group of Dasyuromorphia) and morphological (e.g., Diprotodontia) data.     

Electron acceptor-dependent identification of key anaerobic toluene degraders at a tar-oil-contaminated aquifer by Pyro-SIP

The diversity of archaeal communities growing in four hot springs (65-90 °C, pH 6.5) was assessed with 16S rRNA gene primers specific for the domain Archaea. Overall, mainly uncultured members of the Desulfurococcales, the Thermoproteales and the Korarchaeota, were identified. Based on this diversity, a set of chaperonin heat-shock protein (Hsp60) gene sequences from different archaeal species were aligned to design two degenerate primer sets for the amplification of the chaperonin gene: Ths and Kor (which can also detect the korarchaeotal chaperonin gene from one of the samples). A phylogenetic tree was constructed using the chaperonin sequences retrieved and other sequences from cultured representatives. The Alpha and Beta paralogs of the chaperonin gene were observed within the main clades and orthologs among them. Cultivated representatives from these clades were assigned to either paralog in the chaperonin tree. Uncultured representatives observed in the 16S rRNA gene analysis were found to be related to the Desulfurococcales. The topologies of the 16S rRNA gene and chaperonin phylogenetic trees were compared, and similar phylogenetic relationships were observed. Our results suggest that the chaperonin Hsp60 gene may be used as a phylogenetic marker for the clades found in this extreme environment.