Nucleotide Substitution Process Research Articles

DNA Sequences Are as Useful as Protein Sequences for Inferring Deep Phylogenies.

Inference of deep phylogenies has almost exclusively used protein rather than DNA sequences based on the perception that protein sequences are less prone to homoplasy and saturation or to issues of compositional heterogeneity than DNA sequences. Here, we analyze a model of codon evolution under an idealized genetic code and demonstrate that those perceptions may be misconceptions. We conduct a simulation study to assess the utility of protein versus DNA sequences for inferring deep phylogenies, with protein-coding data generated under models of heterogeneous substitution processes across sites in the sequence and among lineages on the tree, and then analyzed using nucleotide, amino acid, and codon models. Analysis of DNA sequences under nucleotide-substitution models (possibly with the third codon positions excluded) recovered the correct tree at least as often as analysis of the corresponding protein sequences under modern amino acid models. We also applied the different data-analysis strategies to an empirical dataset to infer the metazoan phylogeny. Our results from both simulated and real data suggest that DNA sequences may be as useful as proteins for inferring deep phylogenies and should not be excluded from such analyses. Analysis of DNA data under nucleotide models has a major computational advantage over protein-data analysis, potentially making it feasible to use advanced models that account for among-site and among-lineage heterogeneity in the nucleotide-substitution process in inference of deep phylogenies.

Phylogenetic relationships among Sanguinolaria species in the coastal waters of China

PDF HTML阅读 XML下载 导出引用 引用提醒 中国沿海紫蛤属的系统发育 DOI: 作者: 作者单位: 中国海洋大学 海水养殖教育部重点实验室, 山东 青岛 266003 作者简介: 蒋丽莎(1991-),女,硕士研究生,主要从事贝类遗传育种研究.E-mail:957924763@qq.com 通讯作者: 中图分类号: S917 基金项目: 山东省重点研发计划项目（2016ZDJS06A06）；中央高校基本科研业务费专项（201762014）. Phylogenetic relationships among Sanguinolaria species in the coastal waters of China Author: Affiliation: Key Laboratory of Mariculture, Ministry of Education;Ocean University of China, Qingdao 266003, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:紫蛤属（）贝类具有很高的经济价值，长期以来却存在着物种鉴定错误、种名使用混乱、同物异名等一系列问题，关于其系统分类和演化问题的研究较少。为探究中国沿海紫蛤属的系统发育关系，本研究利用两个线粒体基因（COI和16S rRNA），以及两个核基因（H3和28S rRNA）的部分序列，对采自中国沿海紫蛤属3亚属9种紫蛤61个个体进行系统发育分析。基于4个基因片段联合数据集T12（剔除COI第三位密码子位置）构建系统发育树。结果表明，两个线粒体基因片段GC含量明显低于AT含量，表现出对AT偏斜，两个核基因表现出对GC的偏斜。4个基因片段（COI、16S rRNA、H3和28S rRNA）的颠换与转换比值分别为5.073、3.042、1.564和1.480，均远高于系统分析的临界值0.4，能够提供有效的系统发育信息。基于4个基因片段的遗传多样性分析表明，9种紫蛤均具有较高的核苷酸多样性（Pi<0.05）与单倍型多态性（Hd<0.5）。基于COI基因片段的紫蛤属种内遗传距离为0~0.016，种间的平均遗传距离为0.087~0.331，其中卵紫蛤（）遗传距离最小，仅为0.087。利用最大似然法（maximum likelihood，ML）和贝叶斯法（Bayesian inference，BI）构建的系统发生树拓扑结构一致，分化为3大支，与形态学分类的三个亚属分别对应。中国紫蛤和卵紫蛤在系统树上首先聚为一支，表明其亲缘关系最近。本研究结果阐明了中国沿海紫蛤属贝类的遗传多样性及其系统演化关系，为紫蛤属贝类种质资源的保护及可持续利用提供了科学依据。 Abstract:Fragments of the mitochondrial gene (COI and 16S rRNA) and the nuclear marker (H3 and 28S rRNA) regions are suitable for phylogenetic relationship analysis. The phylogenetic relationships among nine S. diphos, S. olivacea, and ) collected from the coastal areas of China was analyzed through the sequencing of COI, H3, 16S rRNA, and 28S rRNA genes. The two mitochondrial genes, COI 16S rRNA, had an obvious AT-skewness, and their GC content were 41.17% and 41.01%, respectively, showing a certain degree of base preference. The overall transition/transversion bias of the four fragments (COI 5.073, 16S rRNA 3.042, H3 1.564, and 28S rRNA 1.480) were much higher than the critical value (0.4), which could offer effective phylogenetic information. As the genetic diversity parameters of all four markers in nine species had higher values (Pi<0.05, Hd<0.5) than most bivalve species, it is suggested that the species may have long history in the localities, and sufficient phylogenetic information. Among the nine species, the genetic average distances based on the COI gene were 0.087-0.331, and the genetic distances within species were 0-0.016. The minimum genetic distance was between , which was much higher than the average value (0.005) between most species of bivalves. The saturation of the nucleotide substitution process in the four markers was calculated as well. The third codon positions of the COI gene's transition and transversion substitutions reached the point of saturation, which means that the third codon positions of the COI gene could not provide effective information. The combined data set T12, including the four molecular markers without the third codon positions of COI, totaled 1555 bp in length. Phylogenetic trees constructed by the Maximum Likelihood (ML) and Bayesian Inference (BI) methods were consistent, and cluster analysis based on the data set T12 showed three separate lineages, corresponding to three morphologically defined subgenera:. Combining morphological and molecular evidence, the results support that has the closest phylogenetic relationship. The study sheds light on the genetic diversity and phylogenetic relationship of species occurring along the coast of China, which may be useful in genetic resource protection and sustainable use. 参考文献 相似文献 引证文献

Testing evolutionary models to explain the process of nucleotide substitution in gut bacterial 16S rRNA gene sequences

The 16S rRNA gene has been widely used as a marker of gut bacterial diversity and phylogeny, yet we do not know the model of evolution that best explains the differences in its nucleotide composition within and among taxa. Over 46000 good-quality near-full-length 16S rRNA gene sequences from five bacterial phyla were obtained from the ribosomal database project (RDP) by study and, when possible, by within-study characteristics (e.g. anatomical region). Using alignments (RDPX and MUSCLE) of unique sequences, the FINDMODEL tool available at http://www.hiv.lanl.gov/ was utilized to find the model of character evolution (28 models were available) that best describes the input sequence data, based on the Akaike information criterion. The results showed variable levels of agreement (from 33% to 100%) in the chosen models between the RDP-based and the MUSCLE-based alignments among the taxa. Moreover, subgroups of sequences (using either alignment method) from the same study were often explained by different models. Nonetheless, the different representatives of the gut microbiota were explained by different proportions of the available models. This is the first report using evolutionary models to explain the process of nucleotide substitution in gut bacterial 16S rRNA gene sequences.

Bayesian Selection of Nucleotide Substitution Models and Their Site Assignments

Probabilistic inference of a phylogenetic tree from molecular sequence data is predicated on a substitution model describing the relative rates of change between character states along the tree for each site in the multiple sequence alignment. Commonly, one assumes that the substitution model is homogeneous across sites within large partitions of the alignment, assigns these partitions a priori, and then fixes their underlying substitution model to the best-fitting model from a hierarchy of named models. Here, we introduce an automatic model selection and model averaging approach within a Bayesian framework that simultaneously estimates the number of partitions, the assignment of sites to partitions, the substitution model for each partition, and the uncertainty in these selections. This new approach is implemented as an add-on to the BEAST 2 software platform. We find that this approach dramatically improves the fit of the nucleotide substitution model compared with existing approaches, and we show, using a number of example data sets, that as many as nine partitions are required to explain the heterogeneity in nucleotide substitution process across sites in a single gene analysis. In some instances, this improved modeling of the substitution process can have a measurable effect on downstream inference, including the estimated phylogeny, relative divergence times, and effective population size histories.

Computational Design of Centralized HIV-1 Genes

The extreme genetic diversity of the HIV-1 remains as a daunting challenge for the development of an effective AIDS vaccine. One strategy for creating a single vaccine that protects against the HIV-1 expanding population is to reconstruct centralized immunogenic sequences that minimize the genetic distance to circulating strains that the vaccine is targeting. Such centralized genes can be estimated with inferred consensus, ancestral and center-of-tree sequences. Although the increased breadth of antibody and T-cell responses induced by the centralized vaccines to date are encouraging, they are modest and may only be partly effective in combating HIV-1. One of the reasons of this limited success might be that several features of HIV-1 molecular evolution have not been yet taken into account in the design of these centralized vaccines, the most important likely being its high recombination rate and complex nucleotide substitution process. Here we describe evolutionary methodologies for the inference of centralized HIV-1 genes, with particular focus on the sources of error introduced by recombination and the model of evolution, in order to foster the development of more effective immunogens before synthesis and assessment in the lab, and final testing in AIDS vaccine trials.

Quantifying the Stationarity and Time Reversibility of the Nucleotide Substitution Process

Markov models describing the evolution of the nucleotide substitution process, widely used in phylogeny reconstruction, usually assume the hypotheses of stationarity and time reversibility. Although these models give meaningful results when applied to biological data, it is not clear if the 2 assumptions mentioned above hold and, if not, how much sequence evolution processes deviate from them. To this aim, we introduce 2 sets of indices that can be calculated from the nucleotide distribution and the substitution rates. The stationarity indices (STIs) can be used to test the validity of the equilibrium assumption. The irreversibility indices (IRIs) are derived from the Kolmogorov cycle conditions for time reversibility and quantify the degree of nontime reversibility of a process. We have computed STIs and IRIs for the evolutionary process of 2 lineages, Drosophila simulans and Homo sapiens. In the latter case, we use a modified form of the indices that takes into account the CpG decay process. In both cases, we find statistically significant deviations from the ideal case of a process that has reached stationarity and is time reversible.

Identification and measurement of neighbor-dependent nucleotide substitution processes

Neighbor-dependent substitution processes generated specific pattern of dinucleotide frequencies in the genomes of most organisms. The CpG-methylation-deamination process is, e.g. a prominent process in vertebrates (CpG effect). Such processes, often with unknown mechanistic origins, need to be incorporated into realistic models of nucleotide substitutions. Based on a general framework of nucleotide substitutions we developed a method that is able to identify the most relevant neighbor-dependent substitution processes, estimate their relative frequencies and judge their importance in order to be included into the modeling. Starting from a model for neighbor independent nucleotide substitution we successively added neighbor-dependent substitution processes in the order of their ability to increase the likelihood of the model describing given data. The analysis of neighbor-dependent nucleotide substitutions based on repetitive elements found in the genomes of human, zebrafish and fruit fly is presented. A web server to perform the presented analysis is freely available at: http://evogen.molgen.mpg.de/server/substitution-analysis

Should we use model-based methods for phylogenetic inference when we know that assumptions about among-site rate variation and nucleotide substitution pattern are violated?

An often-expressed concern regarding the use ofmaximum likelihoodandothermodelbased methods in phylogenetic analysis is that the assumedmodels are too simple, violating known aspects of sequence evolution in several ways (e.g., Sanderson and Kim, 2000). For example,we know that sites do not truly evolve independently, that the substitution process is not completely homogeneous across sites and through time, and that simple stochastic models fail to adequately represent the complexity of the nucleotide substitution process. Many advances have been made in an effort to model sequence evolution more realistically, including allowance for unequal base composition (Felsenstein, 1981), complex substitution patterns (e.g., Tavare, 1986; Yang, 1994), among-site rate variation (Yang, 1993), compensatory base substitution (Schoniger and Von Haeseler, 1994), and heterogeneity of the substitution process across the tree (Galtier and Gouy, 1998). Nevertheless, even with these improvements, there is no reason to believe that even the most general and parameter-rich evolutionarymodels currently available capture all the nuances of the processes that have generated any particular set of sequences. Furthermore, accurate estimation of the parameters of complex evolutionary models can be very difŽcult, anddata sets containing many taxa and sites may be needed for likelihood estimation to consistently yield reliable parameter estimates (Nielsen, 1997; Sullivan et al., 1999). Fortunately, perfect models are not necessarily a prerequisite for reliable statistical inference (e.g., Burnham and Andersen, 1998). Many statistical methods performwell in the face ofviolationof commondistributional assumptions such as normality (e.g., Boneau, 1960; Donaldson, 1968). However, this observation does not guarantee the robustness of maximum likelihood when applied to the phylogenetic estimation problem. It is therefore important to examine the impact of various model violations on the accuracy of phylogenetic estimation (e.g., Huelsenbeck, 1995). In some cases, biases introduced by violation of a model’s assumptions can actually favor recovery of the true tree rather than an incorrect tree (Yang, 1997; Siddall, 1998). For example, Yang (1997) simulated data under a Jukes–Cantor (JC) model with variable rates across sites (following a gamma distribution) and demonstrated that estimation under the JC model with equal rates could be more efŽcient than estimation in which among-site rate variationwas correctly modeled by a gamma distribution. However, the reasons for the improvement in efŽciency shouldbedisconcerting (Bruno andHalpern, 1999; Swofford et al., 2001). These results suggest that application of increasingly general and complex models would sometimes lead to decreased efŽciency, despite the fact that themore complex models almost always provide signiŽcantly better Žt to real data than the simplermodels. However, in Yang’s study, the improvement

Shared nucleotide composition biases among species and their impact on phylogenetic reconstructions of the Drosophilidae.

Compositional changes are a major feature of genome evolution. Overlooking nucleotide composition differences among sequences can seriously mislead phylogenetic reconstructions. Large compositional variation exists among the members of the family Drosophilidae. Until now, however, base composition differences have been largely neglected in the formulations of the nucleotide substitution process used to reconstruct the phylogeny of this important group of species. The present study adopts a maximum-likelihood framework of phylogenetic inference in order to analyze five nuclear gene regions and shows that (1) the pattern of compositional variation in the Drosophilidae does not match the phylogeny of the species; (2) accounting for the heterogeneous GC content with Galtier and Gouy's nucleotide substitution model leads to a tree that differs in significant aspects from the tree inferred when the nucleotide composition differences are ignored, even though both phylogenetic hypotheses attain strong nodal support in the bootstrap analyses; and (3) the LogDet distance correction cannot completely overcome the distorting effects of the compositional variation that exists among the species of the Drosophilidae. Our analyses confidently place the Chymomyza genus as an outgroup closer than the genus Scaptodrosophila to the Drosophila genus and conclusively support the monophyly of the Sophophora subgenus.

Polytomies, the power of phylogenetic inference, and the stochastic nature of molecular evolution: a comment on Walsh et al. (1999).

Walsh et al. (1999) recently suggested an innovative application of power analysis (Cohen 1977) to explore the nature of polytomies (multifurcating rather than bifurcating relationships) in phylogenetic inference. The frequent recovery of polytomies in phylogenetic analyses has prompted substantial interest in the underlying biological nature of these inferred polytomies. Many authors assume that polytomies simply reflect the inability to resolve bifurcating relationships (soft polytomies) and suggest that additional data or improved analyses will allow the recovery of the underlying relationships (e.g., Maddison 1989; DeSalle et al. 1994). In contrast, other researchers consider polytomous relationships to be valid phylogenetic hypotheses reflecting multiple simultaneous speciation events (a polytomy; Hoelzer and Melnick 1994a,b). Examining the differences between these alternative viewpoints is extremely difficult, because soft polytomies do not exhibit clear differences from hard polytomies in phylogenetic analyses. Specifically, Walsh et al. (1999) sought to determine whether internal branch lengths which were not significantly different from zero simply reflect an insufficient sample size (amount of sequence data). The example developed by Walsh et al. (1999) involved the polytomous relationships among auklets (Charadriiformes: Alcidae) inferred using mitochondrial DNA sequences. Since this inferred polytomy might reflect a soft polytomy (defined by Walsh et al. [1999] as speciation during successive glacial/interglacial periods during the late Pliocene and early Pleistocene) or a hard polytomy (defined as multiple speciations during the same period of climatic oscillation), Walsh et al. (1999) used power analysis to determine whether the number of base pairs (sites) of sequence data obtained was sufficiently large to detect substitutions along the internal branches if the speciation was not simultaneous (a soft polytomy). We found this an innovative application of classical power analysis and an excellent alternative to Monte Carlo simulation (e.g., Saitou and Nei 1986; Hillis et al. 1994; Huelsenbeck et al. 1996). We believe that an extremely important aspect of the work by Walsh et al. (1999) is their clear statement of the alternative and null hypotheses concerning the differentiation between hard and soft polytomies. As they point out, of the biological reality of polytomies is complicated by the fact that a polytomy represents the null hypothesis for phylogenetic reconstruction-all taxa are equally relatedand therefore cannot be proven. By restating the problem in the context of the power necessary to detect short internal branches, Walsh et al. (1999) place the hypothesis of a hard polytomy in a much more testable context. However, we were surprised to find that power analysis suggests only 215 to 1237 base pairs of mitochondrial sequence data are required for resolution of a soft polytomy in the auklets, as defined by Walsh et al. (1999). To further explore the study of polytomies in a statistical context, we examined the implications of the inherent variance exhibited by the nucleotide substitution process. It is clear that molecular evolution is a stochastic process with the number, type, and position of fixed sequence differences being determined by the processes of mutation, selection, and genetic drift. The stochastic nature of molecular evolution may have a profound impact upon the number of differences observed during a specific time period and it is possible that many fewer (or many more) mutations will be fixed during a particular period of time than one expects based upon the total number of substitutions in the phylogenetic tree. Nucleotide substitution can be modeled as a Poisson process (e.g., Wilson et al. 1987), with the probability (P) of N nucleotide substitutions occurring during a given period of time given by:

Evidence for a high ancestral GC content in Drosophila.

Study of the nucleotide composition in Drosophila, focusing on the saltans and willistoni groups, has revealed unanticipated differences in nucleotide composition among lineages. Compositional differences are associated with an accelerated rate of nucleotide substitution in functionally less constrained regions. These observations have been set forth against the extended opinion that the pattern of point mutation has remained constant during the evolution of the genus. A crucial assumption has been that the most recent common ancestor of the subgenus Sophophora had an elevated GC content. Until now, this assumption has been supported by indirect arguments, consisting of extrapolations from closely related outgroups and limited by the robustness of mathematical descriptions concerning the extensive nucleotide composition differences among sequences. The present study seeks to test the assumption of a high ancestral GC content using realistic representations of the nucleotide substitution process to account for potential biases induced by the heterogeneous GC content of the taxa. The analysis of eight nuclear genes unambiguously corroborates that the common ancestor of Sophophora had an elevated GC content.

Phylogenetic relationships among the phrynosomatid sand lizards inferred from mitochondrial DNA sequences generated by heterogeneous evolutionary processes.

Nucleotide sequences of the mitochondrial protein coding cytochrome b (cyt b; 650 bp) and small-subunit 12S ribosomal RNA (approximately 350 bp) genes were used in analyses of phylogenetic relationships among extant phrynosomatid sand lizards, including an examination of competing hypotheses regarding the evolution of "earlessness." Sequences were obtained from all currently recognized species of sand lizards as well as representatives of the first and second outgroups and analyzed using both parsimony and likelihood methods. The cyt b data offer strong support for relationships that correspond with relatively recent divergences and moderate to low support for relationships reflecting more ancient divergences within the clade. These data support monophyly of the "earless" taxa, the placement of Uma as the sister taxon to the other sand lizards, and monophyly of all four taxa traditionally ranked as genera. All well-supported relationships in the 12S phylogeny are completely congruent with well-supported relationships in the cyt b phylogeny; however, the 12S data alone provide very little support for deeper divergences. Phylogenetic relationships within species are concordant with geography and suggest patterns of phylogeographic differentiation, including the conclusion that at least one currently recognized species (Holbrookia maculata) actually consists of more than one species. By independently optimizing likelihood model parameters for various subsets of the data, we found that nucleotide substitution processes vary widely between genes and among the structural and functional regions or classes of sites within each gene. Therefore, we compared competing phylogenetic hypotheses, using parameter estimates specific to those subsets, analyzing the subsets separately and in various combinations. The hypothesis supported by the cyt b data was favored over rival hypotheses in all but one of the five comparisons made with the entire data set, including the set of partitions that best explained the data, although we were unable to confidently reject (P < 0.05) alternative hypotheses. Our results highlight the importance of optimizing models and parameter estimates for different genes or parts thereof--a strategy that takes advantages of the strengths of both combining and partitioning data.

Comparative Genomics of Mitochondrial DNA in Members of the Drosophila melanogaster Subgroup

In this study, a comparative genomics approach is employed to investigate the forces that shape evolutionary change in the mitochondrial DNA (mtDNA) of members of the Drosophila melanogaster subgroup. This approach facilitates differentiation of the patterns of variation resulting from processes acting at a higher level from those acting on a single gene. The mitochondrial genomes of three isofemale lines of D. simulans (siI, -II, and -III), two of D. melanogaster (Oregon R and a line from Zimbabwe), and D. mauritiana (maI and -II), and one of D. sechellia were sequenced and compared with that derived from D. yakuba. Data presented here indicate that at least three broad mechanisms shape the evolutionary dynamics of mtDNA in these taxa. The first set of mechanisms is intrinsic to the molecule. Dominant processes may be interpreted as selection for an increased rate of replication of the mtDNA molecule, biases in DNA repair, and differences in the pattern of nucleotide substitution among strands. In the genes encoded on the major strand (62% of the coding DNA) changes to or from C predominate, whereas on the minor changes to or from G predominate. The second set of mechanisms affects distinct lineages. There are evolutionary rate differences among lineages, possibly owing to population demographic changes or changes in mutational biases. This is supported by the heterogeneity found in synonymous, nonsynonymous, and silent substitutions. The third set of mechanisms differentially affects distinct genes. A maximum-likelihood sliding-window analysis detected four disjunct regions that have a significantly different nucleotide substitution process from that derived from the complete sequence. These data show the potential for comparative genomics to tease apart subtle forces that shape the evolution of DNA.

Molecular evolution and phylogeny of the buzzatii complex (Drosophila repleta group): a maximum-likelihood approach.

The buzzatii complex of the mulleri subgroup (Drosophila repleta group) consists of three clusters of species whose evolutionary relationships are poorly known. We analyzed 2,085 coding nucleotides from the xanthine dehydrogenase (XDH:) gene in the 10 available species of the complex and Drosophila mulleri and Drosophila hydei. We adopted a statistical model-fitting approach within the maximum-likelihood (ML) framework of phylogenetic inference. We first modeled the process of nucleotide substitution using a tree topology which was reasonably accurate. Then we used the most satisfactory description so attained to reconstruct the evolutionary relationships in the buzzatii complex. We found that a minimally realistic description of the substitution process of XDH: should allow six substitution types and different substitution rates for codon positions. Using this description we obtained a strongly supported, fully resolved tree which is congruent with the already-known (yet few) relationships. We also analyzed published data from three mitochondrial cytochrome oxidases (CO I, II, and III). In our analyses, these relatively short DNA sequences failed to discriminate statistically among alternative phylogenies. When the data of these three gene regions are combined with the XDH: sequences, the phylogenetic signal emerging from XDH: becomes reinforced. All four of the gene regions evolve faster in the buzzatii and martensis clusters than in the stalkeri cluster, paralleling the amount of chromosomal evolution.

Examining rates and patterns of nucleotide substitution in plants.

Driven by rapid improvements in affordable computing power and by the even faster accumulation of genomic data, the statistical analysis of molecular sequence data has become an active area of interdisciplinary research. Maximum likelihood methods have become mainstream because of their desirable properties and, more importantly, their potential for providing statistically sound solutions in complex data analysis settings. In this chapter, a review of recent literature focusing on rates and patterns of nucleotide substitution rates in the nuclear, chloroplast, and mitochondrial genomes of plants demonstrates the power and flexibility of these new methods. The emerging picture of the nucleotide substitution process in plants is a complex one. Evolutionary rates are seen to be quite variable, both among genes and among plant lineages. However, there are hints, particularly in the chloroplast, that individual factors can have important effects on many genes simultaneously.

A likelihood method for the detection of selection and recombination using nucleotide sequences.

Different regions along nucleotide sequences are often subject to different evolutionary forces. Recombination will result in regions having different evolutionary histories, while selection can cause regions to evolve at different rates. This paper presents a statistical method based on likelihood for detecting such processes by identifying the regions which do not fit with a single phylogenetic topology and nucleotide substitution process along the entire sequence. Subsequent reanalysis of these anomalous regions may then be possible. The method is tested using simulations, and its application is demonstrated using the primate psi eta-globin pseudogene, the V3 region of the envelope gene of HIV-1, and argF sequences from Neisseria bacteria. Reanalysis of anomalous regions is shown to reveal possible immune selection in HIV-1 and recombination in Neisseria. A computer program which implements the method is available.

Patterns of Nucleotide Substitution in Mitochondrial Protein Coding Genes of Vertebrates

Maximum likelihood methods were used to study the differences in substitution rates among the four nucleotides and among different nucleotide sites in mitochondrial protein-coding genes of vertebrates. In the 1st + 2nd codon position data, the frequency of nucleotide G is negatively correlated with evolutionary rates of genes, substitution rates vary substantially among sites, and the transition/transversion rate bias (R) is two to five times larger than that expected at random. Generally, largest transition biases and greatest differences in substitution rates among sites are found in the highly conserved genes. The 3rd positions in placental mammal genes exhibit strong nucleotide composition biases and the transitional rates exceed transversional rates by one to two orders of magnitude. Tamura-Nei and Hasegawa-Kishino-Yano models with gamma distributed variable rates among sites (gamma parameter, alpha) adequately describe the nucleotide substitution process in 1st+2nd position data. In these data, ignoring differences in substitution rates among sites leads to largest biases while estimating substitution rates. Kimura's two-parameter model with variable-rates among sites performs satisfactorily in likelihood estimation of R, alpha, and overall amount of evolution for 1st+2nd position data. It can also be used to estimate pairwise distances with appropriate values of alpha for a majority of genes.

Phylogenetic analysis using parsimony and likelihood methods.

The assumptions underlying the maximum-parsimony (MP) method of phylogenetic tree reconstruction were intuitively examined by studying the way the method works. Computer simulations were performed to corroborate the intuitive examination. Parsimony appears to involve very stringent assumptions concerning the process of sequence evolution, such as constancy of substitution rates between nucleotides, constancy of rates across nucleotide sites, and equal branch lengths in the tree. For practical data analysis, the requirement of equal branch lengths means similar substitution rates among lineages (the existence of an approximate molecular clock), relatively long interior branches, and also few species in the data. However, a small amount of evolution is neither a necessary nor a sufficient requirement of the method. The difficulties involved in the application of current statistical estimation theory to tree reconstruction were discussed, and it was suggested that the approach proposed by Felsenstein (1981, J. Mol. Evol. 17: 368-376) for topology estimation, as well as its many variations and extensions, differs fundamentally from the maximum likelihood estimation of a conventional statistical parameter. Evidence was presented showing that the Felsenstein approach does not share the asymptotic efficiency of the maximum likelihood estimator of a statistical parameter. Computer simulations were performed to study the probability that MP recovers the true tree under a hierarchy of models of nucleotide substitution; its performance relative to the likelihood method was especially noted. The results appeared to support the intuitive examination of the assumptions underlying MP. When a simple model of nucleotide substitution was assumed to generate data, the probability that MP recovers the true topology could be as high as, or even higher than, that for the likelihood method. When the assumed model became more complex and realistic, e.g., when substitution rates were allowed to differ between nucleotides or across sites, the probability that MP recovers the true topology, and especially its performance relative to that of the likelihood method, generally deteriorates. As the complexity of the process of nucleotide substitution in real sequences is well recognized, the likelihood method appears preferable to parsimony. However, the development of a statistical methodology for the efficient estimation of the tree topology remains a difficult open problem.

Specific compositional patterns of synonymous positions in homologous mammalian genes

All 69 homologous coding sequences that are currently available in four mammalian orders were aligned and the synonymous (ie., third) positions of quartet (fourfold degenerate) codons were divided into three classes (that will be called conserved, intermediate, and variable), according to whether they show no change, one change, and more than one change, respectively. The three classes were analyzed in their compositional patterns. In the majority of GC-rich genes, the three classes of positions (but especially conserved positions) exhibited significantly different base compositions compared to expectations based on a "random" substitution process from the "ancestral" (consensus) sequence to the present-day (actual) sequences. Significant differences were rare in GC-poor genes. An analysis of the present results indicates that natural selection plays a role in the synonymous nucleotide substitution process, especially in GC-rich genes which represent the vast majority of mammalian genes.

Weak male-driven molecular evolution in rodents.

In humans and rodents the male-to-female ratio of mutation rate (alpha m) has been suggested to be extremely large, so that the process of nucleotide substitution is almost completely male-driven. However, our sequence data from the last intron of the X chromosome-linked (Zfx) and Y chromosome-linked (Zfy) zinc finger protein genes suggest that alpha m is only approximately 2 in rodents with a 95% confidence interval from 1 to 3. Moreover, from published data on oogenesis and spermatogenesis we estimate the male-to-female ratio of the number of germ cell divisions per generation to be approximately 2 in rodents, confirming our estimate of alpha m and suggesting that errors in DNA replication are the primary source of mutation. As the estimated alpha m for rodents is only one-third of our previous estimate of approximately 6 for higher primates, there appear to be generation-time effects--i.e., alpha m decreases with decreasing generation time.