Construction of phylogenetic trees for proteins and nucleic acids: Empirical evaluation of alternative matrix methods

Summary The methods of Fitch and Margoliash and of Farris for the construction of phylogenetic trees were compared. A phenetic clustering technique - the UPGMA method — was also considered.The three methods were applied to difference matrices obtained from comparison of macromolecules by immunological, DNA hybridization, electrophoretic, and amino acid sequencing techniques. To evaluate the results, we used the goodness-of-fit criterion. In some instances, the F-M and Farris methods gave a comparably good fit of the output to the input data, though in most cases the F-M procedure gave a much better fit. By the fit criterion, the UPGMA procedure was on the average better than the Farris method but not as good as the F-M procedure.On the basis of the results given in this report and the goodness-of-fit criterion, it is suggested that where input data are likely to include overestimates as well as true estimates and underestimates of the actual distances between taxonomic units, the F-M method is the most reasonable to use for constructing phylogenies from distance matrices. Immunological, DNA hybridization, and electrophoretic data fall into this category. By contrast, where it is known that each input datum is indeed either a true estimate or an underestimate of the actual distance between 2 taxonomic units, the Farris procedure appears, on theoretical grounds, to be the matrix method of choice. Amino acid and nucleotide sequence data are in this category.The following abbreviations are used in this work F-M Fitch-Margoliash - UPGMA unweighted pair-group method using arithmetic averages - SD percent standard deviation     

Bayesian Network Expansion Identifies New ROS and Biofilm Regulators

Signaling and regulatory pathways that guide gene expression have only been partially defined for most organisms. However, given the increasing number of microarray measurements, it may be possible to reconstruct such pathways and uncover missing connections directly from experimental data. Using a compendium of microarray gene expression data obtained from Escherichia coli, we constructed a series of Bayesian network models for the reactive oxygen species (ROS) pathway as defined by EcoCyc. A consensus Bayesian network model was generated using those networks sharing the top recovered score. This microarray-based network only partially agreed with the known ROS pathway curated from the literature and databases. A top network was then expanded to predict genes that could enhance the Bayesian network model using an algorithm we termed ‘BN+1’. This expansion procedure predicted many stress-related genes (e.g., dusB and uspE), and their possible interactions with other ROS pathway genes. A term enrichment method discovered that biofilm-associated microarray data usually contained high expression levels of both uspE and gadX. The predicted involvement of gene uspE in the ROS pathway and interactions between uspE and gadX were confirmed experimentally using E. coli reporter strains. Genes gadX and uspE showed a feedback relationship in regulating each other''s expression. Both genes were verified to regulate biofilm formation through gene knockout experiments. These data suggest that the BN+1 expansion method can faithfully uncover hidden or unknown genes for a selected pathway with significant biological roles. The presently reported BN+1 expansion method is a generalized approach applicable to the characterization and expansion of other biological pathways and living systems.     

DISTANCE METHODS: A REPLY TO FARRIS

Abstract— Farris (1985) claimed that my assertions about unbiasedness and consistency of estimates of a phylogeny obtained by least squares fitting are in error. The counterexample he constructed violates the assumptions of additivity and independence of distances which were clearly stated in my earlier paper. As such it is not a valid counterexample. It is argued, contrary to Farris's claims, that one need not avoid nonmetric distances, and that one should avoid negative branch lengths in estimates of phylogenies from distance data. Statistical tests of clockness, and, to a limited extent, of alternative phylogenies can be constructed, and these are demonstrated by example. A computer program to infer phylogenies from distance matrices has been in free distribution by me for several years; it seems as effective as the program recently announced by Farris. Information on phylogenies is present in distance data, as in other kinds of data, and statistical methods can be developed to extract it.     

Measuring Topological Congruence by Extending Character Techniques

A measure of topological congruence which is an extension of the Mickevich–Farris character incongruence metric ( i.e. , ILD; Mickevich and Farris, 1981) is proposed. Group inclusion characters (1 = member of a clade; 0 = not a member) are constructed for each topology to be considered. The sets of characters derived from the topologies are then compared for character incongruence due to data set combination. Each homoplasy signifies a disagreement among topological statements. The value is normalized for potential maximum incongruence to adjust values for unresolved topologies. This measure is compared to other topological and character congruence techniques and explored in test data.     

Accuracy of phylogenetic trees estimated from DNA sequence data

The relative merits of four different tree-making methods in obtaining the correct topology were studied by using computer simulation. The methods studied were the unweighted pair-group method with arithmetic mean (UPGMA), Fitch and Margoliash's (FM) method, thd distance Wagner (DW) method, and Tateno et al.'s modified Farris (MF) method. An ancestral DNA sequence was assumed to evolve into eight sequences following a given model tree. Both constant and varying rates of nucleotide substitution were considered. Once the DNA sequences for the eight extant species were obtained, phylogenetic trees were constructed by using corrected (d) and uncorrected (p) nucleotide substitutions per site. The topologies of the trees obtained were then compared with that of the model tree. The results obtained can be summarized as follows: (1) The probability of obtaining the correct rooted or unrooted tree is low unless a large number of nucleotide differences exists between different sequences. (2) When the number of nucleotide substitutions per sequence is small or moderately large, the FM, DW, and MF methods show a better performance than UPGMA in recovering the correct topology. The former group of methods is particularly good for obtaining the correct unrooted tree. (3) When the number of substitutions per sequence is large, UPGMA is at least as good as the other methods, particularly for obtaining the correct rooted tree. (4) When the rate of nucleotide substitution varies with evolutionary lineage, the FM, DW, and MF methods show a better performance in obtaining the correct topology than UPGMA, except when a rooted tree is to be produced from data with a large number of nucleotide substitutions per sequence.(ABSTRACT TRUNCATED AT 250 WORDS)      

Accuracy of estimated phylogenetic trees from molecular data

Summary The accuracies and efficiencies of four different methods for constructing phylogenetic trees from molecular data were examined by using computer simulation. The methods examined are UPGMA, Fitch and Margoliash's (1967) (F/M) method, Farris' (1972) method, and the modified Farris method (Tateno, Nei, and Tajima, this paper). In the computer simulation, eight OTUs (32 OTUs in one case) were assumed to evolve according to a given model tree, and the evolutionary change of a sequence of 300 nucleotides was followed. The nucleotide substitution in this sequence was assumed to occur following the Poisson distribution, negative binomial distribution or a model of temporally varying rate. Estimates of nucleotide substitutions (genetic distances) were then computed for all pairs of the nucleotide sequences that were generated at the end of the evolution considered, and from these estimates a phylogenetic tree was reconstructed and compared with the true model tree. The results of this comparison indicate that when the coefficient of variation of branch length is large the Farris and modified Farris methods tend to be better than UPGMA and the F/M method for obtaining a good topology. For estimating the number of nucleotide substitutions for each branch of the tree, however, the modified Farris method shows a better performance than the Farris method. When the coefficient of variation of branch length is small, however, UPGMA shows the best performance among the four methods examined. Nevertheless, any tree-making method is likely to make errors in obtaining the correct topology with a high probability, unless all branch lengths of the true tree are sufficiently long. It is also shown that the agreement between patristic and observed genetic distances is not a good indicator of the goodness of the tree obtained.     

The neighbor-joining method: a new method for reconstructing phylogenetic trees

A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.      

An electrophoretic investigation of six species of darters in the subgenus Catonotus

The electrophoretic patterns of lactate dehydrogenase, phosphoglucomutase and total muscle proteins distinguished the species Etheostoma flabellare, E. kennicotti and E. squamiceps from each other and from three species of the E. virgatum group (E. virgatum, E. obeyense and E. barbouri), which all possessed identical patterns. Etheostoma flabellare, the species with the widest geographic range, possessed the greatest amount of genetic variation in all three protein systems studied. Patristic distances were calculated via an unrooted Wagner network. Using this method it was shown that E. flabellare was the most divergent species, E. kennicotti and E. squamiceps were less divergent, and the members of the E. virgatum group clustered closely.     

A new method for the rapid separation of cell organelles

A rapid procedure is described for the separation of plant cell organelles from castor bean endosperm (Ricinus communis). This method is based on the reorientation of sucrose density gradients during centrifugation in a vertical rotor, thus resulting in a shorter path length and drastically reduced run times. Comparison to a separation by a standard procedure shows that, by using this method, equal resolution is possible in less than 10% spin time.     

Model of the coordination site for cobalt-substituted cytochrome P450cam

Cobalt-substituted cytochrome P450_cam was recently reconstituted by Wagner et al (J. Biol. Chem. 256, 6266, (1981)). A model of its coordination site was constructed to determine the mode of axial coordination of the native enzyme. Complexes were prepared from cobalt porphyrins (cobalt-protoporphyrin IX (CoPPIX), cobalt-meso-tetraphenylporphyrin, cobalt-γ-laurylpyridyl triphenylporphyrin, and cobalt-octaethylporphyrin), thioglycolate ester, and tetramethylammonium hydroxide in organic solvents. Complexes prepared in an organic solvent such as CHC1₃ under air at room temperature exhibited a stable Soret hyperporphyrin spectrum characterized by split Soret bands, especially like that of the thiol-Co-P450_cam complex Comparison of the spectra of the hyperporphyrin spectral complexes titrated with various types of alcohol and imidazole, with the spectrum of Co-P450_cam in the oxidized state support the idea that an axial thiolate at the fifth position and a hydroxyl group of alcohol at the sixth position of the heme form the coordination site of Co-P450_cam The CoPPIX-thiolate-ethanol complex retaining S⁻-Co(III)-OH coordination is thought to be a possible model of Co-P450_cam in the oxidized state.     

Accuracy of estimated phylogenetic trees from molecular data

The accuracies and efficiencies of three different methods of making phylogenetic trees from gene frequency data were examined by using computer simulation. The methods examined are UPGMA, Farris' (1972) method, and Tateno et al.'s (1982) modified Farris method. In the computer simulation eight species (or populations) were assumed to evolve according to a given model tree, and the evolutionary changes of allele frequencies were followed by using the infinite-allele model. At the end of the simulated evolution five genetic distance measures (Nei's standard and minimum distances, Rogers' distance, Cavalli-Sforza's f theta, and the modified Cavalli-Sforza distance) were computed for all pairs of species, and the distance matrix obtained for each distance measure was used for reconstructing a phylogenetic tree. The phylogenetic tree obtained was then compared with the model tree. The results obtained indicate that in all tree-making methods examined the accuracies of both the topology and branch lengths of a reconstructed tree (rooted tree) are very low when the number of loci used is less than 20 but gradually increase with increasing number of loci. When the expected number of gene substitutions (M) for the shortest branch is 0.1 or more per locus and 30 or more loci are used, the topological error as measured by the distortion index (dT) is not great, but the probability of obtaining the correct topology (P) is less than 0.5 even with 60 loci. When M is as small as 0.004, P is substantially lower. In obtaining a good topology (small dT and high P) UPGMA and the modified Farris method generally show a better performance than the Farris method. The poor performance of the Farris method is observed even when Rogers' distance which obeys the triangle inequality is used. The main reason for this seems to be that the Farris method often gives overestimates of branch lengths. For estimating the expected branch lengths of the true tree UPGMA shows the best performance. For this purpose Nei's standard distance gives a better result than the others because of its linear relationship with the number of gene substitutions. Rogers' or Cavalli-Sforza's distance gives a phylogenetic tree in which the parts near the root are condensed and the other parts are elongated. It is recommended that more than 30 loci, including both polymorphic and monomorphic loci, be used for making phylogenetic trees. The conclusions from this study seem to apply also to data on nucleotide differences obtained by the restriction enzyme techniques.     

PHYLOGENETICS OF THE LISIANTHIUS SKINNERI (GENTIANACEAE) SPECIES COMPLEX IN PANAMA UTILIZING DNA RESTRICTION FRAGMENT ANALYSIS

The well-delimited and evolutionary interesting tropical shrub group, the Lisianthius skinneri (Gentianaceae) species complex, was analyzed for variation in nuclear ribosomal DNA and chloroplast DNA by restriction endonuclease fragment analysis. A most parsimonious tree using variations in both DNAs was constructed for seven populations in the group by including an appropriate outgroup. This phylogeny is significantly more compatible with the DNA data than most, but not all, less parsimonious phylogenies. At least two distinct lineages have independently evolved geographically restricted, cloud forest species from the putative ancestral, widespread, and lower elevation L. skinneri. Lisianthius skinneri itself is shown to be paraphyletic with populations derived separately from the two distinct lineages. Except for a switch in the placement of two populations, this DNA-based phylogeny is congruent with an isozyme-based Wagner network depicting relationships in the species complex. Relative rates of divergence, in terms of nuclear ribosomal DNA, chloroplast DNA, isozymes, and morphology, differ markedly within and between lineages. The non-transcribed spacer region of ribosomal DNA is shown to evolve in a manner that is not in accord with a molecular clock hypothesis. Small population sizes, restricted and isolated nature of populations, and probable founder events are suggested as instrumental in causing this lack of concerted divergence within and between lineages of the L. skinneri species complex.     

Minimal Network Topologies for Signal Processing during Collective Cell Chemotaxis

Cell-cell communication plays an important role in collective cell migration. However, it remains unclear how cells in a group cooperatively process external signals to determine the group’s direction of motion. Although the topology of signaling pathways is vitally important in single-cell chemotaxis, the signaling topology for collective chemotaxis has not been systematically studied. Here, we combine mathematical analysis and simulations to find minimal network topologies for multicellular signal processing in collective chemotaxis. We focus on border cell cluster chemotaxis in the Drosophila egg chamber, in which responses to several experimental perturbations of the signaling network are known. Our minimal signaling network includes only four elements: a chemoattractant, the protein Rac (indicating cell activation), cell protrusion, and a hypothesized global factor responsible for cell-cell interaction. Experimental data on cell protrusion statistics allows us to systematically narrow the number of possible topologies from more than 40,000,000 to only six minimal topologies with six interactions between the four elements. This analysis does not require a specific functional form of the interactions, and only qualitative features are needed; it is thus robust to many modeling choices. Simulations of a stochastic biochemical model of border cell chemotaxis show that the qualitative selection procedure accurately determines which topologies are consistent with the experiment. We fit our model for all six proposed topologies; each produces results that are consistent with all experimentally available data. Finally, we suggest experiments to further discriminate possible pathway topologies.     

Statistical properties of molecular tree construction methods under the neutral mutation model

Summary The statistical properties of three molecular tree construction methods—the unweighted pair-group arithmetic average clustering (UPG), Farris, and modified Farris methods—are examined under the neutral mutation model of evolution. The methods are compared for accuracy in construction of the topology and estimation of the branch lengths, using statistics of these two aspects. The distribution of the statistic concerning topological construction is shown to be as important as its mean and variance for the comparison.Of the three methods, the UPG method constructs the tree topology with the least variation. The modified Farris method, however, gives the best performance when the two aspects are considered simultaneously. It is also shown that a topology based on two genes is much more accurate than that based on one gene.There is a tendency to accept published molecular trees, but uncritical acceptance may lead one to spurious conclusions. It should always be kept in mind that a tree is a statistical result that is affected strongly by the stochastic error of nucleotide substitution and the error intrinsic to the tree construction method itself.     

PCR-RFLP Analysis on Phylogeny of Allium

The molecular phylogeny of the genus Allium which includes eighteen species selected from nine sections was investigated through PCR-RFLP analysis of two chloroplast DNA fragments, including trak gene (approximately 2 520 bp) and rpL16 gene (approximately 1 230 bp). Digestion of these two fragments by 26 restriction endonucleases yielded 303 polymorphic recognition sites, of which 163 were informative sites. The restriction site data matrix were analyzed following the parsimonious Wagner and parsimonious Dollo principle of PAUP ( version 3.1.1 ). Topologically, the most parsimonious Wagner tree constructed by branch-and-bound and heuristic search was similar to the most parsimonious Dollo tree. All the taxa of Allium form a monophyletic group, and five sections based on morphological characters were supported strongly by this result. Sect. Augninum is closely related to Sect. Bromatorrhiza, Sect. Molium is closely related to Sect. Caloscordum. Their reliability was farther confirmed by the bootstrap test very well. In morphology, A. pallasii is closely related to A. caeruleum and belongs to Sect. Haplostemen, A. cepa is closely related to A. galanthum and belongs to Sect. Cepa. But evidence from cladistics of parsimonious tree based on 163 informative sites of PCR-RFLPs showed that they are neither confined to a monophyletic group nor to a natural taxon.     

Characterization and genetic mapping of a new blood-flesh trait controlled by the single dominant locus DBF in peach

Anthocyanin-rich peaches, because of their antioxidant properties and their strong attractiveness to consumers, are increasingly considered in French peach varietal innovation programs that integrate plant genomics and classical breeding. In this study, we describe a new blood-flesh trait identified in the ‘Wu Yue Xian’ peach accession from China. ‘Wu Yue Xian’ exhibits a fully red mesocarp during the later stages of fruit development, both with green midrib leaf and normal growth of the tree. This blood-flesh phenotype clearly differs from that determined by a single recessive locus (bf) in ‘Harrow Blood’, a clone showing blood-flesh in both immature and mature fruit associated with red midrib leaf and reduced tree height. We have then provided genetic evidence that blood-flesh phenotype of ‘Wu Yue Xian’ was controlled by a single dominant locus, designated DBF (Dominant Blood-Flesh), in four successive families derived from this accession. A genetic linkage map of the blood-flesh parent (‘D6090’) of the fourth population was constructed, including 102 SSRs spanning a total distance of 562.3 cM in eight linkage groups. Whereas the bf locus is located to linkage group 4, we mapped DBF to the top of linkage group 5, thus proving that DBF and bf loci are not alleles. Among 64 predicted genes in the DBF region (505 kbp), three genes of the dihydroflavonol-4-reductase family were identified as good candidates for the control of the DBF trait. Furthermore, SSR markers flanking DBF, such as AMPP157 and AMPPG178, supply a good basis to implement marker-assisted selection for this trait.     

葱属系统发育的PCR—RFLP分析初报

葱属（Ａｌｉｕｍ）为广义百合科（Ｌｉｌｉａｃｅａｅ）葱族（Ａｌｉｅａｅ）的一个重要类群。该属种类丰富、分布极广，关于它的系统发育和进化问题，目前尚存在不少分歧［１］。我国有葱属１１０种（含变种和引进外来种），主要分布于东北、华北、西北和西南地区。我们...