Ellen M. Prager

Mitochondrial DNA sequences of primates: Tempo and mode of evolution

SummaryWe cloned and sequenced a segment of mitochondrial DNA from human, chimpanzee, gorilla, orangutan, and gibbon. This segment is 896 bp in length, contains the genes for three transfer RNAs and parts of two proteins, and is homologous in all 5 primates. The 5 sequences differ from one another by base substitutions at 283 positions and by a deletion of one base pair. The sequence differences range from 9 to 19% among species, in agreement with estimates from cleavage map comparisons, thus confirming that the rate of mtDNA evolution in primates is 5 to 10 times higher than in nuclear DNA. The most striking new finding to emerge from these comparisons is that transitions greatly outnumber transversions. Ninety-two percent of the differences among the most closely related species (human, chimpanzee, and gorilla) are transitions. For pairs of species with longer divergence times, the observed percentage of transitions falls until, in the case of comparisons between primates and non-primates, it reaches a value of 45. The time dependence is probably due to obliteration of the record of transitions by multiple substitutions at the same nucleotide site. This finding illustrates the importance of choosing closely related species for analysis of the evolutionary process. The remarkable bias toward transitions in mtDNA evolution necessitates the revision of equations that correct for multiple substitutions at the same site. With revised equations, we calculated the incidence of silent and replacement substitutions in the two protein-coding genes. The silent substitution rate is 4 to 6 times higher than the replacement rate, indicating strong functional constraints at replacement sites. Moreover, the silent rate for these two genes is about 10% per million years, a value 10 times higher than the silent rate for the nuclear genes studied so far. In addition, the mean substitution rate in the three mitochondrial tRNA genes is at least 100 times higher than in nuclear tRNA genes. Finally, genealogical analysis of the sequence differences supports the view that the human lineage branched off only slightly before the gorilla and chimpanzee lineages diverged and strengthens the hypothesis that humans are more related to gorillas and chimpanzees than is the orangutan.

Tempo and mode of sequence evolution in mitochondrial DNA of HawaiianDrosophila

SummarySequence comparisons were made for up to 667 bp of DNA cloned from 14 kinds of HawaiianDrosophila and five other dipteran species. These sequences include parts of the genes for NADH dehydrogenase (subunits 1, 2, and 5) and rRNA (from the large ribosomal subunit). Because the times of divergence among these species are known approximately, the sequence comparisons give insight into the evolutionary dynamics of this molecule. Transitions account for nearly all of the differences between sequences that have diverged by less than 2%; for these sequences the mean rate of divergence appears to be about 2%/Myr. In comparisons involving greater divergence times and greater sequence divergence, relatively more of the sequence differences are due to transversions. Specifically, the fraction of these differences that are counted as transversions rises from an initial value of less than 0.1 to a plateau value of nearly 0.6. The time required to reach half of the plateau value, about 10 Myr, is similar to that for mammalian mtDNA. The mtDNAs of flies and mammals are also alike in the shape of the curve relating the percentage of positions at which there are differences in protein-coding regions to the time of divergence. For both groups of animals, the curve has a steep initial slope ascribable to fast accumulation of synonymous substitutions and a shallow final slope resulting from the slow accumulation of substitutions causing amino acid replacements. However, the percentage of all sites that can experience a high rate of substitution appears to be only about 8% for fly mtDNA compared to about 20% for mammalian mtDNA. The low percentage of hypervariable sites may be a consequence of a functional constraint associated with the low content of guanine and cytosine in fly mtDNA.

Ancient origin of lactalbumin from lysozyme: Analysis of DNA and amino acid sequences

SummaryParsimony trees relating DNA sequences coding for lysozymesc and α-lactalbumins suggest that the gene duplication that allowed lactalbumin to evolve from lysozyme preceded the divergence of mammals and birds. Comparisons of the amino acid sequences of additional lysozymes and lactalbumins are consistent with this view. When all base positions are considered, the probability that the duplication leading to the lactalbumin gene occurred after the start to mammalian evolution is estimated to be 0.05–0.10. Elimination of the phylogenetic noise generated by fast evolution and compositional bias at third positions of codons reduced this probability to 0.002–0.03. Thus the gene duplication may have long preceded the acquisition of lactalbumin function.

Molecular time scale for evolution

Abstract Molecular evolutionary clocks have ticked at much the same rate per year in many eubacterial genes as in the nuclear genes of animals and plants. This implication emerges from comparative studies of ribosomal RNA and protein-coding genes. The existence of nearly universal molecular clocks in cellular genomes provides a challenge to explain them as well as an opportunity to use them for putting knowledge of biological diversity on a temporal framework.

Congruency of phylogenies derived from different proteins

SummaryThis communication examines the question of phylogenetic congruency- i.e., whether or not the branching order of evolutionary trees is independent of the protein studied. It was found that trees constructed for birds on the basis of immunological comparison of their transferrins, albumins, and ovalbumins agree approximately with a published tree based on the amino acid sequences of their lysozymesc. This congruency is especially noteworthy with respect to the phylogenetic position of the chachalaca, a Mexican bird classified on morphological grounds in the family Cracidae of the order Galliformes. At the protein level, this species differs as much from non-cracid galliform birds as does the duck, which belongs to another order. Despite the organismal similarity between cracid and non-cracid galliform birds, the molecular relationship is remote. If this contrast between organismal and molecular results had been based on comparative studies with only lysozyme, one could have ascribed the contrast to the possibility that chachalaca lysozyme was paralogous, rather than orthologous, to the other bird lysozymesc. Examination of several proteins is thus desirable in cases of possible paralogy.

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

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

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin.

SummaryThe complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymesc and α-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymesc and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.

Slow evolution of transferrin and albumin in birds according to micro-complement fixation analysis

SummaryRabbit antisera were prepared to purified ovotransferrin from chicken (order Galliformes) and red-winged blackbird (order Passeriformes) and to purified serum albumin from chicken and rhea (order Rheiformes). Quantitative microcomplement fixation was used to compare these proteins immunologically with those of representatives of all 27 orders of birds. The average interordinal immunological distances were 123 units for transferrin and 53 units for albumin.Extensive intraordinal comparisons of transferrin among 51 species within the order Galliformes and 33 species within the order Passeriformes were also carried out. Values ranging from 0–75 immunological distance units were found within each order.Rabbit antisera to purified alligator albumin were also prepared and shown to react with representatives of all 27 orders of birds, the average immunological distance being 166 units.When the data presented here are considered in relation to the fossil record of birds, it appears that transferrin and albumin have evolved more slowly in birds than in other vertebrates. If prevailing interpretations of the fossil record are correct, transferrin has evolved 2–4 times as fast in mammals and snakes as in birds, while serum albumin has evolved about 3 times as fast in mammals, iguanids, crocodilians, and frogs as in birds. Published immunological and sequence comparisons of lysozyme and cytochromec are also consistent with a slower rate of evolution in birds than in other vertebrates. The implications of a general slowdown in the evolution of bird proteins are discussed.

RATES OF EVOLUTION IN CONIFERS (PINACEAE)

Pines and other conifers provide striking examples of evolutionary conservatism. The genus Pinus, for instance, is well represented by fossils over 100 million years old (Florin, 1963). Thus conifers present an opportunity to examine the genetic basis of evolutionary change at the organismal level. Any type of genetic change that has evolved unusually slowly in conifers could be at the basis of organismal evolution. Our comparative immunological studies of proteins indicate that structural genes in conifers of the family Pinaceae have evolved at roughly the standard rate. By contrast, information on chromosome numbers and the fossil record indicates that evolutionary changes in gene arrangement have proceeded extremely slowly in Pinaceae; this has been recognized by many authors (Saylor and Smith, 1966 and references therein). Thus point mutations in structural genes may be less important than chromosomal rearrangements for evolution at the organismallevel. This hypothesis has also emerged from similar studies with various groups of animals (Wilson, Sarich, and Maxson, 1974; Prager and

Evolution of flightless land birds on southern continents: Transferrin comparison shows monophyletic origin of ratites

SummaryA biochemical approach was used to study the evolution of ratite birds, i.e., the ostriches, rheas, cassowaries, emus, and kiwis. Quantitative immunological comparison of transferrin from ratites, tinamous, and other flying birds indicates that all the ratites and tinamous are allied phylogenetically and that they are of monophyletic origin relative to other birds. To explain the current geographic distribution of ratites and the magnitude of the transferrin distances, it is supposed that the ancestors of these flightless birds walked across land bridges between the southern continents during Cretaceous times.