Phylogeny Inference Package Research Articles

A new proposal for Min subgrouping based on a maximum-parsimony algorithm for generating phylogenetic trees

The Min dialect group is easily characterized, in contrast to other dialect groups in China. Min varieties do, however, exhibit a considerable degree of diversity. The internal relationship among these dialects has remained a puzzle, and the study of Min classification is still considered one of the most challenging tasks in Chinese dialectology. The present article applies a new approach, subgrouping, to solve the problem of classifying the 12 representative Min dialects. The study begins with a review of Norman's (1973, 1974, 1981) Proto-Min system and revises this system based on more comprehensive data. The revised Proto-Min system is then compared with the modern Min dialects to arrive at 39 shared innovations that constitute criteria for subgrouping. A phylogenetic tree model processed by the PENNY program (from PHYLIP, a package of computer programs for inferring phylogenies; Phylogeny Inference Package, version 3.695) displays the result of Min subgrouping based on maximum parsimony. The phylogenetic results derived through this method are shown to differ significantly from the traditional classification of Min dialects. The model also pinpoints the position of some controversial dialects within the Min family.

Phylogenetic position of the yeast-like symbiotes of <i>Tagosodes orizicolus</i> (Homoptera: Delphacidae) based on 18S ribosomal DNA partial sequences

Tagosodes orizicolus Muir (Homoptera: Delphacidae), the endemic delphacid species of tropical America carries yeast-like symbiotes (YLS) in the abdominal fat bodies and the ovarial tissues, like other rice planthoppers of Asia. These YLS are obligate symbiotes, which are transmitted transovarially, and maintain a mutualistic relationship with the insect host. This characteristic has made in vitro culture and classification of YLS rather difficult using conventional methods. Nevertheless, microorganisms of similar characteristics have been successfully classified by using molecular taxonomy. In the present work, the YLS of Tagosodes orizicolus (YLSTo) were purified on Percoll gradients, and specific segments of 18S rDNA were amplified by PCR, cloned and sequenced. Sequences were aligned by means of the CLUSTAL V (DNASTAR) program; phylogenetic trees were constructed with the Phylogeny Inference Package (PHYLIP), showing that YLSTo belong to the fungi class Pyrenomycetes, phylum Ascomycota. Similarities between 98% and 100% were observed among YLS of the rice delphacids Tagosodes orizicolus, Nilaparvata lugens, Laodelphax striatellus and Sogatella fur cifera, and between 89.8% and 90.8% when comparing the above to YLS of the aphid Hamiltonaphis styraci. These comparisons revealed that delphacid YLS are a highly conserved monophyletic group within the Pyrenomycetes and are closely related to Hypomyces chrysospermus.

Phylogeny of the selected members of family Cornaceae as inferred from PCR-RFLP analysis of chloroplast DNA (cpDNA) and internal transcribed spacer (ITS)

PCR-RFLP (polymerase chain reaction–restriction fragment length polymorphism) analysis of cpDNA and PCR-RFLP of rDNA were performed to assess the phylogenetic relationship in seven species of the family Cornaceae. Four pairs of primers, trnD/trnT, trnH/trnK, psaA/trnS and rbcL/ORF106, and one restriction enzyme (HinfI) were used for RFLP-PCR of cpDNA, and two primer pairs ITS5/ITS2 and ITS3/ITS4 with one restriction enzyme (HhaI) were used for ITS1 and ITS2 regions of rDNA, successively. Restriction products produced by trnH/trnK-HinfI and psaA/trnS-HinfI for cpDNA and ITS5/ITS2-HhaI and ITS3/ITS4-HhaI for rDNA, generated very informative bands from all of the seven species studied. A total of 147 scores were recorded and used in PHYLIP (phylogeny inference package) to generate a combined phylogenetic tree. The family was divided into two clades: the Aucuba clade and the cornaceous clade. The cornaceous clade further divided into two subclades: the previously suggested blue- or white-fruited dogwoods (C. walteri and C. macrophylla) and the Cornelian cherries (C. officinalis), and the big-bracted dogwoods (C. kousa and C. florida). The opposite-leaved blue- or white-fruited dogwoods were found to be close sisters to the alternate-leaved blue- or white-fruited dogwoods and all sisters to the Cornelian cherries. C. kousa and C. florida were found to be close sisters under big-bracted dogwoods. Based on the topology portrayed by the phylogenetic tree obtained, it is possible to conclude that big-bracted dogwoods are the most primitive member followed by the intermediates – the cornelian cherries, and the blue- or white-fruited dogwoods are the most advanced members, while the Aucuba clade is the most primitive in the family.

JCoDA: a tool for detecting evolutionary selection

BackgroundThe incorporation of annotated sequence information from multiple related species in commonly used databases (Ensembl, Flybase, Saccharomyces Genome Database, Wormbase, etc.) has increased dramatically over the last few years. This influx of information has provided a considerable amount of raw material for evaluation of evolutionary relationships. To aid in the process, we have developed JCoDA (Java Codon Delimited Alignment) as a simple-to-use visualization tool for the detection of site specific and regional positive/negative evolutionary selection amongst homologous coding sequences.ResultsJCoDA accepts user-inputted unaligned or pre-aligned coding sequences, performs a codon-delimited alignment using ClustalW, and determines the dN/dS calculations using PAML (Phylogenetic Analysis Using Maximum Likelihood, yn00 and codeml) in order to identify regions and sites under evolutionary selection. The JCoDA package includes a graphical interface for Phylip (Phylogeny Inference Package) to generate phylogenetic trees, manages formatting of all required file types, and streamlines passage of information between underlying programs. The raw data are output to user configurable graphs with sliding window options for straightforward visualization of pairwise or gene family comparisons. Additionally, codon-delimited alignments are output in a variety of common formats and all dN/dS calculations can be output in comma-separated value (CSV) format for downstream analysis. To illustrate the types of analyses that are facilitated by JCoDA, we have taken advantage of the well studied sex determination pathway in nematodes as well as the extensive sequence information available to identify genes under positive selection, examples of regional positive selection, and differences in selection based on the role of genes in the sex determination pathway.ConclusionsJCoDA is a configurable, open source, user-friendly visualization tool for performing evolutionary analysis on homologous coding sequences. JCoDA can be used to rapidly screen for genes and regions of genes under selection using PAML. It can be freely downloaded at http://www.tcnj.edu/~nayaklab/jcoda.

Proteomics Analysis of the Excretory/Secretory Component of the Blood-feeding Stage of the Hookworm, Ancylostoma caninum

Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting approximately 600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three beta-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.

Motif Decomposition of the Phosphotyrosine Proteome Reveals a New N-terminal Binding Motif for SHIP2

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. From 20 clusters we extracted 16 known and four new interaction motifs. Using quantitative mass spectrometry we pulled down Tyr(P)-specific binding partners for peptides corresponding to the extracted motifs. We confirmed numerous previously known interaction motifs and found 15 new interactions mediated by phosphosites not previously known to bind SH2 or PTB. Remarkably, a novel hydrophobic N-terminal motif ((L/V/I)(L/V/I)pY) was identified and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid binding.

Role of the Synthase Domain of Ags1p in Cell Wall α-Glucan Biosynthesis in Fission Yeast

The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential component, consisting mainly of (1,3)-alpha-glucan with approximately 10% (1,4)-linked alpha-glucose residues. The multidomain protein Ags1p is required for alpha-glucan biosynthesis and is conserved among cell wall alpha-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-alpha-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-alpha-glucan constituent of cell wall alpha-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-alpha-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-alpha-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-alpha-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall alpha-glucan.

Dynamic Hepatitis C Virus Genotypic and Phenotypic Changes in Patients Treated With the Protease Inhibitor Telaprevir

Telaprevir (VX-950), a hepatitis C virus (HCV) NS3.4A protease inhibitor, has shown strong antiviral activity in phase 1 clinical studies. Because of high levels of HCV replication and the low fidelity of HCV polymerase, selection of resistant isolates during therapy may occur. A highly sensitive sequencing method was developed in which approximately 80 clones/sample were analyzed to identify mutations in the NS3 protease catalytic domain in HCV genotype-1-infected patients dosed with 450 mg every 8 hours, 750 mg every 8 hours, or 1250 mg every 12 hours of telaprevir for 14 days. Mutations that confer low-level resistance (V36A/M, T54A, R155K/T, and A156S) and high-level resistance (A156V/T, 36+155, 36+156) to telaprevir were detected and correlated with telaprevir exposure and virologic response. Changes in the frequency of mutations after the end of dosing showed an inverse relationship between in vivo viral fitness and resistance. In the absence of telaprevir selective pressure the majority of resistant variants were replaced by wild-type virus within 3-7 months. Resistant HCV isolates are selected rapidly during therapy with the highly active protease inhibitor telaprevir. Combination therapy with pegylated interferon-alfa or other direct antiviral drugs seem mandatory to avoid developing resistance.

Impact of character correlation and variable groupings on modern human population tree resolution.

Relationships among modern human populations are often explored through the use of linear measurements taken on the cranium and expressed in the form of dendrograms. However, craniometric variables are strongly correlated and thereby violate the assumption of independence that most statistical analyses require. This study explores the relationship between differing methods of variable treatment and the statistical robustness of the outcomes they yield, as depicted in interpopulational trees of relatedness among modern humans. Three methods of grouping variables are examined. The first method leaves them ungrouped, the second groups variables on the basis of the developmental and/or functional complex of the cranium to which they are thought to belong, and the last method reduces variables by using principal components analysis. The strength of each of these methods is tested through the use of the Continuous Character Maximum Likelihood (CONTML) program in the PHYLIP phylogeny inference package. This program produces output in the form of trees, and the resolution of the branching topology is given as a log-likelihood value, with statistical confidence intervals supporting each branch placement on the tree. The results indicate that leaving variables ungrouped provides misleadingly strong results by failing to account for character correlation. Of the alternative two grouping methods, the covarying components method yields the best-resolved tree with stronger statistical support for its topology than the approach of grouping variables on the basis of their location on the cranium. Finally, the implications for interpreting population histories based on such methods are discussed.

Using RAPD markers to analyse genetic diversity in Portuguese potato cyst nematode populations

Abstract The two species of potato cyst nematodes (PCN) have different spectra of virulence towards host resistance genes and a proper identification is important in the control of these nematodes by means of resistant potato cultivars. Genetic variability of 32 populations of Globodera rostochiensis and three of G. pallida from different regions of Portugal were analysed and compared using random amplified polymorphic DNA and sixteen primers. In addition, a population of G. pallida (Luffness) from Scotland, UK, was included for comparison. The patterns obtained were evaluated using the Nei coefficient and the UPGMA cluster method. The resulting clusters were expressed as dendrograms in the Phylogeny Inference Package (PHYLIP) program. Separation of the two species of PCN was achieved with an average similarity of 43%. Two populations of G. rostochiensis (PT1 and PT82) appear to be distinct from the main group of this species. One population of G.pallida (PT104) was distinct from the others of this species and the Luffness population was linked most closely with PT5701. Distinct clusters were observed within both species but the clusters could not be related to the geographic proximity of the populations.

Antigenic characterization of bovine viral diarrhoea virus isolates from Spain with a panel of monoclonal antibodies.

A group of 47 bovine viral diarrhoea virus (BVDV) strains isolated from a variety of bovine tissues from eight different geographical areas of Spain and two BVDV strains isolated from a cell line were characterized antigenically with a panel of 23 monoclonal antibodies (mAbs). The mAbs were directed at one of three viral proteins: E2, Erns and NS2-3. A peroxidase-linked assay was used to test the mAbs for reactivity against infected cell monolayers. The data were analysed by two computational methods: the Antigenic Distance Program (MAP) and the Phylogeny Inference Package (PHYLIP), and compared with those obtained previously using the same mAbs with other pestiviruses, including reference strains and UK field isolates. All the Spanish field strains studied appeared to be broadly similar to reference strains of BVDV and were included in the subgroup of classical BVDV, meanwhile the two strains isolated from a cell line were included in the subgroup of atypical pestiviruses.

Transmission of HIV-1 and HCV by head-butting

Two men who had never met before had a fight as a consequence of a motor car accident. One, who was HIV-1 positive, head-butted the other who was wearing metal-framed spectacles on his forehead. The spectacles left imprints on the foreheads of both men, and there was copious bleeding. The man who was head-butted tested negative for HIV-1 3 days after the fight. 14 days later, however, he developed symptoms consistent with primary HIV-1 infection. About 3 months after the assault, he also had acute type B hepatitis; at which time he was found positive for antibodies against all the major HIV-1 structural proteins, although HIV-1 EIA was negative, and with CD4 T-cell counts of 606 10/L. The assailant had a history of intravenous-drug use. In May, 1990, he reported syringe exchange and sexual intercourse with an HIV-seropositive woman. At the time, he tested negative for antibodies against HIV-1 p24 antigen. Positive tests for HIV-1, hepatitis C virus (HCV), and hepatitis B virus (HBV) were found in May, 1991, when his CD4 T-cell count was 720 10/L. His CD4 cell counts were 124 10/L at the time of the fight. Sequencing of a 183 base pair of the gp120 V3 env region of DNA from both men’s peripheral blood mononuclear cells was done 126 days after the fight. Nucleotide sequence distances of the men and four Italian HIV-1 infected mother/infant pairs living in the same region were calculated with the DNADIST program of the 3.572c Phylogeny Inference Package. The average distance of the men (2·80 [SD 0·26]) was similar to those calculated for the mother/infant pairs, which ranged from 1·91 (0·36) to 7·05 (0·66). By contrast, distances between either of the men and mothers and infants were those typical of epidemiologically unlinked infections (variability range from 9·37% to 18·44%). The men’s pairwise distances were significantly different (p<0·0025) from the distances between either of them and the less divergent control, suggesting that the similarity between the men’s HIV-1 DNA was not random. The skin injuries in the man likely provided HIV-1 and HBV with a direct access to the bloodstream. Although the assailant was also infected with HCV, the other man tested negative for HCV 4 months after the accident. Parenteral exposure via blood or blood products leads to HCV infection in most cases, particularly at high levels of viraemia. The head-butter’s HCV viral burden was less than 1000 copies/mL plasma, which may explain the lack of HCV transmission.

Molecular Paleobiology of Protists: Amplification and Direct Sequencing of Foraminiferal DNA

Molecular biological techniques are used to reveal or confirm phylogenies and biogeographic and ecologic histories of fossilizable organisms with great success. Using the PCR (polymerase chain reaction) technique, we have successfully amplified and sequenced ribosomal DNA (1 8S-rDNA) from benthic and planktic foraminifera. DNA is first extracted from single living specimens and DNA templates are prepared. The DNA is then amplified using PCR for single and double strands. The direct methods using primers developed for protists and fungi allow amplification of the 18S-rRNA gene, a relatively conservative gene useful in phylogenetic analyses. The amplified DNA is sequenced on acrylomide gels using the chain-terminaton method. The 18S-rDNA nucleotide sequences may then be analyzed using standard phylogenetic analytical techniques such as PAUP (Phylogenetic Analysis Using Parsimony) or PHYLIP (Phylogeny Inference Package)..

The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiomycota. I. Cladistic analysis of rRNA sequences

To clarify the phylogenetic relationships of the Chytridiomycota and the anaerobic fungi from the rumen and caecum of herbivorous animals, we analyzed the partial 18S rRNA sequences from 28 species ranging from protists to mammals and internal transcribed spacer 1 (ITS1) and its adjacent sequences from four gut fungi and one chytrid by using three algorithms from the Phylogeny Inference Package (PHYLIP). To get the confidence limits for each branch, we applied bootstrapping for each algorithm. Our analysis on partial 18S rRNA sequences shows that the Chytridiomycota are clustered with other fungi with 98, 76, and 30% confidences in the Fitch–Margoliash, neighbour-joining, and maximum parsimony algorithms. None of these three algorithms place any of 17 protists from 12 phyla with the fungi, including the chytrids. The same analysis also shows that the Spizellomycetales and Chytridiales cluster with the gut fungi but does not identify which order is closest to them. These results suggest that the Chytridiomycota, including the gut fungi, are indeed fungi but the gut fungi might not belong to the Spizellomycetales. The phylogenetic trees generated by the above three algorithms, plus the maximum likelihood algorithm, based on ITS1 and its adjacent regions show that Anaeromyces is more distant from Orpinomyces, Neocallimastix, and Piromyces. However, they failed to determine the relationships among the last three genera. Key words: Chytridiomycota, gut fungi, rumen fungi, phylogeny, rRNA sequences.

The taxonomy and phylogenetic relationships of the Pollenia semicinerea species‐group (Diptera: Calliphoridae)

Abstract. Within Pollenia Robineau‐Desvoidy, a semicinerea species‐group is defined. It consists of the redescribed P.semicinerea Villeneuve, P.grunini sp.n., P.mystica sp.n. and P.paragrunini sp.n., and P.pectinata Grunin, for which some new characters are given. A key is provided and the known terminalia of both sexes are illustrated. A lectotype is designated for P.semicinerea Villeneuve. P.semicinerea is known from the Middle East (Israel, Syria, Lebanon), P.grunini, P.mystica and P.paragrunini from the Caucasus and neighbouring areas in the U.S.S.R. and P.pectinata from the temperate parts of the Palaearctic Region except west of Poland and U.S.S.R. It is argued that the semicinerea‐ and racfe‐groups are monophyletic sister‐groups. The tenuiforceps‐group is most probably the monophyletic sister‐group of the composite monophyletic group (rudis‐group+semicinerea‐group). The intermedia‐group and the griseo‐tomentosa‐group are both monophyletic. The composite group (rudis‐group +semicinerea‐group+tenuiforceps‐group + intermedia‐group + griseotomentosa‐group) is probably a monophyletic subgroup of Pollenia. A partly resolved synapomorphy diagram is provided, which is compared to trees generated by various computer programs in the Phylogeny Inference Package (PHYLIP) of Felsenstein. Pollenia alajensis Rodendorf (=sytshevskajae Grunin) is assigned to the tenuiforceps species‐group on the basis of male and female genitalia.