Showing papers in "Molecular Biology and Evolution in 1993"

Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.

Abstract: Examining the pattern of nucleotide substitution for the control region of mitochondrial DNA ( mtDNA ) in humans and chimpanzees, we developed a new mathematical method for estimating the number of transitional and transversional substitutions per site, as well as the total number of nucleotide substitutions. In this method, excess transitions, unequal nucleotide frequencies, and variation of substitution rate among different sites are all taken into account. Application of this method to human and chimpanzee data suggested that the transition / transversion ratio for the entire control region was - 15 and nearly the same for the two species. The 95% confidence interval of the age of the common ancestral mtDNA was estimated to be 80,000-480,000 years in humans and 0.57-2.72 Myr in common chimpanzees.

Maximum-likelihood estimation of phylogeny from DNA sequences when substitution rates differ over sites.

Abstract: Felsenstein's maximum-likelihood approach for inferring phylogeny from DNA sequences assumes that the rate of nucleotide substitution is constant over different nucleotide sites. This assumption is sometimes unrealistic, as has been revealed by analysis of real sequence data. In the present paper Felsenstein's method is extended to the case where substitution rates over sites are described by the gamma distribution. A numerical example is presented to show that the method fits the data better than do previous models.

Theoretical foundation of the minimum-evolution method of phylogenetic inference.

Abstract: The minimum-evolution (ME) method of phylogenetic inference is based on the assumption that the tree with the smallest sum of branch length estimates is most likely to be the true one. In the past this assumption has been used without mathematical proof. Here we present the theoretical basis of this method by showing that the expectation of the sum of branch length estimates for the true tree is smallest among all possible trees, provided that the evolutionary distances used are statistically unbiased and that the branch lengths are estimated by the ordinary least-squares method. We also present simple mathematical formulas for computing branch length estimates and their standard errors for any unrooted bifurcating tree, with the least-squares approach. As a numerical example, we have analyzed mtDNA sequence data obtained by Vigilant et al. and have found the ME tree for 95 human and 1 chimpanzee (outgroup) sequences. The tree was somewhat different from the neighbor-joining tree constructed by Tamura and Nei, but there was no statistically significant difference between them.

Evolution of the Zfx and Zfy genes: rates and interdependence between the genes.

Abstract: A phylogenetic analysis of sex-chromosomal zinc-finger genes (Zfx and Zfy) indicates that the genes have not evolved completely independently since their initial separation. The sequence similarities suggest gene conversion in the last exon between the duplicated Y-chromosomal genes Zfy-1 and Zfy-2 in the mouse. There are also indications of conversion (or recombination) between the X- and Y-chromosomal genes in the crab-eating fox and in the mouse. The method for estimating synonymous and nonsynonymous substitutions is modified by incorporating the substitutions in the twofold-degenerate sites in a novel way. The estimates of synonymous substitutions support the generation-time hypothesis in that the obtained rates are higher in mice (by a factor of 4.7) than in humans and higher in the Y-chromosomal genes (by a factor of 1.9) than in the X-chromosomal genes.

Evolution of the alpha-crystallin/small heat-shock protein family.

Abstract: The common characteristic of the alpha-crystallin/small heat-shock protein family is the presence of a conserved homologous sequence of 90-100 residues. Apart from the vertebrate lens proteins--alpha A- and alpha B-crystallin--and the ubiquitous group of 15-30-kDa heat-shock proteins, this family also includes two mycobacterial surface antigens and a major egg antigen of Schistosoma mansoni. Multiple small heat-shock proteins are especially present in higher plants, where they can be distinguished in at least two classes of cytoplasmic proteins and a chloroplast-located class. The alpha-crystallins have recently been found in many tissues outside the lens, and alpha B-crystallin, in particular, behaves in many respects like a small heat-shock protein. The homologous sequences constitute the C-terminal halves of the proteins and probably represent a structural domain with a more variable C-terminal extension. These domains must be responsible for the common structural and functional properties of this protein family. Analysis of the phylogenetic tree and comparison of the biological properties of the various proteins in this family suggest the following scenario for its evolution: The primordial role of the small heat-shock protein family must have been to cope with the destabilizing effects of stressful conditions on cellular integrity. The alpha-crystallin-like domain appears to be very stable, which makes it suitable both as a surface antigen in parasitic organisms and as a long-living lens protein in vertebrates. It has recently been demonstrated that, like the other heat-shock proteins, the alpha-crystallins and small heat-shock proteins function as molecular chaperones, preventing undesired protein-protein interactions and assisting in refolding of denatured proteins. Many of the small heat-shock proteins are differentially expressed during normal development, and there is good evidence that they are involved in cytomorphological reorganizations and in degenerative diseases. In conjunction with the stabilizing, thermoprotective role of alpha-crystallins and small heat-shock proteins, they may also be involved in signal transduction. The reversible phosphorylation of these proteins appears to be important in this respect.

Taxonomic congruence versus total evidence, and amniote phylogeny inferred from fossils, molecules, and morphology.

Abstract: Taxonomic congruence and total evidence are competing paradigms in phylogenetic inference. Taxonomic congruence focuses on deriving a consensus from the results obtained from separately analyzed data sets, whereas total evidence uses character congruence in the search for the best-fitting hypothesis for all of the available character evidence. Explicit or implicit use of taxonomic congruence is usually employed when an investigator either has both molecular and morphological data sets or has different gene-, rRNA-, or protein-sequence data sets available. Indeed, a taxonomic congruence rationale is frequently used as the basis for exploring classes of data, thus allowing comparison between the phylogenetic signal emerging from a particular data set and those of other such classes. Problematic aspects of employing the taxonomic congruence approach include the potentially misleading and arbitrary choices of both a consensus method and the division of characters into subsets. If the goal of an analysis is to provide the best estimate of genealogy afforded by the available character evidence, then taxonomic congruence is substantially more arbitrary than a total evidence approach. The theoretical advantages of phylogenetic estimates based on total evidence are argued in the present study and are illustrated with an example of amniote relationships. We report conflicting results from total evidence and taxonomic congruence approaches, with analyses of previously reported data from both fossil and living amniotes and from both morphology and molecules, the latter including available 18S rRNA, 28S rRNA, and protein sequences. We conclude that a more highly resolved and robust phylogenetic hypothesis of amniotes, the traditional one, emerges when a total evidence approach is employed.

Prospects for estimating nucleotide divergence with RAPDs.

Abstract: The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.

Evolutionary relationships of human populations on a global scale.

Abstract: Using gene frequency data for 29 polymorphic loci ( 12 1 alleles), we conducted a phylogenetic analysis of 26 representative populations from around the world by using the neighbor-joining (NJ) method. We also conducted a separate analysis of 15 populations by using data for 33 polymorphic loci. These analyses have shown that the first major split of the phylogenetic tree separates Africans from nonAfricans and that this split occurs with a 100% bootstrap probability. The second split separates Caucasian populations from all other non-African populations, and this split is also supported by bootstrap tests. The third major split occurs between Native American populations and the Greater Asians that include East Asians (mongoloids), Pacific Islanders, and Australopapuans (native Australians and Papua New Guineans), but Australopapuans are genetically quite different from the rest of the Greater Asians. The second and third levels of population splitting are quite different from those of the phylogenetic tree obtained by Cavalli-Sforza et al. ( 1988), where Caucasians, Northeast Asians, and Amerindians form the Northeurasian supercluster and the rest of non-Africans form the Southeast Asian supercluster. One of the major factors that caused the difference between the two trees is that Cavalli-Sforza et al. used unweighted pair-group method with arithmetic mean (UPGMA) in phylogenetic inference, whereas we used the NJ method in which evolutionary rate is allowed to vary among different populations. Bootstrap tests have shown that the UPGMA tree receives poor statistical support whereas the NJ tree is well supported. Implications that the phylogenetic tree obtained has on the current controversy over the out-of-Africa and the multiregional theories of human origins are discussed.

Allelic genealogy and human evolution.

Abstract: Genetic variation at most loci examined in human populations indicates that the (effective) population size has been approximately 10(4) for the past 1 Myr and that individuals have been genetically united rather tightly. Also suggested is that the population size has never dropped to a few individuals, even in a single generation. These impose important requirements for the hypotheses for the origin of modern humans: a relatively large population size and frequent migration if populations were geographically subdivided. Any hypothesis that assumes a small number of founding individuals throughout the late Pleistocene can be rejected. Extraordinary polymorphism at some loci of the major histocompatibility complex (Mhc) rules out the past action of severe bottlenecks, or the so-called founder principle, which invokes only a small number of founding individuals when a new species emerges. This conclusion may be extended to the 35-Myr-old history of the human lineage, because some polymorphism at Mhc loci seems to have lasted that long. Furthermore, although the population structure prior to the late Pleistocene is less clear, owing to the insensitivity of Mhc alleles, even to low levels of migration, the nature of Mhc polymorphism suggests that the effective size of populations leading to humans was as large as 10(5). Hence, the effective population size of humans might have become somewhat smaller in most of the late Pleistocene. The reduction could be due either to the then adverse environment in the Old World and/or to the increased migration rate. It is also argued that population explosion fostered by the agriculture revolution has had significant effects on incorporating new alleles into human populations.

Peopling of the Americas, founded by four major lineages of mitochondrial DNA.

Abstract: Nucleotide sequence analysis of the major noncoding region of human mitochondrial DNA from various races was extended with 72 Native Americans from 16 different local populations (nine populations from Chile, four from Colombia, and one each from Brazil and from Maya and Apache Indians). The sequences were determined directly from the polymerase chain reaction products. On the basis of a comparison of the 482-bp sequences in the 72 Native Americans, 43 different types of mitochondrial DNA sequences were observed. The nucleotide diversity within the Native Americans was estimated to be 1.29%, which is slightly less than the value of 1.44% from the total human population including Africans, Europeans, and Asians. Phylogenetic analysis revealed that most Native American lineages are classified into four major distinct clusters. Individuals belonging to each cluster share at least two specific polymorphic sites that are nearly absent in other human populations, indicating a unique phylogenetic position of Native Americans. A phylogenetic tree of 193 individuals including Africans, Europeans, Asians, and Native Americans indicated that the four Native American clusters are distinct and dispersed in the tree. These clusters almost exclusively consist of Native Americans--with only a few Asians, if any. We postulate that four ancestral populations gave rise to different waves of migration to the New World. From the estimated coalescence time of the Asian and Native American lineages, we infer that the first migration across the Bering landbridge took place approximately 14,000-21,000 years ago. Furthermore, sequence differences in all pairwise comparisons of Native Americans showed a bimodal distribution that is significantly different from Poisson. These results suggest that the ancestral Native American population underwent neither a severe bottleneck nor rapid expansion in population size, during the migration of people into the Americas.

Reduced natural selection associated with low recombination in Drosophila melanogaster.

Abstract: Synonymous codons are not used equally in many organisms, and the extent of codon bias varies among loci. Earlier studies have suggested that more highly expressed loci in Drosophila melanogaster are more biased, consistent with findings from several prokaryotes and unicellular eukaryotes that codon bias is partly due to natural selection for translational efficiency. We link this model of varying selection intensity to the population-genetics prediction that the effectiveness of natural selection is decreased under reduced recombination. In analyses of 385 D. melanogaster loci, we find that codon bias is reduced in regions of low recombination (i.e., near centromeres and telomeres and on the fourth chromosome). The effect does not appear to be a linear function of recombination rate; rather, it seems limited to regions with the very lowest levels of recombination. The large majority of the genome apparently experiences recombination at a sufficiently high rate for effective natural selection against suboptimal codons. These findings support models of the Hill-Robertson effect and genetic hitchhiking and are largely consistent with multiple reports of low levels of DNA sequence variation in regions of low recombination.

Ribosomal RNA secondary structure: compensatory mutations and implications for phylogenetic analysis.

Abstract: Using sequence data from the 28s ribosomal RNA (rRNA) genes of selected vertebrates, we investigated the effects that constraints imposed by secondary structure have on the phylogenetic analysis of rRNA sequence data. Our analysis indicates that characters from both base-pairing regions (stems) and non-base-pairing regions (loops) contain phylogenetic information, as judged by the level of support of the phylogenetic results compared with a well-established tree based on both morphological and molecular data. The best results (the greatest level of support of wellaccepted nodes) were obtained when the complete data set was used. However, some previously supported nodes were resolved using either the stem or loop bases alone. Stem bases sustain a greater number of compensatory mutations than would be expected at random, but the number is ~40% of that expected under a hypothesis of perfect compensation to maintain secondary structure. Therefore, we suggest that in phylogenetic analyses, the weighting of stem characters be reduced by no more than 20%, relative to that of loop characters. In contrast to previous suggestions, we do not recommend weighting of stem positions by one-half, compared with that of loop positions, because this overcompensates for the constraints that selection imposes on the secondary structure of rRNA.

Evolution of duplicate genes in a tetraploid animal, Xenopus laevis.

Abstract: To understand the evolution of duplicate genes, we compared rates of nucleotide substitution between 17 pairs of nonallelic duplicated genes in the tetraploid frog Xenopus luevis with rates between the orthologous loci of human and rodent. For all duplicated X. laevis genes, the number of synonymous substitutions per site (ds) was greater than the number of nonsynonymous substitutions per site ( dN), indicating that these genes are subject to purifying selection. There was also a significant positive correlation (Y = 0.9 15) between & for the X. laevis genes and & for the mammalian genes, suggesting that, at the amino acid level, the X. laevis genes and the mammalian genes are under similar constraints. Results of relativerate tests showed nearly equal rates of nonsynonymous substitution in each copy of the X. laevis genes; apparently there are similar constraints on both copies. No correlation was found between ds for the X. laevis genes and ds for the mammalian genes. There was a significant positive correlation both between members of pairs of duplicated X. laevis genes (Y = 0.95 1) and between human and rodent orthologues (r = 0.854) with respect to third-position G + C content but no such relationship between the X . laevis genes and either of their mammalian orthologues. The results indicate that both copies of a duplicate gene can be subject to purifying selection and thus support the hypothesis of selection against all genotypes containing a null allele at either of two duplicate loci.

The vertebrate genome: isochores and evolution.

Abstract: Vertebrate genomes are mosaics of isochores--namely, of long (> 300 kb), compositionally homogeneous DNA segments that can be subdivided into a small number of families characterized by different GC levels. In the human genome (which is representative of a number of mammalian genomes, and, more broadly, of the genomes of warm-blooded vertebrates), the compositional range of isochores is 30%-60% GC, and five families of isochores have been identified: two GC-poor families, L1 and L2, together representing 62% of the genome, and three GC-rich families, H1, H2, and H3, representing 22%, 9%, and 3%, respectively (the remaining 4% of the genome is formed by satellite and ribosomal DNA). Gene concentration is strikingly nonuniform, being highest in the H3 isochore family, lowest in the L1 + L2 families, and intermediate in the H1 + H2 families. The H3 family corresponds to T(elomeric) bands of metaphase chromosomes, and the L1 + L2 families correspond to G(iemsa) bands, whereas R(everse) bands comprise both GC-poor and GC-rich isochores. The compositional distributions of large genome fragments, of exons (and their codon positions), and of introns are correlated with each other. They represent compositional patterns and are very different between the genomes of cold- and warm-blooded vertebrates, mainly in that the former are much less heterogeneous in base composition and never reach the highest GC levels attained by the latter. Only relatively small compositional differences are found among the genomes of either cold- or warm-blooded vertebrates. Compositional patterns allow one to define two modes in genome evolution: a conservative mode, with no compositional change, and a transitional (or shifting) mode, with compositional changes. The conservative mode can be observed among either cold- or warm-blooded vertebrates. The transitional mode comprises both major and minor compositional changes. In vertebrate genomes, the major changes are associated with the appearance of GC-rich and very GC-rich isochores in mammalian and avian genomes. Mutational biases play a role in both modes of compositional evolution. According to one viewpoint, the fixation of compositionally biased mutations is responsible for the transitional mode of evolution of bacterial genomes; in the conservative mode of evolution of vertebrates, they accomplish their role in conjunction with differences either in chromatin structures that modulate replication errors or in chromatin transcriptional activities that may lead to various extents of repair-DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex.

Abstract: Two regions of the genome, a 1 -kbp portion of the zeste locus and a 1.1 -kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolkprotein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D. mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58-0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.

Analysis of a genetic hitchhiking model, and its application to DNA polymorphism data from Drosophila melanogaster.

Abstract: Begun and Aquadro have demonstrated that levels of nucleotide variation correlate with recombination rate among 20 gene regions from across the genome of Drosophila melanogaster. It has been suggested that this correlation results from genetic hitchhiking associated with the fixation of strongly selected mutants. The hitchhiking process can be described as a series of two-step events. The first step consists of a strongly selected substitution wiping out linked variation in a population; this is followed by a recovery period in which polymorphism can build up via neutral mutations and random genetic drift. Genetic hitchhiking has previously been modeled as a steady-state process driven by recurring selected substitutions. We show here that the characteristic parameter of this steady-state model is alpha v, the product of selection intensity (alpha = 2Ns) and the frequency of beneficial mutations v (where N is population size and s is the selective advantage of the favored allele). We also demonstrate that the steady-state model describes the hitchhiking process adequately, unless the recombination rate is very low. To estimate alpha v, we use the data of DNA sequence variation from 17 D. melanogaster loci from regions of intermediate to high recombination rates. We find that alpha v is likely to be > 1.3 x 10(-8). Additional data are needed to estimate this parameter more precisely. The estimation of alpha v is important, as this parameter determines the shape of the frequency distribution of strongly selected substitutions.

Recovering phylogenetic signal from DNA sequences: relationships within the corvine assemblage (class aves) as inferred from complete sequences of the mitochondrial DNA cytochrome-b gene.

Abstract: Phylogenetic analysis of cytochrome-b sequences and cranial osteological characters for nine genera of corvine passerine birds supports the hypothesis that the two major groups of birds of paradise, the manucodines and paradisaeinines, constitute a monophyletic group and that their postulated sister group is the Corvidae (crows, jays, and allies). The data are also consistent with the hypothesis that the bowerbirds are not closely related to the birds of paradise but instead lie near the base of the corvine assemblage. The corvine radiation exemplifies a case of multiple star phylogenies embedded within a major clade, with the branching pattern characterized by very short internodal divergence times. Such histories are difficult to resolve no matter what type of data is employed, because little change accumulates between branching events. With respect to sequence data, reconstructed tree topologies are sensitive to the choice of outgroup and to the method of analysis (e.g., transversion vs. global parsimony). In such cases, assessing the “reliability” of a best-fit or mostparsimonious tree inferred from any particular data set becomes problematic. Statistical tests of tree topologies that depend on random sampling of characters will generally be inconclusive in that all cladistic components will tend to be poorly supported because relatively few character-state changes will be recorded between branching events. It is suggested, on the other hand, that congruence in cladistic signal across different data sets may be a potentially more useful method for evaluating the reliability of the signal of any one data set. Resolution of star phylogenies will probably be possible only if DNA sequence and morphological characters are combined in a single analysis.

Congruence in Control-Region Sequence and Restriction- Site Variation in Mitochondrial DNA of Brook Charr (Salvelinus fontinalis Mitchill)'

Abstract: We compared the congruence in genetic variation and phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes detected with restriction-fragment-length polymorphisms (RFLPs) and sequence analysis in the brook charr, Sulvelinus fontinalis Mitchill. This was accomplished by analyzing variation both at 172 restriction sites subsampling 903 bp over the entire mitochondrial genome and in the sequence of a 300-bp segment of the control region among the same 33 individuals. The sequence and RFLP analyses were highly congruent both in terms of mtDNA variants detected per number of nucleotides sampled and in assessing phylogenetic relationships among haplotypes. Therefore, variation observed in a small segment of the control region was representative of that existing over the entire genome. The major difference between both approaches was in the number of mutational changes observed per nucleotide in the sequence analysis. This number was approximately twice as high as that in the RFLP analysis. Nevertheless, the level of variation observed in the control region of charr was less than expected a priori on the basis of previous observations in other vertebrates. Lower mutation rates may be related to an unusually low transition : transversion ratio ( 8 : 3 ) for mtDNA intraspecific variation in vertebrates. The combined restriction-site and sequence data resolved at least three distinct phylogenetic groupings in S. fontinalis. However, and despite the high-resolution level of the study (> 1,200 bp screened, or 7% of the mitochondrial genome), some major branching patterns in gene tree topology remained unresolved, which stresses the importance of recognizing large standard errors associated with sequence variability when phylogeny is reconstructed.

Intraspecific DNA sequence variation of the mitochondrial control region of white sturgeon (Acipenser transmontanus).

Abstract: Intraspecific sequence variation in the D-loop region of mtDNA in white sturgeon (Acipenser transmontanus), a relict North American fish species, was examined in 27 individuals from populations of the Columbia and Fraser rivers. Thirty-three varied nucleotide positions were present in a 462-nucleotide D-loop sequence, amplified using the polymerase chain reaction. Bootstrapped neighbor-joining and maximum-parsimony trees of sequences from 19 haplotypes suggest that the two populations have recently diverged. This is consistent with the hypothesis that the Columbia River, a Pleistocene refugium habitat, was the source of founders for the Fraser River after the last glacial recession. On the basis of a divergence time of 10-12 thousand years ago, the estimated substitution rate of the white sturgeon D-loop region is 1.1-1.3 x 10(-7) nucleotides/site/year, which is comparable to rates for hypervariable sequences in the human D-loop region. Furthermore, the ratio of mean percent nucleotide differences in the D-loop (2.27%) to that in whole mtDNA (0.54%, as estimated from restriction-enzyme data) is 4.3, which is similar to the fourfold-to-fivefold-higher substitution rate estimated for the human D-loop. The high nucleotide substitution rate of the hypervariable region indicates that the vertebrate D-loop has potential as a genetic marker in molecular population studies.

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire.

Abstract: Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V D J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.

Retention of an ancestral polymorphism in the Mbuna species flock (Teleostei: Cichlidae) of Lake Malawi

Abstract: Molecular genetic variation was examined within and among species of the rock­ dwelling cichlid fishes (mbuna) of Lake Malawi. Phylogenetic relationships among mbuna mitochondrial DNA (mtDNA) haplotypes were estimated by using restric­ tion-fragment-length polymorphisms. The distribution of these lineages among mbuna species is of particular significance for phylogenetic systematic study of this fauna. Some species were found to-he polymorphic for divergent haplotypes that substantially predate their isolation from sister taxa. Repeated speciation events among numerous closely related taxa appear to have been so recent that mtDNA lineage sorting among species is incomplete. Thus, the mtDNA gene tree is not congruent with the putative species tree. These results indicate that analysis of mtDNA alone will not be sufficient for resolution of phylogenetic relationships in the mbuna. Clarification of these relationships will require examination ofmultiple nuclear loci, because many of these new markers are also likely to retain ancestral polymorphisms.

Cetacean mitochondrial DNA control region: sequences of all extant baleen whales and two sperm whale species.

Abstract: The sequence of the mitochondrial control region was determined in all 10 extant species commonly assigned to the suborder Mysticeti (baleen or whalebone whales) and to two odontocete (toothed whale) species (the sperm and the pygmy sperm whale). In the mysticetes, both the length and the sequence of the control region were very similar, with differences occurring primarily in the first approximately 160 bp of the 5' end of the L-strand of the region. There were marked differences between the mysticete and sperm whale sequences and also between the two sperm whales. The control region, less its variable portion, was used in a comparison including the 10 mysticete sequences plus the same region of an Antarctic minke whale specimen and the two sperm whales. The difference between the minke whales from the North Atlantic and the Antarctic was greater than that between any acknowledged species belonging to the same genus (Balaenoptera). The difference was similar to that between the families Balaenopteridae (rorquals) and Eschrichtiidae (gray whales). The findings suggest that the Antarctic minke whale should have a full species status, B. bonaerensis. Parsimony analysis separated the bowhead and the right whale (family Balaenidae) from all remaining mysticetes, including the pygmy right whale. The pygmy right whale is usually included in family Balaenidae. The analysis revealed a close relationship between the gray whale (family Eschrichtiidae) sequence and those of the rorquals (family Balaenopteridae). The gray whale was included in a clade together with the sei, Bryde's, fin, blue, and humpback whales. This clade was separated from the two minke whale types, which branched together.

Relationships in the Drosophila obscura species group, inferred from mitochondrial cytochrome oxidase II sequences.

Abstract: We compare the sequences for the mitochondrial cytochrome oxidase II gene of 13 species of the Drosophila obscura group. The survey includes six members of the D. afinis subgroup, four of the D. pseudoobscura subgroup, and three of the D. obscura subgroup. In all species, the gene is 688 nucleotides in length, encoding a protein of 229 amino acids plus the first position T of the stop codon. The sequences show the typical high-transition bias for closely related species, but that bias is essentially eliminated for species pairs of >5% sequence divergence. The phylogenetic relationships in the species group are inferred using both neighbor-joining and maximum parsimony. The two procedures give comparable results, showing that the D. afinis and D. pseudoobscura subgroups are monophyletic groupings that appear to have closer affinities to one another than either has to the D. obscura subgroup. We use transversion distances to estimate times of divergence, on the basis of three different estimates of the time of separation of the D. obscura species group from the D. melanogaster group. If that event occurred 35 Mya, then we can estimate the origin of the nearctic forms at -22 Mya and the separation of the D. afinis and D. pseudoobscura subgroups at - 17 Mya.

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability.

Abstract: We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Naf and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Saw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.

The phylogenetic affinities of the chaetognaths: a molecular analysis.

Abstract: The chaetognaths, or arrowworms, constitute a small and enigmatic phylum of marine invertebrates whose phylogenetic affinities have long been uncertain. A popular hypothesis is that the chaetognaths are the sister group of the major deuterostome phyla: chordates, hemichordates, and echinoderms. Here we attempt to determine the affinities of the chaetognaths by using molecular sequence data. We describe the isolation and nucleotide sequence determination of 18S ribosomal DNA from one species of chaetognath and one acanthocephalan. Extensive phylogenetic analyses employing a suite of phylogenetic reconstruction methods (maximum parsimony, maximum likelihood, evolutionary parsimony, and two distance methods) suggest that the hypothesized relationship between chaetognaths and the deuterostomes is incorrect. In contrast, we propose that the lineage leading to the chaetognaths arose prior to the advent of the coelomate metazoa.

The lepidopteran mitochondrial control region: structure and evolution.

Abstract: For several species of lepidoptera, most of the -350-bp mitochondrial controlregion sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common-inferred stem loops with higher-than-random folding energies-although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12s rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species.

Abstract: Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio-Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%-4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.

PCR-based assays of mendelian polymorphisms from anonymous single-copy nuclear DNA: techniques and applications for population genetics.

Abstract: This paper outlines a PCR-based approach for population genetics that offers several advantages over conventional Southern blotting methods for revealing restriction-fragment-length polymorphisms (RFLPs) in nuclear DNA. Primers are constructed from clones isolated from a nuclear DNA library, and these primers subsequently are employed in in vitro syntheses of homologous regions. Amplified products are then screened directly for RFLPs by using gel-staining procedures. Population applications for this PCR-based approach, including potential strengths and weaknesses, are exemplified by two RFLP data sets generated to estimate (a) male-mediated gene flow in the green turtle (Chelonia mydas) and (b) geographic population genetic structure in the American oyster (Crassostrea virginica). Restriction assays of amplified products from 14 or 15 independent primer pairs in each species revealed polymorphisms at several loci that proved highly informative in the population genetic analyses. In general, the Mendelian polymorphisms produced by this PCR-based approach will provide useful genetic markers for population studies, particularly in situations where simpler and less expensive allozyme methods have failed, for whatever reason, to provide adequate information.

Mitochondrial phylogeny of the endemic mouthbrooding lineages of cichlid fishes from Lake Tanganyika in eastern Africa.

Abstract: Of the three cichlid species flocks in eastern Africa, Lake Tanganyika harbors the oldest species assemblage, which is also the most diverse morphologically and behaviorally. For 12 species (20 individuals) of 12 genera of the tribe Ectodini, 852 bp from two segments (cytochrome b and control region) of the mitochondrial genome were sequenced. In addition, orthologous sequences were obtained from eight species (11 individuals) representing other mouthbrooding lineages from Lake Tanganyika. Comparisons of sequence divergences revealed that the single Tanganyikan tribe Ectodini appears to be approximately five times older than the whole Lake Malawi cichlid species flock, suggesting that the radiation of the Tanganyikan mouthbrooding lineages took place long before the species flocks of Lakes Malawi and Victoria evolved. Seven of nine surveyed tribes of Tanganyikan cichlids appear to be approximately equally divergent, and this seems to corroborate the hypothesis of a rapid simultaneous formation of lineages at an early stage in the history of the Lake Tanganyika species flock. The close genetic relationship between the endemic Tropheus lineage and a nonendemic "Haplochromine," Astatotilapia burtoni, indicates that members of the tribe Tropheini may be the sister group of the cichlid flocks of Lakes Malawi and Victoria. The phylogenetic analyses demonstrate the monophyly of the Ectodini and identify the Cyprichromini as their sister group among the Tanganyikan cichlids. Within the tribe Ectodini the molecular data suggest both a branching pattern different than that previously proposed and a subdivision of the Ectodini into four clades, instead of the two originally described. The previously postulated model of morphological transformations believed to be responsible for the drastically different types of ecological specialization found among the Ectodini might therefore be in need of reinterpretation. Characters immediately related to foraging and nutrition seem to be particularly prone to homoplasy, even among members of a single lineage of cichlid fishes.

Mitochondrial COII sequences and modern human origins.

Abstract: The aim of this study is to measure human mitochondrial sequence variability in the relatively slowly evolving mitochondrial gene cytochrome oxidase subunit II (COII) and to estimate when the human common ancestral mitochondrial type existed. New COII gene sequences were determined for five humans (Homo sapiens), including some of the most mitochondrially divergent humans known; for two pygmy chimpanzees (Pan paniscus); and for a common chimpanzee (P. troglodytes). COII sequences were analyzed with those from another relatively slowly evolving mitochondrial region (ND4-5). From class 1 (third codon position) sequence data, a relative divergence date for the human mitochondrial ancestor is estimated as 1/27 th of the human-chimpanzee divergence time. If it is assumed that humans and chimpanzees diverged 6 Mya, this places a human mitochondrial ancestor at 222,000 years, significantly different from 1 Myr (the presumed time of an H. erectus emergence from Africa). The mean coalescent time estimated from all 1,580 sites of combined mitochondrial data, when a 6-Mya human-chimpanzee divergence is assumed, is 298,000 years, with 95% confidence interval of 129,000-536,000 years. Neither estimate is compatible with a 1-Myr-old human mitochondrial ancestor. The mitochondrial DNA sequence data from COII and ND4-5 regions therefore do not support this multiregional hypothesis for the emergence of modern humans.