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Molecular phylogenetic dating of asterid flowering plants shows early Cretaceous diversification.

We present a phylogenetic dating of asterids, based on a 111-taxon tree representing all major groups and orders and 83 of the 102 families of asterids, with an underlying data set comprising six chloroplast DNA markers totaling 9914 positions. Phylogenetic dating was done with semiparametric rate smoothing by penalized likelihood. Confidence intervals were calculated by bootstrapping. Six reference fossils were used for calibration. To explore the effects of various sources of error, we repeated the analyses with alternative dating methods (nonparametric rate smoothing and the Langley-Fitch clock-based method), alternative tree topologies, reduced taxon sampling (22 of the 111 taxa deleted), partitioning the data into three genes and three noncoding regions, and calibrating with single reference fossils. The analyses with alternative topologies, reduced taxon sampling, and coding versus noncoding sequences all yielded small or in some cases no deviations. The choice of method influenced the age estimates of a few nodes considerably. Calibration with reference fossils is a critical issue, and use of single reference fossils yielded different results depending on the fossil. The bootstrap confidence intervals were generally small. Our results show that asterids and their major subgroups euasterids, campanulids, and lamiids diversified during the Early Cretaceous. Cornales, Ericales, and Aquifoliales also have crown node ages from the Early Cretaceous. Dipsacales and Solanales are from the Mid-Cretaceous, the other orders of core campanulids and core lamiids from the Late Cretaceous. The considerable diversity exhibited by asterids almost from their first appearance in the fossil record also supports an origin and first phase of diversification in the Early Cretaceous.

DNA, Chloroplast↗

Frequentist properties of Bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models.

What does the posterior probability of a phylogenetic tree mean?This simulation study shows that Bayesian posterior probabilities have the meaning that is typically ascribed to them; the posterior probability of a tree is the probability that the tree is correct, assuming that the model is correct. At the same time, the Bayesian method can be sensitive to model misspecification, and the sensitivity of the Bayesian method appears to be greater than the sensitivity of the nonparametric bootstrap method (using maximum likelihood to estimate trees). Although the estimates of phylogeny obtained by use of the method of maximum likelihood or the Bayesian method are likely to be similar, the assessment of the uncertainty of inferred trees via either bootstrapping (for maximum likelihood estimates) or posterior probabilities (for Bayesian estimates) is not likely to be the same. We suggest that the Bayesian method be implemented with the most complex models of those currently available, as this should reduce the chance that the method will concentrate too much probability on too few trees.

Animals↗

Fundamental differences between the methods of maximum likelihood and maximum posterior probability in phylogenetics.

Using a four-taxon example under a simple model of evolution, we show that the methods of maximum likelihood and maximum posterior probability (which is a Bayesian method of inference) may not arrive at the same optimal tree topology. Some patterns that are separately uninformative under the maximum likelihood method are separately informative under the Bayesian method. We also show that this difference has impact on the bootstrap frequencies and the posterior probabilities of topologies, which therefore are not necessarily approximately equal. Efron et al. (Proc. Natl. Acad. Sci. USA 93:13429-13434, 1996) stated that bootstrap frequencies can, under certain circumstances, be interpreted as posterior probabilities. This is true only if one includes a non-informative prior distribution of the possible data patterns, and most often the prior distributions are instead specified in terms of topology and branch lengths. [Bayesian inference; maximum likelihood method; Phylogeny; support.].

Bayes Theorem↗

Weighted least-squares likelihood ratio test for branch testing in phylogenies reconstructed from distance measures.

A variety of analytical methods is available for branch testing in distance-based phylogenies. However, these methods are rarely used, possibly because the estimation of some of their statistics, especially the covariances, is not always feasible. We show that these difficulties can be overcome if some simplifying assumptions are made, namely distance independence. The weighted least-squares likelihood ratio test (WLS-LRT) we propose is easy to perform, using only the distances and some of their associated variances. If no variances are known, the use of the Felsenstein F-test, also based on weighted least squares, is discussed. Using simulated data and a data set of 43 mammalian mitochondrial sequences we demonstrate that the WLS-LRT performs as well as the generalized least-squares test, and indeed better for a large number of taxa data set. We thus show that the assumption of independence does not negatively affect the reliability or the accuracy of the least-squares approach. The results of the WLS-LRT are no worse than the results of the bootstrap methods, such as the Felsenstein bootstrap selection probability test and the Dopazo test. We also show that WLS-LRT can be applied in instances where other analytical methods are inappropriate. This point is illustrated by analyzing the relationships between human immunodeficiency virus type 1 (HIV-1) sequences isolated from various organs of different individuals.

Animals↗

Data sets, partitions, and characters: philosophies and procedures for analyzing multiple data sets.

We compared four approaches for analyzing three data sets derived from staphylinoid beetles, a superfamily whose known species diversity is roughly comparable to that of vertebrates. One data set is derived from adult morphology and the two molecular data sets are from 12S ribosomal RNA and cytochrome b mitochondrial DNA. We found that taxonomic congruence following conditional data combination, herein called compatible evidence (CE), resolved more nodes compatible with an initial conservative hypothesis than did total evidence (TE), conditional data combination (CDC), or taxonomic congruence (TC). CE sets a base of nodes obtained by CDC analysis and then investigates what further agreement may arise in a universe where these nodes are accepted as given. We suggest that CE75-75 may be appropriate for future studies that aim to both generate a well-corroborated tree and investigate conflicts between data sets, partitions, and characters. CE75-75 is a 75% bootstrap consensus CDC tree followed by combinable-component consensus of a 75% bootstrap consensus of each homogeneous set of partitions having hierarchical structure.

Animals↗

Inferring complex phylogenies using parsimony: an empirical approach using three large DNA data sets for angiosperms.

To explore the feasibility of parsimony analysis for large data sets, we conducted heuristic parsimony searches and bootstrap analyses on separate and combined DNA data sets for 190 angiosperms and three outgroups. Separate data sets of 18S rDNA (1,855 bp), rbcL (1,428 bp), and atpB (1,450 bp) sequences were combined into a single matrix 4,733 bp in length. Analyses of the combined data set show great improvements in computer run times compared to those of the separate data sets and of the data sets combined in pairs. Six searches of the 18S rDNA + rbcL + atpB data set were conducted; in all cases TBR branch swapping was completed, generally within a few days. In contrast, TBR branch swapping was not completed for any of the three separate data sets, or for the pairwise combined data sets. These results illustrate that it is possible to conduct a thorough search of tree space with large data sets, given sufficient signal. In this case, and probably most others, sufficient signal for a large number of taxa can only be obtained by combining data sets. The combined data sets also have higher internal support for clades than the separate data sets, and more clades receive bootstrap support of > or = 50% in the combined analysis than in analyses of the separate data sets. These data suggest that one solution to the computational and analytical dilemmas posed by large data sets is the addition of nucleotides, as well as taxa.

Classification↗

Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant.

We look at the higher-order phylogeny of mammals, analyzing in detail the complete mtDNA sequences of more than 40 species. We test the support for several proposed superordinal relationships. To this end, we apply a number of recently programmed methods and approaches, plus better-established methods. New pairwise tests show highly significant evidence that amino acid frequencies are changing among nearly all the genomes studied when unvaried sites are ignored. LogDet amino acid distances, with modifications to take into account invariant sites, are combined with bootstrapping and the Neighbor Joining algorithm to account for these violations of standard models. To weight the more slowly evolving sites, we exclude the more rapidly evolving sites from the data by using "site stripping". This leads to changing optimal trees with nearly all methods. The bootstrap support for many hypotheses varies widely between methods, and few hypotheses can claim unanimous support from these data. Rather, we uncover good evidence that many of the earlier branching patterns in the placental subtree could be incorrect, including the placement of the root. The tRNA genes, for example, favor a split between the group hedgehog, rodents, and primates versus all other sequenced placentals. Such a grouping is not ruled out by the amino acid sequence data. A grouping of all rodents plus rabbit, the old Glires hypothesis, is also feasible with stripped amino acid data, and rodent monophyly is also common. The elephant sequence allows confident rejection of the older taxon Ferungulata (Simpson, 1945). In its place, the new taxa Scrotifera and Fereuungulata are defined. A new likelihood ratio test is used to detect differences between the optimal tree for tRNA versus that for amino acids. While not clearly significant as made, some results indicate the test is tending towards significance with more general models of evolution. Individual placement tests suggest alternative positions for hedgehog and elephant. Congruence arguments to support elephant and armadillo together are striking, suggesting a superordinal group composed of Xenarthra and African endemic mammals, which in turn may be near the root of the placental subtree. Thus, while casting doubt on some recent conclusions, the analyses are also unveiling some interesting new possibilities.

Animals↗

Phylogenetic assessment of molecular and morphological data for eutherian mammals.

The interordinal relationships of eutherian (placental) mammals were evaluated by a phylogenetic analysis of four published data sets (three sequences and one morphological). The nature and degree of support and conflict for particular groups were assessed by separate bootstrap and homogeneity tests, which were followed by combined analyses of the sequence and morphological data. Between orders, strong support (i.e., > or = 95% bootstrap scores) was found for a paraphyletic Artiodactyla (relative to Cetacea) and a monophyletic Cetartiodactyla (Artiodactyla and Cetacea) and Paenungulata (Hyracoidea, Proboscidea, and Sirenia). In turn, some reasonable to strong evidence (> or = 85%) was obtained for Hyracoidea with Sirenia, Dermoptera with Scandentia, Glires (Lagomorpha with Rodentia), and Afrotheria (Amblysomus, Macroscelidea, Paenungulata, and Tubulidentata). Otherwise, no other interordinal clades were supported at these reasonable to strong levels. This overall lack of resolution for eutherian interordinal clusters agrees with other studies that suggest further progress will continue to be slow and difficult. Further resolution will require the integration of more recently published data, the continued sampling of taxa and characters, and the use of more powerful methods of data analysis.

Animals↗

Additional support for Afrotheria and Paenungulata, the performance of mitochondrial versus nuclear genes, and the impact of data partitions with heterogeneous base composition.

We concatenated sequences for four mitochondrial genes (12S rRNA, tRNA valine, 16S rRNA, cytochrome b) and four nuclear genes [aquaporin, alpha 2B adrenergic receptor (A2AB), interphotoreceptor retinoid-binding protein (IRBP), von Willebrand factor (vWF)] into a multigene data set representing 11 eutherian orders (Artiodactyla, Hyracoidea, Insectivora, Lagomorpha, Macroscelidea, Perissodactyla, Primates, Proboscidea, Rodentia, Sirenia, Tubulidentata). Within this data set, we recognized nine mitochondrial partitions (both stems and loops, for each of 12S rRNA, tRNA valine, and 16S rRNA; and first, second, and third codon positions of cytochrome b) and 12 nuclear partitions (first, second, and third codon positions, respectively, of each of the four nuclear genes). Four of the 21 partitions (third positions of cytochrome b, A2AB, IRBP, and vWF) showed significant heterogeneity in base composition across taxa. Phylogenetic analyses (parsimony, minimum evolution, maximum likelihood) based on sequences for all 21 partitions provide 99-100% bootstrap support for Afrotheria and Paenungulata. With the elimination of the four partitions exhibiting heterogeneity in base composition, there is also high bootstrap support (89-100%) for cow + horse. Statistical tests reject Altungulata, Anagalida, and Ungulata. Data set heterogeneity between mitochondrial and nuclear genes is most evident when all partitions are included in the phylogenetic analyses. Mitochondrial-gene trees associate cow with horse, whereas nuclear-gene trees associate cow with hedgehog and these two with horse. However, after eliminating third positions of A2AB, IRBP, and vWF, nuclear data agree with mitochondrial data in supporting cow + horse. Nuclear genes provide stronger support for both Afrotheria and Paenungulata. Removal of third positions of cytochrome b results in improved performance for the mitochondrial genes in recovering these clades.

Animals↗

Testing hybridization hypotheses based on incongruent gene trees.

Hybridization is an important evolutionary mechanism in plants and has been increasingly documented in animals. Difficulty in reconstruction of reticulate evolution, however, has been a long-standing problem in phylogenetics. Consequently, hybrid speciation may play a major role in causing topological incongruence between gene trees. The incongruence, in turn, offers an opportunity to detect hybrid speciation. Here we characterized certain distinctions between hybridization and other biological processes, including lineage sorting, paralogy, and lateral gene transfer, that are responsible for topological incongruence between gene trees. Consider two incongruent gene trees with three taxa, A, B, and C, where B is a sister group of A on gene tree 1 but a sister group of C on gene tree 2. With a theoretical model based on the molecular clock, we demonstrate that time of divergence of each gene between taxa A and C is nearly equal in the case of hybridization (B is a hybrid) or lateral gene transfer, but differs significantly in the case of lineage sorting or paralogy. After developing a bootstrap test to test these alternative hypotheses, we extended the model and test to account for incongruent gene trees with numerous taxa. Computer simulation studies supported the validity of the theoretical model and bootstrap test when each gene evolved at a constant rate. The computer simulation also suggested that the model remained valid as long as the rate heterogeneity was occurring proportionally in the same taxa for both genes. Although the model could not test hypotheses of hybridization versus lateral gene transfer as the cause of incongruence, these two processes may be distinguished by comparing phylogenies of multiple unlinked genes.

Biological Evolution↗

Classification ensembles for unbalanced class sizes in predictive toxicology.

This paper investigates the effects of the ratio of positive-to-negative samples on the sensitivity, specificity, and concordance. When the class sizes in the training samples are not equal, the classification rule derived will favor the majority class and result in a low sensitivity on the minority class prediction. We propose an ensemble classification approach to adjust for differential class sizes in a binary classifier system. An ensemble classifier consists of a set of base classifiers; its prediction rule is based on a summary measure of individual classifications by the base classifiers. Two re-sampling methods, augmentation and abatement, are proposed to generate different bootstrap samples of equal class size to build the base classifiers. The augmentation method balances the two class sizes by bootstrapping additional samples from the minority class, whereas the abatement method balances the two class sizes by sampling only a subset of samples from the majority class. The proposed procedure is applied to a data set to predict estrogen receptor binding activity and to a data set to predict animal liver carcinogenicity using SAR (structure-activity relationship) models as base classifiers. The abatement method appears to perform well in balancing sensitivity and specificity.

Discriminant Analysis↗

Variability in respiratory protection and the assigned protection factor.

The workplace protection factor (WPF) for a given respirator wearer shows substantial variability from wearing to wearing; this variability is commonly assumed to be lognormal in nature. Further, when multiple WPFs are measured for each of multiple wearers, the aggregated WPFs appear to follow a lognormal distribution. However, the analysis typically applied to WPF data does not apportion variability within versus between wearers. We present an analytical framework based on a normal random effects model of log-transformed penetration P values (P = 1/WPF). Data from seven studies of negative-pressure air-purifying half-mask respirators, and from two studies of hemlet-and-visor type powered air-purifying respirators were analyzed by the method of maximum likelihood in the context of the model. More specifically, analyses were performed for log-transformed P values and for logit-transformed P values. Parameter estimates included within-wearer and between-wearer variance components. In general, the within-wearer component dominated the between-wearer component. We also propose a method for establishing an assigned protection factor, APF, that properly accounts for these variance components. Our method provides an APF satisfying two criteria: (1) for a given wearer, an acceptable WPF distribution has no more than 5% of WPFs below the APF value; and (2) for a wearer population, no more than 5% of wearers have unacceptable WPF distributions. The method incorporates an one-sided confidence limit to account for sampling variability. Alternative confidence limits were computed based on large sample variance estimates of random effects model parameters versus a bootstrap method. In general, there was good agreement between the APF values based on log-transformed versus logit-transformed P data, and between APF values based on the large sample variance estimates versus the bootstrap method. Based on large sample variance estimates for the logit-transformed P data from the seven half-mask studies, estimated APFs ranged from 1.4 to 250, with 5/7 studies yielding an APF </= 5.3. Given these results and related considerations, we recommend that the current half-mask APF be reduced from 10 to 5.

Air Pollution, Indoor↗

An individualized nomogram for predicting progression-free survival in systemic anaplastic large cell lymphoma: a multicenter, retrospective, and internally validated study.

OBJECTIVES: To develop an individualized nomogram for predicting disease progression risk in systemic anaplastic large cell lymphoma (sALCL). METHODS: Independent predictors of progression-free survival (PFS) were identified using Cox regression in a multicenter retrospective cohort of 109 sALCL patients (2010-2022). These were incorporated into a three-factor nomogram, evaluated via bootstrapped internal validation (1000 resamples), ROC analysis, C-index, decision curve analysis (DCA), and clinical impact curve (CIC). RESULTS: A total of 29 PFS events occurred during a median follow-up of 31 months. Multivariable modelling selected serum &#x3b2;2-microglobulin elevation, extranodal disease, and front-line chemotherapy choice (CHOP versus CHOPE or BV+CHP) as autonomous progression drivers. Upon internal bootstrap validation, the nomogram yielded strong prognostic accuracy, achieving AUCs of 0.81, 0.85 and 0.87 for 1-, 3- and 5-year progression-free survival, alongside a corrected C-index of 0.779 (95% CI: 0.699 - 0.861). Calibration plots showed close agreement between predicted and observed outcomes, while DCA confirmed superior net clinical benefit versus conventional IPI or Ann Arbor stratification across multiple decision thresholds. CONCLUSION: This first sALCL-specific nomogram integrates clinical and treatment variables to provide personalized PFS risk estimation. While internally validated, this exploratory, observation-based tool requires external validation and recalibration in prospective cohorts before clinical implementation.

Humans↗

Design and analysis of trials with quality of life as an outcome: a practical guide.

Health Related Quality of Life (HRQoL) measures are becoming more frequently used in clinical trials, as both primary and secondary endpoints. Investigators are now asking statisticians for advice on how to plan (e.g., sample size) and analyze studies using HRQoL measures. HRQoL measures such as the SF-36 are usually measured on an ordered categorical (ordinal) scale. In the designing stages and when analyzing, the scales are often scored and the scores treated as if they were continuous and normally distributed. However the ordinal scaling of HRQoL measures leads to problems in determining sample size, and conventional parametric methods of estimation and hypothesis testing may not be appropriate for such outcomes. We present practical guidelines for the design and analysis of trials with HRQoL measures as outcomes. We used conventional statistical methods (i.e., t-tests and multiple regression), various ordinal regression models (proportional odds, continuation ratio, polytomous and stereotype) and bootstrap methods to analyze an HRQoL dataset. To illustrate the various methods we used HRQoL data on the SF-36 Role Limitations Emotional dimension for two groups of patients with leg ulcers. The bootstrap, t-test, and multiple regression methods gave similar results. The various ordinal regression models also gave similar results. If the HRQoL measure has a large number of ordered categories, most of which are occupied, and the underlying scale really is continuous but measured imperfectly by an instrument with a limited number of discrete values, then an informal rule of thumb is that this discrete scale should be treated as continuous if it has seven or more categories and as ordinal otherwise.

Algorithms↗

Methods for comparison of parameters from longitudinal rhythmometric models with multiple components.

Multiple components linear least-squares methods have been proposed for the detection of periodic components in nonsinusoidal longitudinal time series. However, a proper test for comparison of parameters obtained from this method for two or more time series is not yet available. Accordingly, we propose two methods, one parametric and one nonparametric, to compare parameters from rhythmometric models with multiple components. The parametric method is based on techniques commonly and generally employed in linear regression analysis. The comparison of parameters among two or more time series is accomplished by the use of so-called dummy variables. The nonparametric method is based on bootstrap techniques. This approach basically tests if the difference in any given parameter obtained by fitting a model with the same periods to two different longitudinal time series differs from zero. This method calculates a confidence interval for the difference in the tested parameter. If this interval does not contain zero, it can be concluded that the parameters obtained from the two time series are different with high probability. An estimation of the p-value for the corresponding test can also be calculated. By the use of similar bootstrap techniques, confidence intervals can also be obtained for any parameter derived from the multiple component fit of several periods to nonsinusoidal longitudinal time series, including the orthophase (peak time), bathyphase (trough time), and global amplitude (difference between the maximum and the minimum) of the fitted model waveform. These methods represent a valuable tool for the comparison of rhythm parameters obtained by multiple component analysis, and they render this approach as a generally applicable one for waveform representation and detection of periodicities in nonsinusoidal, sparse, and noisy longitudinal time series sampled with either equidistant or unequidistant observations.

Blood Pressure↗

Comparison of parameters from rhythmometric models with multiple components on hybrid data.

Population multiple components is a statistical tool useful for the analysis of time-dependent hybrid data. With a small number of parameters, it is possible to model and to predict the periodic behavior of a population. In this article, we propose two methods to compare among populations rhythmometric parameters obtained by multiple component analysis. The first is a parametric method based in the usual statistical techniques for comparison of mean vectors in multivariate normal populations. The method, through MANOVA analysis, allows comparison of the MESOR and amplitude-acrophase pair of each component among two or more populations. The second is a nonparametric method, based in bootstrap techniques, to compare parameters from two populations. This test allows one to compare the MESOR, the amplitude, and the acrophase of each fitted component, as well as the global amplitude, orthophase, and bathyphase estimated when all fitted components are harmonics of a fundamental period. The idea is to calculate a confidence interval for the difference of the parameters of interest. If this interval does not contain zero, it can be concluded that the parameters from the two models are different with high probability. An estimation of p-value for the corresponding test can also be calculated. Both methods are illustrated with an example, based on clinical data. The nonparametric test can also be applied to paired data, a special situation of great interest in practice. By the use of similar bootstrap techniques, we illustrate how to construct confidence intervals for any rhythmometric parameter estimated from population multiple components models, including the orthophase, bathyphase, and global amplitude. These tests for comparison of parameters among populations are a needed tool when modeling the nonsinusoidal rhythmic behavior of hybrid data by population multiple component analysis.

Chronobiology Phenomena↗

Acute renal failure needing dialysis in the intensive care unit and prognostic scores.

BACKGROUND: Generic prognostic scores used in acute renal failure (ARF) give imprecise results; disease-specific indices applied to distinct populations or intensive care practices becomes inaccurate. The current study evaluates the adequacy of prognostic scores, in patients with severe ARF needing dialysis. METHODS: Known generic (APACHE II) and disease-specific (ATN-ISS) indices were applied to a cohort (n = 280) with ARF needing dialysis, under intensive care. Possible risk factors as causal factors, organ dysfunctions and clinical variables were examined, and a local index assembled by multivariate logistic regression analysis. Area under the receiver operating characteristics (ROC) curves evaluated the indices discriminating capacity. Goodness-of-fit testing and linear regression analysis appraised calibration. Validation was accomplished by the bootstrapping technique. The end-point was hospital mortality. RESULTS: Overall mortality was 85%. Female gender < 44 years (OR: 0.29; 95% CI: 0.10-0.84), liver/obstructive biliary disease (OR: 6.03; 95% CI: 1.65-22.08), being conscious (OR: 0.49; 95% CI: 0.21-1.14), use of vasoactive drug (OR: 3.13; 95% CI: 1.25-7.83), respiratory dysfunction (OR: 5.20; 95% CI: 1.25-7.83) or sepsis (OR: 2.62; 95% CI: 1.14-6.02) were associated with outcome. Areas under the ROC curve of 0.815, 0.652 and 0.814; Goodness-of-fit test P = 0.593, P < 0.001 and P = 0.002; and linear regression R2 = 0.973, R2 = 0.526 and R2 = 0.919 for the local index, APACHE II and ATN-ISS, respectively, indicate better performance by the local index. The local index median area under the ROC curve, by bootstrapping, was 0.820 (95% CI: 0.741-0.907). CONCLUSIONS: APACHE II score was inaccurate, and ATN-ISS poorly calibrated. When mortality or intensive care practices significantly deviate, local scores may better evaluate prognosis in severe ARF.

Acute Kidney Injury↗

Near-full-length genome sequencing of divergent African HIV type 1 subtype F viruses leads to the identification of a new HIV type 1 subtype designated K.

We recently reported a high divergence among African subtype F strains. Three well-separated groups (F1, F2, and F3) have been shown based on the phylogenetic analysis of the p24 gag and envelope sequences with genetic distances similar to those observed for known subtypes. In this study, we characterized the near-full-length genomes of two strains from epidemiological unlinked individual belonging to each of the subgroups: F1 (96FR-MP411), F2 (95CM-MP255 and 95CM-MP257), and F3 (96CM-MP535 and 97ZR-EQTB11). Phylogenetic analysis of the near-full-length sequences and for each of the genes separately showed the same three groups, supported by high bootstrap values. Diversity plotting, BLAST subtyping, and bootstrap plotting confirmed that the divergent F strains correspond to nonrecombinant viruses. The divergence between F1 and F2 is consistently lower than that seen in any other intersubtype comparison, with the exception of subtypes B and D. Based on all the different analyses, we propose to divide subtype F into two subclades, with F1 gathering the known subtype F strains from Brazil and Finland, and our African strain (96FR-MP411), and F2 containing the 95CM-MP255 and 95CM-MP257 strains from Cameroon. The F3 strains, 97ZR-EQTB11 from the Democratic Republic of Congo and 96CM-MP535 from Cameroon, meet the criteria of a new subtype designated as K. The equidistance of subtype K to the other subtypes of HIV-1 suggests that this subtype existed as long as the others, the lower distance between B and D, and between F1 and F2 suggest a more recent subdivision for these latter strains.

Africa↗