Telling the Evolutionary Time

Molecular Clocks and the Fossil Record

Philip C J Donoghue, M. Paul Smith

• Publisher: CRC Press
• Year: 2003
• Print ISBN: 9780415275248
• eBook ISBN: 9781134477296
• Topic: Biological Sciences
• Subtopic: Biology
• Index: Biological Sciences

Determining the precise timing for the evolutionary origin of groups of organisms has become increasingly important as scientists from diverse disciplines attempt to examine rates of anatomical or molecular evolution and correlate intrinsic biological events to extrinsic environmental events. Molecular clock analyses indicate that many major groups

Chapter 1
Molecular clocks: whence and whither?

Francisco Rodríguez-Trelles, Rosa Tarrío and Francisco J.Ayala

ABSTRACT

The neutrality theory of molecular evolution predicts that the rate of molecular evolution is constant over time, and thus that there is a molecular clock that can be used for timing evolutionary events. Experimental data have shown that the variance of the rate of evolution is generally larger than expected according to the neutrality theory. This raises the question of how reliable the molecular clock is or, indeed, whether there is a molecular clock. We have carried out an extensive investigation of nine proteins in organisms belonging to the three multicellular kingdoms, namely ADH, AMD, DDC, GPDH, G6PD, PGD, SOD, TPI, and XDH. We observe that the nine proteins evolve erratically through time and across lineages. The observations are inconsistent with the neutrality theory and also with various subsidiary hypotheses proposed to account for the overdispersion of the molecular clock.

Introduction: the hypothesis of a molecular clock

Biological evolution is a time-dependent process, by and large unidirectional. Some degree of correlation is, therefore, expected between the biological differentiation of two organisms and the time elapsed since their separation, by the comparison between an organism and its ancestor, or between two organisms sharing a common ancestor. The correlation, however, need not be exact, if only because organisms evolve in response to the vagaries of environmental change in time and space. It is well known that some organisms have evolved fast morphologically, at least with respect to some traits, whereas others have changed but little over millions of years (see, e.g. Dobzhansky et al. 1977, pp. 327–31). Zuckerkandl and Pauling (1962, 1965; see also Margoliash, 1963) proposed that the time-change correlation might be more approximately precise if change were measured in the protein and nucleic acid components of organisms, indeed that there might be a molecular clock of evolution.

The hypothesis of the molecular clock was advanced on the grounds that most amino acid substitutions in a protein (or nucleotides in a gene) occur between functionally equivalent residues, so that their replacement along evolving lineages would be determined by mutation rate and time elapsed, rather than by natural selection (Zuckerkandl and Pauling 1965). Natural selection is rather fickle, subject to the vagaries of environmental change and organism interactions, whereas mutation rate for a given gene is likely to remain constant through time and across lineages. The number of amino acid replacements (or nucleotide substitutions) between species would, then, reflect the time elapsed since their last common ancestor. The time of remote events, as well as the degree of relationship among contemporary lineages could, thus, be determined on the basis of amino acid (or nucleotide) differences. A notable feature of the hypothesis of the molecular evolutionary clock is multiplicity: every one of the thousands of proteins or genes of an organism is an independent clock, each ticking at a different rate but all measuring the same events (Ayala 1986; Gillespie 1991; Li 1997).

Early investigations showed that the evolution of the globins in vertebrates conformed fairly well to the clock hypothesis, which allowed reconstruction of the history of globin gene duplications (Zuckerkandl and Pauling 1965). Fitch and Margoliash (1967) would soon provide a ‘genetic distance’ method that was effectively used for reconstruction of the history of 20 organisms, from yeast to moth to human, based on the amino acid sequence of a small protein, cytochrome c. A theoretical foundation for the clock was provided by Kimura (1968), who developed a ‘neutral theory of molecular evolution’, with great mathematical simplicity; notably, the theory states that the rate of substitution of adaptively equivalent (‘neutral’) alleles, k, is precisely the rate of mutation, u, of neutral alleles, k=u. The neutrality theory predicts that molecular evolution behaves like a stochastic clock, such as radioactive decay, with the properties of a Poisson distribution, in which the mean, M, and variance, V, are expected to be identical, so that V/M=1. The ‘index of dispersion’, measuring the deviation of this ratio from the expected value of 1, is a way to test whether observations fit the theory.

Experimental data have shown that often the rate of molecular evolution is ‘overdispersed’, that is, that the index of dispersion is often significantly greater than 1 (Gillespie 1991; Li 1997). Deviations from rate constancy occur between lineages, for example between rodents and mammals, as well as at different times along a given lineage, both factors having significant effects (Langley and Fitch 1974). Consequently, several modifications of the neutral theory have been proposed, seeking to account for the excess variance of the molecular clock. It has been proposed, for example, that most protein evolution involves ‘slightly deleterious’ replacements rather than strictly neutral ones (Ohta 1973); or that certain ‘biological properties’, such as the effectiveness of the error-correcting polymerases, vary among organisms, so that mutation rates for a given gene vary from one organism to another (Kimura and Ohta 1972; Kimura 1980, 1983; Gillespie 1991; Li and Graur 1997). A ‘population size’ hypothesis proposes that organisms with larger effective population size have a slower rate of evolution than organisms with smaller population size, because the time required to fix new mutations increases with population size (Ohta 1972; Kimura 1983). Another supplementary hypothesis invokes a generation-time effect. Protein evolution has been extensively investigated in primates and rodents with the common observation that the number of replacements is greater in the rodents (Kohne 1970; Li et al. 1996). In plants, the overall rate at the rbcL locus is more than five times greater in annual grasses than in palms, which have much longer generations (Gaut et al. 1992). These rate differences could be accounted for, according to the generation-effect hypothesis, by assuming that the time-rate of evolution depends on the number of germ-line replications per year, which is several times greater for the short-generation rodents and grasses than for the long-generation primates and palms. The rationale of the assumption is that the larger the number of replication cycles, the greater the number of mutational errors that will occur.

From a theoretical, as well as operational, perspective, these and other supplementary hypotheses have the discomforting consequence that they involve additional empirical parameters, often not easy to estimate. It is of great epistemological significance that the original proposal of the neutral theory is (i) highly predictive and therefore, (ii) eminently testable. The supplementary hypotheses lead, nevertheless, to certain predictions that can be tested. The ‘generation-time’, ‘population size’, and ‘biological properties’ hypotheses uniformly predict that rate variations observed between lineages or at different times will equally affect (in direction and magnitude) all genes of any particular organism, since these attributes are common to all genes of the same species. The ‘slightly deleterious’ hypothesis predicts that the rate of evolution will be inversely related to population size, and thus reduces to the ‘population size’ hypothesis (Ohta 1973).

In this chapter, we present an analysis of nine genes undertaken as a test of the four supplementary hypotheses, as well as of the neutrality theory, the more general or ‘null’ hypothesis underlying the molecular clock hypothesis. We have, in the past, reported results for three of these genes, showing that they exhibit overdispersed patterns of molecular evolution that are incompatible with the proposed supplementary hypotheses (Rodríguez-Trelles et al. 2001a, and references therein). The additional tests reported here lead to the same conclusion. We surmise that inferences about the timing of past events (and about phylogenetic relationships among species) based on molecular evolution are subject to sources of error not altogether disparate from inferences based on anatomy, embryology, or other phenotypic characteristics. Nevertheless, molecular investigations have two obvious advantages over phenotypic traits, in degree if not completely in kind; namely, that the number of ‘traits’ is very large, that is, every one of the thousands of genes in the make-up of each organism, and that differences can be more precisely quantified, measured as they are in terms of distinct units, such as amino acids or nucleotides. There are many evolutionary issues concerning both timing and phylogenetic relationships between species for which molecular sequence data provide the best, if not the only, dependable evidence. The large-scale reconstruction of the ‘universal tree’ of life is a case in point: the phylogenetic relationships among Archaea and bacterial prokaryotes and between them and the eukaryotes have best been determined with DNA sequences encoding ribosomal RNA genes. The multiplicity of genes opens up the possibility of combining data for numerous genes in assessing the timing of particular evolutionary events, or the phylogeny of species. Because of the time-dependence of the evolutionary process, the multiplicity of independent results would probably tend to converge (by the so-called ‘law of large numbers’) on average values reflecting, with reasonable accuracy, the time elapsed since the divergence of species.

The nine genes and their protein products

We have investigated the following nine nuclear genes: (1) alcohol dehydrogenase (Adh; E.C. 1.1.1.1), (2) aromatic-L-amino acid decarboxylase (Ddc; E.C.4.1.1.28), and its paralogue (3)α-methyl-dopa (Amd; E.C.4.1.1.-), (4) glycerol-3-phosphate dehydrogenase (Gpdh; E.C.1.1.1. 8), (5) glucose-6-phosphate dehydrogenase (G6pd; E.C.1.1.1.49), (6) phosphogluconate dehydrogenase (Pgd; E.C.1.1.1.44), (7) superoxide dismutase (Sod; E.C. 1.15.1.1), (8) triosephosphate isomerase (tpi; E.C.5.3.1.1), and (9) xanthine dehydrogenase (Xdh; E.C.1.1.1. 204) (Table 1.1 and Figure 1.1). Six genes (1–4, 7, 9) have been analysed in 34 species of Diptera, comprising representatives of the families Drosophilidae and Tephritidae (‘Diptera’ dataset in Table 1.1; see also Figure 1.1) and the last six (genes 4–9) in 95 species that include representatives of the three multicellular eukaryote kingdoms, i.e. animals, plants, and fungi (‘Global’ dataset, Table 1.1; see Figure 1.1).

Table 1.1 Likelihood-ratio test carried out on the amino acid data sequences for various genes

The enzymes encoded by the nine genes (i.e. ADH, AMD, DDC, GPDH, G6PD, PGD, SOD, TPI, and XDH) are globular soluble proteins generally composed of two identical subunits with approximate molecular masses of 28, 54, 54, 40, 60, 53, 15, 25, and 145 KDa, respectively. The encoded enzymes are oxidoreductases, except for DDC and AMD, which are carboxylases, and TPI, which is an isomerase.

ADH is a member of the insect-type, or ‘short-chain’ dehydrogenase/reductase family (SDR). In Drosophila melanogaster the Adh coding sequence is interrupted by two introns. The protein consists of two domains for binding the coenzyme (NAD) and the substrate (Benyajati et al. 1981). The crystal structure and reaction mechanisms of ADH have been elucidated in Scaptodrosophila lebanonensis (Benach et al. 1998). The enzyme plays a key role in adaptation to substrates undergoing alcoholic fermentation. It has been intensively investigated in Drosophila from a variety of perspectives (Powell 1997).

Figure 1.1 Tree topology for the species used in this study. Labels and numbers on the branches represent taxonomic categories and divergence times, respectively. Nicotiana and Scaptomyza species are N. tabacum for G6pd and N. plumbaginifolia for Sod; S. adusta for Amd, Ddc, and Sod; and S. albovittata for Adh. a: Gpdh sequences from Kwiatowski and Ayala (1999); b: Ddc sequences from Tatarenkov et al. (1999).

AMD and DDC are paralogues that arose as a result of a gene duplication, holding structural and functional relationships. They code for two decarboxylases involved in morphological differentiation, being essential for the sclerotization and melanization of the newly moulted cuticle of Diptera. In addition, DDC is required for the production of th...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Contributors
• Introduction: Molecular clocks and the fossil record— towards consilience?
• Chapter 1: Molecular clocks: whence and whither?
• Chapter 2: Molecular clocks and a biological trigger for neoproterozoic snowball earth events and the cambrian Explosion
• Chapter 3: Phylogenetic fuses and evolutionary ‘explosions’: conflicting evidence and critical tests
• Chapter 4: The quality of the fossil record
• Chapter 5: Ghost ranges
• Chapter 6: Episodic evolution of nuclear small subunit ribosomal RNA gene in the stem-lineage of Foraminifera
• Chapter 7: Dating the origin of land plants
• Chapter 8: Angiosperm divergence times: congruence and incongruence between fossils and sequence divergence estimates
• Chapter 9: The limitations of the fossil record and the dating of the origin of the bilateria
• Chapter 10: The origin and early evolution of chordates: molecular clocks and the fossil record
• Chapter 11: Bones, molecules, and crown-tetrapod origins
• Chapter 12: The fossil record and molecular clocks: basal radiations within the neornithes
• Systematics association publications