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A 40-year-old man with a left frontotemporal grade II oligodendroglioma developed seizures that were refractory to 14 antiepileptic medications, the ketogenic diet, and epilepsy surgery. With temozolomide therapy, his seizure frequency gradually changed from 30 partial seizures per day to a single simple partial seizure in 6 months. No additional therapeutic measures were introduced during this time. This reduction in seizure frequency appears attributable solely to temozolomide therapy.

The natural killer (NK) receptor gene complex (NKC) encodes a large number of C-type lectin-like receptors, which are expressed on NK and other immune-related cells. These receptors play an important role in regulating NK-cell cytolytic activity, protecting cells against virus infection and tumorigenesis. To understand the evolutionary history of the NKC, we characterized the C-type lectin-like NKC genes and their organization from four major orders of placental mammals, primates (human), rodents (mouse and rat), carnivores (dog), and artiodactyls (cattle) and then conducted phylogenetic analysis of these genes. The results indicate that the NKC of placental mammals is highly heterogeneous in terms of the gene content and rates of birth and death of different gene lineages, but the NKC is also remarkably conserved in its gene organization and persistence of orthologous gene lineages. Among the 28 identified NKC gene lineages, 4, KLRA1, KLRB1, CLEC2D, and CLEC4A/B/C, have expanded rapidly in rodents only. The high birth and death rate of these 4 gene families might be due to functional differentiation driven by positive selection. Identification of putative NKC sequences in opossum and chicken genomes implies that the expansion of the NKC gene families might have occurred before the radiation of placental mammals but after the divergence of birds from mammals.

We investigated the value of non-invasive data for predicting the outcome of intracranial EEG and anterior temporal lobectomy (ATL) (follow-up>1 year) in patients who have bitemporal independent seizures in the scalp EEG. No previous report has dealt with this patient group. Independent variables were duration of epilepsy, febrile seizures, interictal and ictal scalp EEG, ictal behavior, MRI, [18F]-fluorodeoxyglucose-PET (PET) and Wada test and dependent variables were surgical outcome (seizure free or not) and localized on intracranial EEG (finding all symptomatic seizures from one temporal lobe). Non-parametric statistics were used. Of 24 patients, 20 patients had IEEG, of which 12 were localized and 8 were not. Sixteen patients had ATL and, of these, 13 (81%) became seizure free and the remaining three improved. Lateralized findings on MRI and PET, a history of febrile convulsions and shorter duration of epilepsy were all associated with a focal onset on intracranial EEG, while there was a non-significant trend with ictal behavior. The non-invasive data did not predict surgical outcome. We conclude that some of these patients can do well with surgery. In most cases, intracranial EEG is necessary for localization of seizure focus, but if PET and MRI show focal abnormalities and there is a history of febrile convulsions no further evaluation could be needed. These findings need confirmation.

Purpose: The vagal nerve stimulator (VNS) and corpus callosotomy can reduce seizure frequency when seizures are refractory to medications. However, the efficacy and safety of these two procedures have not been compared. This study evaluates the two procedures for generalized seizures., Methods: All patients with refractory generalized seizures (generalized tonic-clonic, tonic, or atonic) who underwent a corpus callosotomy (anterior or complete) (n = 53) without other forms of epilepsy surgery and those who underwent VNS placement (n = 25) were evaluated for this study. Seizure response and procedure complications were evaluated., Results: For those with a corpus callosotomy and generalized tonic-clonic seizures (n = 50), 79.5% had >or=50% decrease in the frequency of generalized tonic-clonic seizures, and 60% had >or=80% seizure reduction. For those with a VNS and generalized tonic-clonic seizures (n = 21), 50% had >or=50% seizure reduction, and 33% had >or=80% seizure reduction. Tonic and atonic seizures decreased after either VNS or a corpus callosotomy. The complication rate for corpus callosotomy was higher (21% all complications, 3.8% permanent) than that for VNS (8%; none permanent), but complications for both corpus callosotomy and VNS were rarely permanent., Conclusions: Both corpus callosotomy and VNS are effective in reducing generalized seizures. Corpus callosotomy is associated with greater efficacy but higher risk for complications, although these were generally transient.

The numbers of functional olfactory receptor (OR) genes in humans and mice are about 400 and 1,000 respectively. In both humans and mice, these genes exist as genomic clusters and are scattered over almost all chromosomes. The difference in the number of genes between the two species is apparently caused by massive inactivation of OR genes in the human lineage and a substantial increase of OR genes in the mouse lineage after the human-mouse divergence. Compared with mammals, fishes have a much smaller number of OR genes. However, the OR gene family in fishes is much more divergent than that in mammals. Fishes have many different groups of genes that are absent in mammals, suggesting that the mammalian OR gene family is characterized by the loss of many group genes that existed in the ancestor of vertebrates and the subsequent expansion of specific groups of genes. Therefore, this gene family apparently changed dynamically depending on the evolutionary lineage and evolved under the birth-and-death model of evolution. Study of the evolutionary changes of two gene families for vomeronasal receptors and two gene families for taste receptors, which are structurally similar, but remotely related to OR genes, showed that some of the gene families evolved in the same fashion as the OR gene family. It appears that the number and types of genes in chemosensory receptor gene families have evolved in response to environmental needs, but they are also affected by fortuitous factors.

It has been known that the conservation or diversity of homeobox genes is responsible for the similarity and variability of some of the morphological or physiological characters among different organisms. To gain some insights into the evolutionary pattern of homeobox genes in bilateral animals, we studied the change of the numbers of these genes during the evolution of bilateral animals. We analyzed 2,031 homeodomain sequences compiled from 11 species of bilateral animals ranging from Caenorhabditis elegans to humans. Our phylogenetic analysis using a modified reconciled-tree method suggested that there were at least about 88 homeobox genes in the common ancestor of bilateral animals. About 50-60 genes of them have left at least one descendant gene in each of the 11 species studied, suggesting that about 30-40 genes were lost in a lineage-specific manner. Although similar numbers of ancestral genes have survived in each species, vertebrate lineages gained many more genes by duplication than invertebrate lineages, resulting in more than 200 homeobox genes in vertebrates and about 100 in invertebrates. After these gene duplications, a substantial number of old duplicate genes have also been lost in each lineage. Because many old duplicate genes were lost, it is likely that lost genes had already been differentiated from other groups of genes at the time of gene loss. We conclude that both gain and loss of homeobox genes were important for the evolutionary change of phenotypic characters in bilateral animals.

Olfaction, which is an important physiological function for the survival of mammals, is controlled by a large multigene family of olfactory receptor (OR) genes. Fishes also have this gene family, but the number of genes is known to be substantially smaller than in mammals. To understand the evolutionary dynamics of OR genes, we conducted a phylogenetic analysis of all functional genes identified from the genome sequences of zebrafish, pufferfish, frogs, chickens, humans, and mice. The results suggested that the most recent common ancestor between fishes and tetrapods had at least nine ancestral OR genes, and all OR genes identified were classified into nine groups, each of which originated from one ancestral gene. Eight of the nine group genes are still observed in current fish species, whereas only two group genes were found from mammalian genomes, showing that the OR gene family in fishes is much more diverse than in mammals. In mammals, however, one group of genes, gamma, expanded enormously, containing approximately 90% of the entire gene family. Interestingly, the gene groups observed in mammals or birds are nearly absent in fishes. The OR gene repertoire in frogs is as diverse as that in fishes, but the expansion of group gamma genes also occurred, indicating that the frog OR gene family has both mammal- and fish-like characters. All of these observations can be explained by the environmental change that organisms have experienced from the time of the common ancestor of all vertebrates to the present.

In mammals many natural killer (NK) cell receptors, encoded by the leukocyte receptor complex (LRC), regulate the cytotoxic activity of NK cells and provide protection against virus-infected and tumor cells. To investigate the origin of the Ig-like domains encoded by the LRC genes, a subset of C2-type Ig-like domain sequences was compiled from mammals, birds, amphibians, and fish. Phylogenetic analysis of these sequences generated seven monophyletic groups in mammals (MI, MII, and FcI, FcIIa, FcIIb, FcIII, FcIV), two in chicken (CI, CII), four in frog (FI-FIV), and five in zebrafish (ZI-ZV). The analysis of the major groups supported the following order of divergence: ZI [or a common ancestor of ZI and F (a cluster composed of the FcIII and FIII groups)], F, CII (or a common ancestor of CII and MII), MII, and MI-CI. The relationships of the remaining groups were unclear, since the phylogenetic positions of these groups were not supported by high bootstrap values. Two main conclusions can be drawn from this analysis. First, the two groups of mammalian LRC sequences must diverged before the separation of the avian and mammalian lineages. Second, the mammalian LRC sequences are most closely related to the Fc receptor sequences and these two groups diverged before the separation of birds and mammals.

In mammals, the cell surface receptors encoded by the leukocyte receptor complex (LRC) regulate the activity of T lymphocytes and B lymphocytes, as well as that of natural killer cells, and thus provide protection against pathogens and parasites. The chicken genome encodes many Ig-like receptors that are homologous to the LRC receptors. The chicken Ig-like receptor (CHIR) genes are members of a large monophyletic gene family and are organized into genomic clusters, which are in conserved synteny with the mammalian LRC. One-third of CHIR genes encode polypeptide molecules that contain both activating and inhibitory motifs. These genes are present in different phylogenetic groups, suggesting that the primordial CHIR gene could have encoded both types of motifs in a single molecule. In contrast to the mammalian LRC genes, the CHIR genes with similar function (inhibition or activation) are evolutionarily closely related. We propose that, in addition to recombination, single nucleotide substitutions played an important role in the generation of receptors with different functions. Structural models and amino acid analyses of the CHIR proteins reveal the presence of different types of Ig-like domains in the same phylogenetic groups, as well as sharing of conserved residues and conserved changes of residues between different CHIR groups and between CHIRs and LRCs. Our data support the notion that the CHIR gene clusters are regions homologous to the mammalian LRC gene cluster and favor a model of evolution by repeated processes of birth and death (expansion-contraction) of the Ig-like receptor genes.

The gene family of killer cell immunoglobulin-like receptors (KIRs) in primates provides the first line of defense against virus infection and tumor transformation. Interacting with MHC class I molecules, KIRs can regulate the cytotoxic activity of natural killer (NK) cells and distinguish the tumor and virus infected cells from normal body cells. Phylogenetic analysis and comparison of domain structures identified three major groups of KIR genes (group I, II, and III genes). These groups of KIR genes, generated by a series of gene duplications, have acquired different MHC-binding specificity. Inference of ancestral KIR sequences suggested that the functional divergence of group I genes from group II genes occurred by positive selection at the MHC-binding sites after duplication. Our evolutionary study has shown that group I genes diverged from group II genes about 17 million years ago (Mya) apparently after separation of hominoids from Old World (OW) monkeys. Around the same time, gene duplication generating the class I MHC-C locus appears to have occurred. These findings suggest that KIR and MHC class I genes have coevolved as an interacting system. The KIR gene family has experienced a rapid expansion in primate species. The rate of expansion of this gene family seems to be one of the highest among all hominoid gene families. The KIR gene family is also subject to birth-and-death evolution.

The chimpanzee is our closest living relative. The morphological differences between the two species are so large that there is no problem in distinguishing between them. However, the nucleotide difference between the two species is surprisingly small. The early genome comparison by DNA hybridization techniques suggested a nucleotide difference of 1-2%. Recently, direct nucleotide sequencing confirmed this estimate. These findings generated the common belief that the human is extremely close to the chimpanzee at the genetic level. However, if one looks at proteins, which are mainly responsible for phenotypic differences, the picture is quite different, and about 80% of proteins are different between the two species. Still, the number of proteins responsible for the phenotypic differences may be smaller since not all genes are directly responsible for phenotypic characters.

The numbers of functional olfactory receptor (OR) genes are quite variable among mammalian species. Previously we have reported that humans have 388 functional OR genes and 414 pseudogenes, while mice have 1037 functional genes and 354 pseudogenes. These observations suggest either that humans lost many functional OR genes after the human-mouse divergence (HMD) or that mice gained many functional genes. To distinguish between these two hypotheses, we devised a new method of inferring the number of functional OR genes in the most recent common ancestor (MRCA) of humans and mice. An application of this method suggested that the MRCA had approximately 750 functional OR genes and that mice acquired approximately 350 new OR genes after the HMD whereas approximately 430 OR genes in the MRCA have become pseudogenes or eliminated in the human lineage. Therefore, the two evolutionary hypotheses mentioned above are not mutually exclusive and both are nearly equally responsible for the difference in the number of OR genes between humans and mice.

Olfactory receptor (OR) genes form the largest multigene family in mammalian genomes. Humans have approximately 800 OR genes, but >50% of them are pseudogenes. By contrast, mice have approximately 1400 OR genes and pseudogenes are approximately 25%. To understand the evolutionary processes that shaped the difference of OR gene families between humans and mice, we studied the genomic locations of all human and mouse OR genes and conducted a detailed phylogenetic analysis using functional genes and pseudogenes. We identified 40 phylogenetic clades with high bootstrap supports, most of which contain both human and mouse genes. Interestingly, a particular clade contains approximately 100 pseudogenes in humans, whereas the numbers of pseudogenes are <20 for most of the mouse clades. We also found that the organization of OR genomic clusters is well conserved between humans and mice in many chromosomal locations. Despite the difference in the numbers of genes, the numbers of large genomic clusters are nearly the same for humans and mice. These observations suggest that the greater OR gene repertoire in mice has been generated mainly by tandem gene duplication within each genomic cluster.

A simple statistical method for predicting the functional differentiation of duplicate genes was developed. This method is based on the premise that the extent of functional differentiation between duplicate genes is reflected in the difference in evolutionary rate because the functional change of genes is often caused by relaxation or intensification of functional constraints. With this idea in mind, we developed a window analysis of protein sequences to identify the protein regions in which the significant rate difference exists. We applied this method to MIKC-type MADS-box proteins that control flower development in plants. We examined 23 pairs of sequences of floral MADS-box proteins from petunia and found that the rate differences for 14 pairs are significant. The significant rate differences were observed mostly in the K domain, which is important for dimerization between MADS-box proteins. These results indicate that our statistical method may be useful for predicting protein regions that are likely to be functionally differentiated. These regions may be chosen for further experimental studies.

Until around 1990, most multigene families were thought to be subject to concerted evolution, in which all member genes of a family evolve as a unit in concert. However, phylogenetic analysis of MHC and other immune system genes showed a quite different evolutionary pattern, and a new model called birth-and-death evolution was proposed. In this model, new genes are created by gene duplication and some duplicate genes stay in the genome for a long time, whereas others are inactivated or deleted from the genome. Later investigations have shown that most non-rRNA genes including highly conserved histone or ubiquitin genes are subject to this type of evolution. However, the controversy over the two models is still continuing because the distinction between the two models becomes difficult when sequence differences are small. Unlike concerted evolution, the model of birth-and-death evolution can give some insights into the origins of new genetic systems or new phenotypic characters.

Mortality rates are higher in people with refractory epilepsy than in the general population. We assessed mortality rates in a prospectively followed cohort who had epilepsy surgery, to examine the factors related to mortality and to assess the relationship between seizure control and mortality. Five hundred eighty-three patients were evaluated. Mortality was strongly related to seizure control (p = 0.001), with 18 deaths observed in patients with recurrent seizures (mortality rate = 11.4 per 1,000 person-years) and 1 death in patients with no recurrent seizures (mortality rate = 0.85 deaths per 1,000 person-years). Patients with generalized epilepsy who had corpus callosotomy had a higher mortality rate than patients who had resective or transective surgery. The side of surgery and gender did not influence mortality rates. The standardized mortality ratio was 5.75 for patients with recurrent seizures and was significantly higher for females than males. These data show that the excess mortality associated with refractory epilepsy is eliminated after epilepsy surgery when seizures are abolished and suggest that epilepsy surgery reduces the risk of epilepsy-associated death.

Background: Most paraneoplastic pemphigus (PNP) cases reported to date have been associated with lymphoproliferative neoplasms. Patients with PNP have autoantibodies against the plakin family (eg, envoplakin and periplakin). Antibodies against desmoglein 3 (Dsg3) and Dsg1, antigens for classic types of pemphigus, have also been reported to play an important role in the initial stage of PNP., Observations: We describe a patient with PNP associated with follicular dendritic cell sarcoma. Antibodies to envoplakin and periplakin were detected. When only mucosal lesions were observed at the early stage, the antibody to Dsg3 but not to Dsg1 was detected by enzyme-linked immunosorbent assay. After skin lesions appeared, antibodies to Dsg1 and Dsg3 were detected. These titers were elevated, with exacerbation of skin lesions. Although the patient received corticosteroid therapy, double-filtration plasmapheresis, and intravenous human immunoglobulin therapy after surgical resection of follicular dendritic cell sarcoma, she died of fungal infective lung embolisms. A direct immunofluorescence study of autopsy samples showed IgG deposition in the epidermis of the skin and oral mucosal membrane, but not in the lungs and kidneys and follicular dendritic cell sarcoma of the para-aortic area., Conclusion: In this patient with PNP and follicular dendritic cell sarcoma, there was an association between the clinical phenotype and the anti-Dsg antibody profile, as seen in pemphigus vulgaris.

Purpose: Temporal lobe epilepsy patients are well known to present deficits on explicit verbal memory procedures (e.g., recall, recognition). The integrity of implicit memory procedures in these patients is not established. Previous studies in this area used implicit memory measures contaminated by the effects of explicit memory., Methods: We examined the integrity of verbal implicit and explicit memory in left temporal lobe epilepsy (LTLE) patients and hypothesized that a clear dissociation in performance would be found with a relative preservation of implicit memory. TLE patients (n = 15) and age- and education-matched healthy normal patients (n = 15) were shown a 40-word study list, followed by a test phase requiring completion of word stems based on the study words or new/unseen words. Experimental conditions involved instructions to provide either the old (study) words or novel/nonlist words when completing the stem. Measures of automaticity and recollection provided uncontaminated indices of implicit and explicit memory, respectively., Results: The data showed a significant difference (p < 0.001) between the patients (Recollection, 0.12; SD, 0.18) and controls (0.50, SD, 0.15) on the measure of explicit memory. In contrast, the patients (Automaticity, 0.51; SD, 0.11) and controls (0.45, SD, 0.18) performed similarly on the implicit memory measure, with patient scores clearly at normative levels based on other Process Dissociation Procedure data., Conclusions: The data demonstrate the integrity of implicit memory in LTLE patients. Finding a dissociation between the two forms of verbal memory in LTLE patients provides evidence that they rely on different neuroanatomic systems., (Copyright 2004 International League Against Epilepsy)

Ly49 genes regulate the cytotoxic activity of natural killer (NK) cells in rodents and provide important protection against virus-infected or tumor cells. About 15 Ly49 genes have been identified in mice, but only a few genes have been reported to date in rats. Here we studied all Ly49 genes in the entire rat genome sequence and identified 17 putative functional and 16 putative non-functional genes together with their genomic locations in a 1.8-Mb region of chromosome 4. Phylogenetic analysis of these genes indicated that the Ly49 gene family expanded rapidly in recent years, and this expansion was mediated by both tandem and genomic block duplication. The joint phylogenetic analysis of mouse and rat genes suggested that the most recent common ancestor of the two species had at least several Ly49 genes, but that the majority of current duplicate genes were generated after divergence of the two species. In both species Ly49 genes are apparently subject to birth-and-death evolution, but the birth and death rates of Ly49 genes are higher in rats than in mice. The rate of gene expansion in the Ly49 gene family in rats is one of the highest among all mammalian multigene families so far studied. The biochemical function of Ly49 genes is essentially the same as that of KIR genes in primates, but the molecular structures of the two groups of NK cell receptors are very different. A hypothesis was presented to explain the origin of the differential use of Ly49 and KIR genes in rodents and primates.

Current efforts to reconstruct the tree of life and histories of multigene families demand the inference of phylogenies consisting of thousands of gene sequences. However, for such large data sets even a moderate exploration of the tree space needed to identify the optimal tree is virtually impossible. For these cases the neighbor-joining (NJ) method is frequently used because of its demonstrated accuracy for smaller data sets and its computational speed. As data sets grow, however, the fraction of the tree space examined by the NJ algorithm becomes minuscule. Here, we report the results of our computer simulation for examining the accuracy of NJ trees for inferring very large phylogenies. First we present a likelihood method for the simultaneous estimation of all pairwise distances by using biologically realistic models of nucleotide substitution. Use of this method corrects up to 60% of NJ tree errors. Our simulation results show that the accuracy of NJ trees decline only by approximately 5% when the number of sequences used increases from 32 to 4,096 (128 times) even in the presence of extensive variation in the evolutionary rate among lineages or significant biases in the nucleotide composition and transition/transversion ratio. Our results encourage the use of complex models of nucleotide substitution for estimating evolutionary distances and hint at bright prospects for the application of the NJ and related methods in inferring large phylogenies.

With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.

Sexually induced gene 1 (Sig1) in the centric diatom Thalassiosira weissflogii is considered to encode a gamete recognition protein. Sorhannus (2003) analyzed nucleotide sequences of Sig1 using parsimony analysis and the maximum-likelihood (ML)-based Bayesian method for inferring positive selection at single amino acid sites and reported that positively selected sites were detected by the latter method but not by the former. He then concluded that for this type of study, the ML-based method is more reliable than parsimony analysis. Here we show that his results apparently represent false-positive cases of the ML-based method and that there is no solid evidence that this gene contains positively selected sites. We further demonstrate that in the tax gene of human T-cell lymphotropic virus type I (HTLV-I), all codon sites, including invariable sites, can be inferred as positively selected sites by the ML-based method. These observations indicate that the ML-based method may produce many false-positive sites. One of the main reasons for the occurrence of false positives is that in the ML-based method, codon sites are grouped into several categories, with different nonsynonymous/synonymous rate ratios (omegas), on a purely statistical basis, and positive selection is inferred indirectly by examining whether the average omega for each category is greater than 1. In parsimony analysis, however, the evolutionary change of nucleotides at each codon site is examined. For this reason, parsimony-based methods rarely produce false positives and are safer than ML-based methods for detecting positive selection at individual codon sites, although a large number of sequences are necessary.

Purpose: Sudden unexpected death in epilepsy (SUDEP) is a major cause of mortality for patients with epilepsy. Cardiac factors may be involved and were evaluated in this study., Methods: EEG and ECG data for 21 patients with definite (n = 6) or probable (n = 15) SUDEP were compared with data from a group of 43 patients with refractory partial epilepsy. ECG abnormalities and heart rate (HR) changes were correlated with clinical data., Results: Fourteen patients died in their sleep; two were awake. Ictal maximal HR (90 seizures from 16 of 21 patients) was significantly higher in SUDEP (mean, 149 beats/min, BPM) than in comparison patients (mean, 126 BPM; p < 0.001). Greater increases in HR were associated with seizures arising from sleep (78 BPM increase) than from wakefulness (47 BPM; p < 0.001) in SUDEP, as compared with the non-SUDEP group (52 BPM in sleep, 43 BPM in wakefulness; p = 0.27). Ictal cardiac repolarization and rhythm abnormalities occurred in 56% of SUDEP (including two atrial fibrillation, two ventricular premature depolarizations, two marked sinus arrhythmia, two atrial premature depolarizations, one junctional escape, one ST-segment elevation), and 39% of comparison patients (p = 0.39). No specific seizure onset (laterality or lobe) was associated with SUDEP., Conclusions: This study reveals, for the first time, evidence of increased autonomic stimulation (as measured by HR) associated with seizures, particularly in sleep, in patients with SUDEP, as compared with a clinically similar group of patients with refractory epilepsy.

We have identified the Hsp70 gene superfamily of the nematode Caenorhabditis briggsae and investigated the evolution of these genes in comparison with Hsp70 genes from C. elegans, Drosophila, and yeast. The Hsp70 genes are classified into three monophyletic groups according to their subcellular localization, namely, cytoplasm (CYT), endoplasmic reticulum (ER), and mitochondria (MT). The Hsp110 genes can be classified into the polyphyletic CYT group and the monophyletic ER group. The different Hsp70 and Hsp110 groups appeared to evolve following the model of divergent evolution. This model can also explain the evolution of the ER and MT genes. On the other hand, the CYT genes are divided into heat-inducible and constitutively expressed genes. The constitutively expressed genes have evolved more or less following the birth-and-death process, and the rates of gene birth and gene death are different between the two nematode species. By contrast, some heat-inducible genes show an intraspecies phylogenetic clustering. This suggests that they are subject to sequence homogenization resulting from gene conversion-like events. In addition, the heat-inducible genes show high levels of sequence conservation in both intra-species and inter-species comparisons, and in most cases, amino acid sequence similarity is higher than nucleotide sequence similarity. This indicates that purifying selection also plays an important role in maintaining high sequence similarity among paralogous Hsp70 genes. Therefore, we suggest that the CYT heat-inducible genes have been subjected to a combination of purifying selection, birth-and-death process, and gene conversion-like events.

Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADS-box genes from the Arabidopsis and rice genomes. Plant MADS-box genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in Arabidopsis. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and approximately 15-20 type II genes in the most recent common ancestor of Arabidopsis and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes.

Olfactory receptor (OR) genes form the largest known multigene family in the human genome. To obtain some insight into their evolutionary history, we have identified the complete set of OR genes and their chromosomal locations from the latest human genome sequences. We detected 388 potentially functional genes that have intact ORFs and 414 apparent pseudogenes. The number and the fraction (48%) of functional genes are considerably larger than the ones previously reported. The human OR genes can clearly be divided into class I and class II genes, as was previously noted. Our phylogenetic analysis has shown that the class II OR genes can further be classified into 19 phylogenetic clades supported by high bootstrap values. We have also found that there are many tandem arrays of OR genes that are phylogenetically closely related. These genes appear to have been generated by tandem gene duplication. However, the relationships between genomic clusters and phylogenetic clades are very complicated. There are a substantial number of cases in which the genes in the same phylogenetic clade are located on different chromosomal regions. In addition, OR genes belonging to distantly related phylogenetic clades are sometimes located very closely in a chromosomal region and form a tight genomic cluster. These observations can be explained by the assumption that several chromosomal rearrangements have occurred at the regions of OR gene clusters and the OR genes contained in different genomic clusters are shuffled.

MADS-box genes in plants control various aspects of development and reproductive processes including flower formation. To obtain some insight into the roles of these genes in morphological evolution, we investigated the origin and diversification of floral MADS-box genes by conducting molecular evolutionary genetics analyses. Our results suggest that the most recent common ancestor of today's floral MADS-box genes evolved roughly 650 MYA, much earlier than the Cambrian explosion. They also suggest that the functional classes T (SVP), B (and Bs), C, F (AGL20 or TM3), A, and G (AGL6) of floral MADS-box genes diverged sequentially in this order from the class E gene lineage. The divergence between the class G and E genes apparently occurred around the time of the angiosperm/gymnosperm split. Furthermore, the ancestors of three classes of genes (class T genes, class B/Bs genes, and the common ancestor of the other classes of genes) might have existed at the time of the Cambrian explosion. We also conducted a phylogenetic analysis of MADS-domain sequences from various species of plants and animals and presented a hypothetical scenario of the evolution of MADS-box genes in plants and animals, taking into account paleontological information. Our study supports the idea that there are two main evolutionary lineages (type I and type II) of MADS-box genes in plants and animals.

The major histocompatibility complex (MHC) is a multigene family that mediates the host immune response by helping T lymphocytes to recognize and respond to foreign antigens. The high degree of polymorphism and a quick turnover of the genetic loci make the evolution of MHC genes an intriguing subject of study. To understand the evolutionary pattern of this multigene family, we studied the phylogeny and divergence times of six functional MHC class I loci from primate species. On the phylogenetic trees, locus F occupies the most basal position among these loci. Our results suggest that the F locus diverged from the other MHC class I loci about 46-66 MYA. The major diversification of the other class I loci was estimated to have occurred at about 35-49 MYA, which is before the time of separation of Old World-New World monkeys. The gene duplication leading to the classical C locus in great apes appears to have occurred about 21-28 MYA. At approximately the same time the duplication of the B locus occurred in macaques. The oldest allelic lineages of A, B, and C loci in humans seem to have appeared at least 14-19, 10-15, and 13-17 MYA, respectively. Our phylogenetic analysis supports the hypothesis that the nonclassical locus F has diverged from the rest of class I loci very early in primate evolution. The overall phylogenetic pattern observed among class I genes is consistent with the model of birth-and-death evolution.

Although the phylogenetic relationships of major lineages of primate species are relatively well established, the times of divergence of these lineages as estimated by molecular data are still controversial. This controversy has been generated in part because different authors have used different types of molecular data, different statistical methods, and different calibration points. We have therefore examined the effects of these factors on the estimates of divergence times and reached the following conclusions: (1) It is advisable to concatenate many gene sequences and use a multigene gamma distance for estimating divergence times rather than using the individual gene approach. (2) When sequence data from many nuclear genes are available, protein sequences appear to give more robust estimates than DNA sequences. (3) Nuclear proteins are generally more suitable than mitochondrial proteins for time estimation. (4) It is important first to construct a phylogenetic tree for a group of species using some outgroups and then estimate the branch lengths. (5) It appears to be better to use a few reliable calibration points rather than many unreliable ones. Considering all these factors and using two calibration points, we estimated that the human lineage diverged from the chimpanzee, gorilla, orangutan, Old World monkey, and New World monkey lineages approximately 6 MYA (with a range of 5-7), 7 MYA (range, 6-8), 13 MYA (range, 12-15), 23 MYA (range, 21-25), and 33 MYA (range 32-36).

Murphy and colleagues reported that the mammalian phylogeny was resolved by Bayesian phylogenetics. However, the DNA sequences they used had many alignment gaps and undetermined nucleotide sites. We therefore reanalyzed their data by minimizing unshared nucleotide sites and retaining as many species as possible (13 species). In constructing phylogenetic trees, we used the Bayesian, maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ) methods with different substitution models. These trees were constructed by using both protein and DNA sequences. The results showed that the posterior probabilities for Bayesian trees were generally much higher than the bootstrap values for ML, MP, and NJ trees. Two different Bayesian topologies for the same set of species were sometimes supported by high posterior probabilities, implying that two different topologies can be judged to be correct by Bayesian phylogenetics. This suggests that the posterior probability in Bayesian analysis can be excessively high as an indication of statistical confidence and therefore Murphy et al.'s tree, which largely depends on Bayesian posterior probability, may not be correct.

Bayesian phylogenetics has recently been proposed as a powerful method for inferring molecular phylogenies, and it has been reported that the mammalian and some plant phylogenies were resolved by using this method. The statistical confidence of interior branches as judged by posterior probabilities in Bayesian analysis is generally higher than that as judged by bootstrap probabilities in maximum likelihood analysis, and this difference has been interpreted as an indication that bootstrap support may be too conservative. However, it is possible that the posterior probabilities are too high or too liberal instead. Here, we show by computer simulation that posterior probabilities in Bayesian analysis can be excessively liberal when concatenated gene sequences are used, whereas bootstrap probabilities in neighbor-joining and maximum likelihood analyses are generally slightly conservative. These results indicate that bootstrap probabilities are more suitable for assessing the reliability of phylogenetic trees than posterior probabilities and that the mammalian and plant phylogenies may not have been fully resolved.

Inferring positive selection at single amino acid sites is of biological and medical importance. Parsimony-based and likelihood-based methods have been developed for this purpose, but the reliabilities of these methods are not well understood. Because the evolutionary models assumed in these methods are only rough approximations to reality, it is desirable that the methods are not very sensitive to violation of the assumptions made. In this study we show by computer simulation that the likelihood-based method is sensitive to violation of the assumptions and produces many false-positive results under certain conditions, whereas the parsimony-based method tends to be conservative. These observations, together with those from previous studies, suggest that the positively selected sites inferred by the parsimony-based method are more reliable than those inferred by the likelihood-based method.

Endosymbionts, which are widely observed in nature, have undergone reductive genome evolution because of their long-term intracellular lifestyle. Here we compared the complete genome sequences of two different endosymbionts, Buchnera and a protist mitochondrion, with their close relatives to study the evolutionary rates of functional genes in endosymbionts. The results indicate that the rate of amino acid substitution is two times higher in symbionts than in their relatives. This rate increase was observed uniformly among different functional classes of genes, although strong purifying selection may have counterbalanced the rate increase in a few cases. Our data suggest that, contrary to current views, neither the Muller's ratchet effect nor the slightly deleterious mutation theory sufficiently accounts for the elevated evolutionary rate. Rather, the elevated evolutionary rate appears to be mainly due to enhanced mutation rate, although the possibility of relaxation of purifying selection cannot be ruled out.

Histones are small basic proteins encoded by a multigene family and are responsible for the nucleosomal organization of chromatin in eukaryotes. Because of the high degree of protein sequence conservation, it is generally believed that histone genes are subject to concerted evolution. However, purifying selection can also generate a high degree of sequence homogeneity. In this study, we examined the long-term evolution of histone H4 genes to determine whether concerted evolution or purifying selection was the major factor for maintaining sequence homogeneity. We analyzed the proportion (p(S)) of synonymous nucleotide differences between the H4 genes from 59 species of fungi, plants, animals, and protists and found that p(S) is generally very high and often close to the saturation level (p(S) ranging from 0.3 to 0.6) even though protein sequences are virtually identical for all H4 genes. A small proportion of genes showed a low level of p(S) values, but this appeared to be caused by recent gene duplication. Our findings suggest that the members of this gene family evolve according to the birth-and-death model of evolution under strong purifying selection. Using histone-like genes in archaebacteria as outgroups, we also showed that H1, H2A, H2B, H3, and H4 histone genes in eukaryotes form separate clusters and that these classes of genes diverged nearly at the same time, before the eukaryotic kingdoms diverged.

Influenza A, B, and C viruses are the etiological agents of influenza. Hemagglutinin (HA) is the major envelope glycoprotein of influenza A and B viruses, and hemagglutinin-esterase (HE) in influenza C viruses is a protein homologous to HA. Because influenza A virus pandemics in humans appear to occur when new subtypes of HA genes are introduced from aquatic birds that are known to be the natural reservoir of the viruses, an understanding of the origin and evolution of HA genes is of particular importance. We therefore conducted a phylogenetic analysis of HA and HE genes and showed that the influenza A and B virus HA genes diverged much earlier than the divergence between different subtypes of influenza A virus HA genes. The rate of amino acid substitution for A virus HAs from duck, a natural reservoir, was estimated to be 3.19 x 10(-4) per site per year, which was slower than that for human and swine A virus HAs but similar to that for influenza B and C virus HAs (HEs). Using this substitution rate from the duck, we estimated that the divergences between different subtypes of A virus HA genes occurred from several thousand to several hundred years ago. In particular, the earliest divergence time was estimated to be about 2,000 years ago. Also, the A virus HA gene diverged from the B virus HA gene about 4,000 years ago and from the C virus HE gene about 8,000 years ago. These time estimates are much earlier than the previous ones.

A typical immunoglobulin (Ig) molecule is composed of four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains. This tetrameric structure is conserved in almost all jawed vertebrate species. However, it has been discovered that camels and llamas (family: Camelidae) possess a type of dimeric Ig that consists of two H chains only. These H chains do not associate with L chains, and they do not have the first constant region (CH1), which is present in the conventional Ig. In spite of these changes, the dimeric Ig maintains the normal immune function. To understand the evolution of the dimeric Ig, we studied the phylogenetic relationships of the variable region (V(H)H) genes of the dimeric Ig from Camelidae and those (V(H)) of the conventional Ig from mammals. The results showed that the V(H)H genes form a monophyletic cluster within one of the mammalian V(H) groups, group C. We examined the type of selective force in complementarity-determining regions (CDRs) and framework regions (FRs) by comparing the rate of synonymous (dS) and nonsynonymous (dN) substitutions. We found that the results obtained from V(H)H genes were similar to those from V(H) genes in that CDRs showed an excess of dN over dS (indicating positive selection), whereas the reverse was true for FRs (purifying selection). However, when the extent of positive selection or purifying selection was investigated at each codon site, three major differences between V(H)H and V(H) genes were found. That is, very different types of selective force were observed between V(H)H and V(H) genes (1) at the sites that contact the L chain in the conventional Ig, (2) at the sites that interact with the CH1 region in the conventional Ig, and (3) in the H1 loop. Our findings suggest that adaptive evolution has occurred in the functionally important sites of the V(H)H genes to maintain the normal immune function in the dimeric Ig.

In some species, histone gene clusters consist of tandem arrays of each type of histone gene, whereas in other species the genes may be clustered but not arranged in tandem. In certain species, however, histone genes are found scattered across several different chromosomes. This study examines the evolution of histone 3 (H3) genes that are not arranged in large clusters of tandem repeats. Although H3 amino acid sequences are highly conserved both within and between species, we found that the nucleotide sequence divergence at synonymous sites is high, indicating that purifying selection is the major force for maintaining H3 amino acid sequence homogeneity over long-term evolution. In cases where synonymous-site divergence was low, recent gene duplication appeared to be a better explanation than gene conversion. These results, and other observations on gene inactivation, organization, and phylogeny, indicated that these H3 genes evolve according to a birth-and-death process under strong purifying selection. Thus, we found little evidence to support previous claims that all H3 proteins, regardless of their genome organization, undergo concerted evolution. Further analyses of the structure of H3 proteins revealed that the histones of higher eukaryotes might have evolved from a replication-independent-like H3 gene.

The reliabilities of parsimony-based and likelihood-based methods for inferring positive selection at single amino acid sites were studied using the nucleotide sequences of human leukocyte antigen (HLA) genes, in which positive selection is known to be operating at the antigen recognition site. The results indicate that the inference by parsimony-based methods is robust to the use of different evolutionary models and generally more reliable than that by likelihood-based methods. In contrast, the results obtained by likelihood-based methods depend on the models and on the initial parameter values used. It is sometimes difficult to obtain the maximum likelihood estimates of parameters for a given model, and the results obtained may be false negatives or false positives depending on the initial parameter values. It is therefore preferable to use parsimony-based methods as long as the number of sequences is relatively large and the branch lengths of the phylogenetic tree are relatively small.

The diversity of T-cell receptors is generated primarily by the variable-region gene families, each of which is composed of a large number of member genes. The entire genomic sequence of the variable region (VB) of the T- cell receptor beta chain from humans and mice has become available. To understand the evolutionary dynamics of the VB gene family, we conducted a phylogenetic analysis of all VB genes from humans and mice, as well as a detailed analysis of internal DNA duplications in the human genomic VB region. The phylogenetic tree obtained shows that human and mouse VB genes intermingle extensively rather than forming two separate clusters and that many gene duplications occurred both before and after the divergence between primates and rodents. Analyzing the genomic maps of transposable elements (e.g., LINEs and SINEs) and relic VB genes in the VB gene region, we present evidence that a 20-kb VB region duplicated tandemly four times in the human lineage during the last 32 Myr, and 6 out of the 15 VB genes in this region have become nonfunctional during this period. Our results show that the VB gene family is subject to evolution by a birth-and-death process rather than to concerted evolution.

When many protein sequences are available for estimating the time of divergence between two species, it is customary to estimate the time for each protein separately and then use the average for all proteins as the final estimate. However, it can be shown that this estimate generally has an upward bias, and that an unbiased estimate is obtained by using distances based on concatenated sequences. We have shown that two concatenation-based distances, i.e., average gamma distance weighted with sequence length (d(2)) and multiprotein gamma distance (d(3)), generally give more satisfactory results than other concatenation-based distances. Using these two distance measures for 104 protein sequences, we estimated the time of divergence between mice and rats to be approximately 33 million years ago. Similarly, the time of divergence between humans and rodents was estimated to be approximately 96 million years ago. We also investigated the dependency of time estimates on statistical methods and various assumptions made by using sequence data from eubacteria, protists, plants, fungi, and animals. Our best estimates of the times of divergence between eubacteria and eukaryotes, between protists and other eukaryotes, and between plants, fungi, and animals were 3, 1.7, and 1.3 billion years ago, respectively. However, estimates of ancient divergence times are subject to a substantial amount of error caused by uncertainty of the molecular clock, horizontal gene transfer, errors in sequence alignments, etc.

Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (p(S)) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown that p(S) is generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level of p(S) values, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-and-death evolution at the DNA level.

In phylogenetic inference by maximum-parsimony (MP), minimum-evolution (ME), and maximum-likelihood (ML) methods, it is customary to conduct extensive heuristic searches of MP, ME, and ML trees, examining a large number of different topologies. However, these extensive searches tend to give incorrect tree topologies. Here we show by extensive computer simulation that when the number of nucleotide sequences (m) is large and the number of nucleotides used (n) is relatively small, the simple MP or ML tree search algorithms such as the stepwise addition (SA) plus nearest neighbor interchange (NNI) search and the SA plus subtree pruning regrafting (SPR) search are as efficient as the extensive search algorithms such as the SA plus tree bisection-reconnection (TBR) search in inferring the true tree. In the case of ME methods, the simple neighbor-joining (NJ) algorithm is as efficient as or more efficient than the extensive NJ+TBR search. We show that when ME methods are used, the simple p distance generally gives better results in phylogenetic inference than more complicated distance measures such as the Hasegawa-Kishino-Yano (HKY) distance, even when nucleotide substitution follows the HKY model. When ML methods are used, the simple Jukes-Cantor (JC) model of phylogenetic inference generally shows a better performance than the HKY model even if the likelihood value for the HKY model is much higher than that for the JC model. This indicates that at least in the present case, selecting of a substitution model by using the likelihood ratio test or the AIC index is not appropriate. When n is small relative to m and the extent of sequence divergence is high, the NJ method with p distance often shows a better performance than ML methods with the JC model. However, when the level of sequence divergence is low, this is not the case.

Purpose: This study assessed the frequency and character of ictal cardiac rhythm and conduction abnormalities in intractable epilepsy. Sudden unexpected death in epilepsy (SUDEP) is a major cause of excess mortality in people with refractory epilepsy, and cardiac arrhythmias during seizures may be responsible. The frequency of cardiac abnormalities during seizures in patients with refractory epilepsy must be determined., Methods: Fifty-one seizures in 43 patients with intractable partial epilepsy were analyzed prospectively from CCTV-EEG monitoring with one ECG channel. Arrhythmias, repolarization abnormalities, and PR and QTc intervals were determined for preictal (3 min), ictal, and postictal (3 min) periods for one or more seizures per patient. Parametric statistics were used for continuous variables, and nonparametric statistics were used for categoric variables., Results: Of the patients, 39% had one or more abnormalities of rhythm and/or repolarization during or immediately after seizures. Abnormalities included asystole (one), atrial fibrillation (one), marked or moderate sinus arrhythmia (six), supraventricular tachycardia (one), atrial premature depolarizations (APDs; eight), ventricular premature depolarizations (VPDs; two), and bundle-branch block (three). Mean seizure duration was longer in patients with abnormalities than in those without (204 vs. 71 s; p < 0.001). Generalized tonic-clonic seizures were also associated with increased occurrence of ictal ECG abnormalities (p = 0.006) as compared with complex partial seizures. There were no clinically significant differences in mean preictal and ictal/postictal PR and QTc intervals., Conclusions: Cardiac rhythm and conduction abnormalities are common during seizures, particularly if they are prolonged or generalized, in intractable epilepsy. These abnormalities may contribute to SUDEP.

Protamines are small, highly basic DNA-binding proteins found in the sperm of animals. Interestingly, the proportion of arginine residues in one type of protamine, protamine P1, is about 50% in mammals. Upon closer examination, it was found that both the total number of amino acids and the positions of arginine residues have changed considerably during the course of mammalian evolution. This evolutionary pattern suggests that protamine P1 is under an unusual form of purifying selection, in which the high proportion of arginine residues is maintained but the positions may vary. In this case, we would expect that the rate of nonsynonymous substitution is not particularly low compared with that of synonymous substitution, despite purifying selection. We would also expect that the selection for a high arginine content results in a high frequency of the nucleotide G in the coding region of this gene, because all six arginine codons contain at least one G. These expectations were confirmed in our study of mammalian protamine genes. Analysis of nonmammalian vertebrate genes also showed essentially the same patterns of evolutionary changes, suggesting that this unusual form of purifying selection has been active since the origin of bony vertebrates. The protamine gene of an insect species shows similar patterns, although its purifying selection is less intense. These observations suggest that arginine-rich selection is a general feature of protamine evolution. The driving force for arginine-rich selection appears to be the DNA-binding function of protamine P1 and an interaction with a protein kinase in the fertilized egg.

The Ig heavy chain variable region (V(H)) genes encode the antigen-binding regions of antibodies. The rabbit genome contains more than 100 V(H) genes, but only one (V(H)1) is preferentially used in the VDJ gene rearrangement. Three highly divergent alleles occur at this V(H)1 locus in most rabbit populations. These three V(H) alleles are also present in snowshoe hare populations, indicating that the polymorphism of the V(H)1 alleles is trans-specific. Here we report the results of a phylogenetic analysis of rabbit Ig germ-line V(H) genes (alleles) together with V(H) genes from humans and mice. We have found that all rabbit V(H) genes belong to one mammalian V(H) group (group C), which also includes various human and mouse V(H) genes. Using the rate of nucleotide substitution obtained from human and mouse V(H) sequences, we have estimated that the V(H)1 polymorphism in the rabbit lineage has been maintained for about 50 million years. This extremely long persistence of V(H)1 polymorphism is apparently caused by overdominant selection, though the real mechanism is unclear.

Purpose: To examine the relation between specific EEG features and clinical outcome, determine whether a predictable sequence of EEG patterns exists during status epilepticus (SE), and examine the relation between periodic epileptiform discharges (PEDs) and SE., Methods: EEG records of 50 patients with SE admitted to Graduate Hospital between January 1990 and July 1995 were reviewed. Ictal EEGs were available in 72%; 28% had only postictal EEGs. Poor outcome was defined as death or persistent vegetative state, and good outcome as all others. Fisher's Exact test, chi2, and t tests were performed for data analysis., Results: Of 50 patients, 72% had a good outcome and 28%, a poor outcome. If PEDs were present at any time during or after SE, outcome tended to be worse (p = 0.053). With PEDs, eight (44%) of 18 had a poor outcome; without PEDs, six (19%) of 32 had a poor outcome. Etiologies for SE did not substantially differ in patients with or without PEDs, and structural abnormalities were not more associated with the presence of PEDs. PEDs were seen both early and late, during and after SE. Other EEG characteristics (lateralized vs. bilateral symmetric ictal EEG, discrete vs. continuous ictal activity, and postictal focal slowing) did not relate to outcome. No predictable sequence of EEG changes was found during SE., Conclusions: PEDs are the only EEG feature related to outcome in SE and are associated with poor outcome independent of etiology.

In the maximum parsimony (MP) and minimum evolution (ME) methods of phylogenetic inference, evolutionary trees are constructed by searching for the topology that shows the minimum number of mutational changes required (M) and the smallest sum of branch lengths (S), respectively, whereas in the maximum likelihood (ML) method the topology showing the highest maximum likelihood (A) of observing a given data set is chosen. However, the theoretical basis of the optimization principle remains unclear. We therefore examined the relationships of M, S, and A for the MP, ME, and ML trees with those for the true tree by using computer simulation. The results show that M and S are generally greater for the true tree than for the MP and ME trees when the number of nucleotides examined (n) is relatively small, whereas A is generally lower for the true tree than for the ML tree. This finding indicates that the optimization principle tends to give incorrect topologies when n is small. To deal with this disturbing property of the optimization principle, we suggest that more attention should be given to testing the statistical reliability of an estimated tree rather than to finding the optimal tree with excessive efforts. When a reliability test is conducted, simplified MP, ME, and ML algorithms such as the neighbor-joining method generally give conclusions about phylogenetic inference very similar to those obtained by the more extensive tree search algorithms.