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Page 1: The insect molecular clock

Overview

The insect molecular clock

Simon Y W Ho* and Nathan Lo

School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia.

Abstract Estimating the timescale of evolution forms an important component of many molecular studies of insects. Byobtaining date estimates for significant evolutionary events, we can gain a better understanding of biologicalprocesses such as diversification and adaptation. Evolutionary timescales can be estimated from geneticsequence data using the molecular clock, which was proposed five decades ago and has subsequentlyundergone considerable development. In this article, we provide a summary of the theoretical basis of themolecular clock, including its relationship to the neutral and nearly neutral theories of molecular evolution. Weexplain how the clock can be used to estimate evolutionary timescales from DNA and protein sequence data.Finally, we describe some of the key challenges facing users of the molecular clock in studies of insects,including the problems of rate variation among lineages and over time.

Key words calibration, neutral theory, phylogenetics, relaxed clock, time-dependent rate.

INTRODUCTION

In evolutionary studies of insects it is often useful to have anestimate of the timescale. Traditionally, this has been obtainedfrom the fossil record, which can provide an indicationof when different lineages first appeared. With the ever-increasing availability of molecular data, however, it hasbecome commonplace to estimate evolutionary timescalesusing molecular-clock analyses of DNA or protein sequences.The molecular clock, which refers to a constancy of evolution-ary rates among lineages, has undergone considerable devel-opment since its proposal in the early 1960s by Zuckerkandland Pauling (1962). Fifty years after its birth, we take theopportunity to examine the current use of the molecular clockin entomology.

Insects provide an excellent opportunity for testing evolu-tionary hypotheses across diverse timescales, owing to theirhigh species diversity, ecological impact and 400 Myr evolu-tionary history (Grimaldi & Engel 2005; Yeates et al. 2012).The molecular clock can aid the understanding of a range ofentomological questions, such as the time frame of evolution-ary radiations (Wiegmann et al. 2011), the age of dispersal intonew niches or territories (Leys et al. 2003), the age of host–symbiont relationships (Thézé et al. 2011) and populationdynamics during periods of past environmental change(Gompert et al. 2008; Garrick et al. 2012). In particular themolecular clock is useful for studying insect groups that havepoor fossil records and for shedding light on the evolutionaryorigin of insects (Gaunt & Miles 2002).

In this overview we introduce the theoretical background tothe molecular clock and explain how it can be used to estimateevolutionary timescales. We then review the use of the molecu-lar clock in studies of insects and describe some of the keychallenges.

THE MOLECULAR CLOCK

A brief history

The molecular clock describes a simple but powerful relation-ship between evolutionary change and time. It has an intimateassociation with the neutral theory of molecular evolution,which states that most mutations have a negligible impact onthe fitness of an organism. Under the neutral theory, mutationsare either strongly deleterious or lethal, such that they disap-pear very rapidly, or have such a small effect on fitness thattheir fate is determined by stochastic genetic drift. Kimura(1968) demonstrated that, when these assumptions are met, therate at which spontaneous mutations occur is equivalent to therate at which mutations become fixed in the population.Assuming that rates of spontaneous mutation remain constantamong lineages, we would expect different species to share thesame rate of evolutionary change. Importantly, this resultdepends on the generation time of each species but is inde-pendent of population size (Kimura 1968). As a consequencespecies with shorter times between successive generations areexpected to evolve more quickly than those with longer gen-eration times (assuming the same number of germ-line celldivisions between generations, and similar levels of replicationfidelity).

Subsequent empirical studies demonstrated that the rate ofprotein evolution is largely independent of generation time*[email protected]

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Australian Journal of Entomology (2013) 52, 101–105

© 2013 The AuthorsAustralian Journal of Entomology © 2013 Australian Entomological Society doi:10.1111/aen.12018

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(Laird et al. 1969; Kohne 1970). This motivated the develop-ment of the ‘nearly neutral’ theory (Ohta 1972, 1973), whichstates that a large proportion of mutations have small positiveor negative impacts on fitness. In this framework populationsize is an important factor and determines the relative impor-tance of natural selection and genetic drift. Selection is moreeffective in large populations, whereas drift dominates the fateof mutations in small populations. The nearly neutral theorypredicts that evolutionary rates are constant among lineagesand are independent of generation time. The neutral and nearlyneutral theories provide the theoretical groundwork for themodern molecular clock.

Estimating timescales using the clock

Apart from its fundamental importance in molecular evolu-tionary theory, the molecular clock has long been appreciatedfor its immense utility in estimating evolutionary timescales. Ifthe rate of evolution for a particular gene is constant amonglineages, and if this rate is known, we can estimate the timingof evolutionary events from genetic data. Different genes mayhave different rates of evolution, depending on whether theyare in the nucleus or in organellar genomes, and on otherfactors such as functional constraints at the protein level.

The simplest way to use a molecular clock is to infer a ratebased on a fossil-dated evolutionary divergence, such as thatbetween Drosophila and Anopheles around 240 Ma (Bentonet al. 2009). By dividing the genetic difference between Dro-sophila and Anopheles by this value, we obtain an estimateof the rate of evolutionary divergence. This value is dividedby two to give the per-lineage evolutionary rate, which is thestandard quantity used in molecular studies of timescales. Ifwe assume that the rate of evolution has been constant amonglineages, we can extrapolate our estimated rate in order to infertimes of divergence among other species. Molecular clocks aretypically used in phylogenetic analysis to produce chrono-grams – evolutionary trees in which branch lengths are meas-ured in units of time.

In reality estimating evolutionary timescales from geneticdata is much more complex and challenging. Early in thehistory of the molecular clock, researchers found convincingevidence of rate variation among lineages, in conflict withpredictions from the nearly neutral theory (e.g. Ohta &Kimura 1971; Langley & Fitch 1974). It now appears thatmost data sets fail tests for rate homogeneity among lineages(for reviews, see Bromham & Penny 2003; Kumar 2005).Such observations have inspired the development of ‘relaxed’molecular clocks, which are able to estimate evolutionarytimescales even when rates vary among lineages. Relaxedclocks have been implemented in various phylogeneticmethods, including likelihood (Sanderson 2002) and Baye-sian approaches (Thorne et al. 1998; Drummond et al. 2006;dos Reis & Yang 2011). There have been several comprehen-sive reviews and tests of these methods (e.g. Ho et al. 2005;Renner 2005; Welch & Bromham 2005; Rutschmann 2006).

As illustrated by the Drosophila–Anopheles exampleearlier, we need to calibrate the molecular clock in order to

estimate the evolutionary rate. This represents one of the mostimportant steps of a molecular dating analysis. The clock canbe calibrated by including temporal information from an inde-pendent source, which can then be attached to one or morenodes in the phylogenetic tree. The fossil record is a commonsource of calibrations because it provides an indication ofwhen taxa first appeared and when different lineages diverged.A fossil can be useful for calibration if its age and taxonomicaffinity are able to be determined with confidence. Suchcalibrations are often available for taxa that are readily pre-served, including vertebrates, but many groups of insects havea poor fossil record. Fortunately, temporal information can beobtained from other sources. Some molecular clocks are cali-brated on the basis of biogeographic hypotheses, e.g. by tyingthe divergence of two lineages to the appearance of a physicalbarrier or to colonisations of new habitats (e.g. Fleischer et al.1998).

Intraspecific calibrations can come from studies of ancientDNA, whereby the ages of the samples can be used to estimatethe rate of molecular evolution. Little work has been done onancient genetic data from insects (e.g. Goldstein & Desalle2003), with early claims of extremely old DNA from amber-trapped insects shown to be irreproducible (Austin et al.1997). Another quandary has been the need for destructivesampling of valuable ancient specimens (Reiss 2006), butmethodological developments have the potential to overcomethis problem (Thomsen et al. 2009). On very short timescaleswe can estimate the evolutionary rate by tracking mutations inpopulations bred in the laboratory (Halligan & Keightley2009). The mutation rate has been estimated in this way fornuclear and mitochondrial DNA in Drosophila melanogaster(Haag-Liautard et al. 2007, 2008).

In recent years there has been considerable progress inmethods for selecting and implementing calibrations in phy-logenetic analyses. These developments have made it possibleto select fossil calibrations using rigorous criteria (Gandolfoet al. 2008), to examine the effects of calibration choice(Sauquet et al. 2012) and to model the temporal uncertaintyassociated with calibrations (Ho & Phillips 2009).

RATES OF MOLECULAR EVOLUTIONIN INSECTS

There have been relatively few attempts to estimate rates ofmolecular evolution in insects (reviewed by Papadopoulouet al. 2010). This is partly because of the paucity of fossil-based calibrations for insects, whose soft bodies are not readilypreserved. Instead there is a widespread reliance on publishedestimates of substitution rates. Many authors have adopted themitochondrial DNA rate published by Brower (1994), whichsuggests that the molecule evolves at a rate of 1.15 ¥ 10-8

substitutions/site/year (equivalent to 1.15% per million years).As of December 2012, this study has been cited nearly 800times, attesting to its status as a ‘standard’ rate in molecularstudies of arthropods. Annual citations of this article remainhigh (Fig. 1).

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Considered from a modern perspective, Brower’s (1994)rate estimate has severe shortcomings. The rate estimate wasmade from five calibrations, ranging from 300 years to3.25 Myr. The five data points were based on a variety ofarthropod taxa and different regions of the mitochondrialgenome, with only two of the data points being based on DNAsequence data. Brower (1994) did not attempt to quantify theuncertainty in any of these estimates, including the combinedrate estimate from the total data set.

Taking these problems into account, especially in view ofthe significant methodological developments that have takenplace over the past two decades, it is remarkable that theBrower rate continues to form the basis of many molecularestimates of insect evolutionary timescales. However, there isgrowing doubt about the validity and general applicability ofthe ‘standard’ Brower rate. Papadopoulou et al. (2010) con-ducted a detailed study of mitochondrial rates in insects andpresented an estimate of the substitution rate in tenebrionidbeetles, a group with low dispersal ability. Their data setincluded multiple representatives of six genera separated by awell-established biogeographic barrier, the mid-Aegeantrench. By conducting a rigorous analysis using a range ofsophisticated methods, they estimated rates for two commonlyused mitochondrial genes, cytochrome oxidase 1 (cox1) and16S rRNA, and two nuclear genes (Mp20 and 28S rRNA).Papadopoulou et al. (2010) estimated evolutionary rates of1.77 � 0.19% per Myr for cox1, 0.54 � 0.09% per Myr for16S rRNA, 1.84 � 1.52% per Myr for the Mp20 intron and0.06% � 0.03% per Myr for 28S rRNA. Although the com-bined cox1 and 16S mitochondrial rate (1.35% per Myr) wassimilar to the estimate of 1.15% per Myr by Brower (1994),Papadopoulou et al. (2010) also emphasised the importance ofchoosing the right model of DNA sequence evolution; the useof a poorly fitting model can lead to an underestimation of therate.

In spite of the progress in estimating molecular rates ininsects, relying on published ‘standard’ rate estimates carriesat least two major risks. First, rates of molecular evolution canvary substantially among lineages, meaning that any singlerate might be highly inaccurate for a given group of insects.

Second, rates vary with the observational timescale, such thatrates appear to be higher over short periods than over longerperiods of time. We briefly discuss these two problems here.

Rate variation among insect lineages

It has long been known that rates of molecular evolution differamong lineages, and studies of molecular rates in insects havefound appreciable levels of variation among genes and amongspecies (e.g. Carmean et al. 1992; Whiting et al. 1997; Takano1998). In their landmark study of the insect molecular clock,Gaunt and Miles (2002) found that the evolutionary rate of themitochondrial gene cox1 varied almost 40-fold among line-ages. Exceptionally high rates of evolution have been observedin certain insect orders, notably Hymenoptera (Crozier et al.1989) and Phthiraptera (Johnson et al. 2003). Evidence ofsignificant rate variation also exists within orders, includingDiptera (Wiegmann et al. 2003), Lepidoptera (Thomas et al.2006), Hymenoptera (Thomas et al. 2006) and Coleoptera(Pons et al. 2010).

Some proportion of the rate variation among lineages can beexplained by differences in life-history traits. Among inverte-brates, there is evidence of a correlation between rate andgeneration time (Thomas et al. 2010), but not between ratesand body size (Thomas et al. 2006). In addition Bromham andLeys (2005) showed that highly eusocial and parasitic lineageshad elevated rates of evolution compared with their respectivenon-social and eusocial relatives, possibly owing to a reducedeffective population size. This pattern was observed in bees,wasps, ants and termites. Despite our growing understandingof the factors that influence evolutionary rates, it remains dif-ficult to make quantitative predictions about molecular rates inunstudied taxa.

In view of the widespread evidence of rate variation amonginsect lineages, it is advisable to test for a molecular clockwhen analysing sequence data. If a strict molecular clock isrejected, relaxed-clock methods can be used to model the ratevariation among lineages while estimating the evolutionarytimescale.

Rate variation across different timescales

Studies of various taxa have indicated that rates of mitochon-drial evolution are time dependent, with very high rates esti-mated in short-term studies of mutation-accumulation linesand much lower rates inferred in phylogenetic analyses cali-brated using fossil data (Ho & Larson 2006). Such a trend ofdeclining rates over time has been observed in insect mito-chondrial DNA (Haag-Liautard et al. 2008; Papadopoulouet al. 2010) (Fig. 2). The time dependence of rates is consistentwith our understanding of the molecular evolutionary processbecause estimates of rates over short time frames tend toinclude the transient deleterious mutations that have not yetbeen removed by purifying selection. However, there are otherfactors that can produce similar trends in rates, includingvarious biases in estimation methods (for a recent review, seeHo et al. 2011). If the pattern is not due solely to methodo-

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1995 1997 1999 2001 2003 2005 2007 2009 2011

Fig. 1. Annual citations of Brower (1994) and Papadopoulouet al. (2010). The data were obtained from Web of Science(accessed 26 December 2012). ( ) Brower, ( )Papadopoulou.

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logical artefacts, then it is necessary to allow different rates toapply at different taxonomic levels.

The time dependence of rates has considerable implicationsfor estimating evolutionary timescales. In a recent study ofDrosophila, Cutter (2008) used an estimate of the spontaneousmutation rate and corrected it for the effects of selection. Thisallowed the rate to be extrapolated backwards in time so thatthe evolutionary time frame of Drosophila could be inferred.Although this method produced date estimates with a largedegree of uncertainty, it explicitly addressed the problem oftime-dependent rates and enabled the timescale to be estimatedin the absence of reliable fossil calibrations.

A practical solution to the problem of time-dependent ratesis to identify reliable calibrations within the group of interest.Unfortunately, it is rare to find useful fossil data that caninform estimates of evolutionary timescales within species.Researchers must typically rely on biogeographic calibrationsfor population-level studies of insects. Another potential solu-tion is the inclusion of ancient DNA sequences from thespecies of interest. These can provide calibrations that are upto tens of thousands of years old and have typically yieldedhigh estimates of rates (Ho et al. 2007). Improvements inDNA extraction and sequencing technologies make this apromising avenue for future research.

CONCLUDING REMARKS

Molecular clocks offer a useful tool for studying evolutionarytimescales in insects. There are many associated pitfalls, butthese can generally be avoided by careful and consideredanalysis. Good practice involves rigorous selection of

nucleotide substitution models, molecular clock models andcalibrations. Rather than relying on ‘standard’ rates of mito-chondrial evolution for insects or arthropods, which tend torepresent averages across different taxa and genes, a moredesirable alternative is to seek out reliable opportunities forcalibration within the group of interest. The insect fossil recordis better than generally appreciated (Grimaldi & Engel 2005),so options for calibrations should be fully explored. Whendoing so, one should bear in mind that rate estimates are timedependent and will vary with the scale of analysis. By takingthese factors into account, estimates from the molecular clockcan be significantly improved, providing a reliable basis forfurther evolutionary inference and interpretation.

ACKNOWLEDGEMENTS

This work was supported by the Australian Research Council.

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Fig. 2. Estimate of the mitochondrial evolutionary rate(substitutions/site/year) plotted against calibration age (years),showing the time-dependent pattern of rates. Note the log scaleson both axes. The line of best fit (power curve) is shown. Thedata were obtained directly from Papadopoulou et al. (2010),except for the leftmost data point which is from Haag-Liautardet al. (2008) and is based on the assumption that there are 10generations of Drosophila melanogaster per year.

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Accepted for publication 16 January 2013.

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