Diversification Rate is Associated with Rate of Molecular Evolution in Ray-Finned Fish (Actinopterygii).

Understanding the factors that drive diversification of taxa across the tree of life is a key focus of macroevolutionary research. While the effects of life history, ecology, climate and geography on diversity have been studied for many taxa, the relationship between molecular evolution and diversification has received less attention. However, correlations between rates of molecular evolution and diversification rate have been detected in a range of taxa, including reptiles, plants and birds. A correlation between rates of molecular evolution and diversification rate is a prediction of several evolutionary theories, including the evolutionary speed hypothesis which links variation in mutation rates to differences in speciation rates. If it is widespread, such correlations could also have significant practical impacts, if they are not adequately accounted for in phylogenetic inference of evolutionary rates and timescales. Ray-finned fish (Actinopterygii) offer a prime target to test for this relationship due to their extreme variation in clade size suggesting a wide range of diversification rates. We employ both a sister-pairs approach and a whole-tree approach to test for correlations between substitution rate and net diversification. We also collect life history and ecological trait data and account for potential confounding factors including body size, latitude, max depth and reef association. We find evidence to support a relationship between diversification and synonymous rates of nuclear evolution across two published backbone phylogenies, as well as weak evidence for a relationship between mitochondrial nonsynonymous rates and diversification at the genus level.

Interdisciplinary research has consistently lower funding success.

Interdisciplinary research is widely considered a hothouse for innovation, and the only plausible approach to complex problems such as climate change. One barrier to interdisciplinary research is the widespread perception that interdisciplinary projects are less likely to be funded than those with a narrower focus. However, this commonly held belief has been difficult to evaluate objectively, partly because of lack of a comparable, quantitative measure of degree of interdisciplinarity that can be applied to funding application data. Here we compare the degree to which research proposals span disparate fields by using a biodiversity metric that captures the relative representation of different fields (balance) and their degree of difference (disparity). The Australian Research Council's Discovery Programme provides an ideal test case, because a single annual nationwide competitive grants scheme covers fundamental research in all disciplines, including arts, humanities and sciences. Using data on all 18,476 proposals submitted to the scheme over 5 consecutive years, including successful and unsuccessful applications, we show that the greater the degree of interdisciplinarity, the lower the probability of being funded. The negative impact of interdisciplinarity is significant even when number of collaborators, primary research field and type of institution are taken into account. This is the first broad-scale quantitative assessment of success rates of interdisciplinary research proposals. The interdisciplinary distance metric allows efficient evaluation of trends in research funding, and could be used to identify proposals that require assessment strategies appropriate to interdisciplinary research.

A phylogenomic approach reveals a low somatic mutation rate in a long-lived plant.

Somatic mutations can have important effects on the life history, ecology, and evolution of plants, but the rate at which they accumulate is poorly understood and difficult to measure directly. Here, we develop a method to measure somatic mutations in individual plants and use it to estimate the somatic mutation rate in a large, long-lived, phenotypically mosaic Eucalyptus melliodora tree. Despite being 100 times larger than Arabidopsis, this tree has a per-generation mutation rate only ten times greater, which suggests that this species may have evolved mechanisms to reduce the mutation rate per unit of growth. This adds to a growing body of evidence that illuminates the correlated evolutionary shifts in mutation rate and life history in plants.

Macroevolutionary and macroecological approaches to understanding the evolution of stress tolerance in plants.

Environmental stress response in plants has been studied using a wide range of approaches, from lab-based investigation of biochemistry and genetics, to glasshouse studies of physiology and growth rates, to field-based trials and ecological surveys. It is also possible to investigate the evolution of environmental stress responses using macroevolutionary and macroecological analyses, analysing data from many different species, providing a new perspective on the way that environmental stress shapes the evolution and distribution of biodiversity. "Macroevoeco" approaches can produce intriguing results and new ways of looking at old problems. In this review, we focus on studies using phylogenetic analysis to illuminate macroevolutionary patterns in the evolution of environmental stress tolerance in plants. We follow a particular thread from our own research-evolution of salt tolerance-as a case study that illustrates a macroevolutionary way of thinking that opens up a range of broader questions on the evolution of environmental stress tolerances. We consider some potential future applications of macroevolutionary and macroecological analyses to understanding how diverse groups of plants evolve in response to environmental stress, which may allow better prediction of current stress tolerance and a way of predicting the capacity of species to adapt to changing environmental stresses over time.

Rate of language evolution is affected by population size.

The effect of population size on patterns and rates of language evolution is controversial. Do languages with larger speaker populations change faster due to a greater capacity for innovation, or do smaller populations change faster due to more efficient diffusion of innovations? Do smaller populations suffer greater loss of language elements through founder effects or drift, or do languages with more speakers lose features due to a process of simplification? Revealing the influence of population size on the tempo and mode of language evolution not only will clarify underlying mechanisms of language change but also has practical implications for the way that language data are used to reconstruct the history of human cultures. Here, we provide, to our knowledge, the first empirical, statistically robust test of the influence of population size on rates of language evolution, controlling for the evolutionary history of the populations and formally comparing the fit of different models of language evolution. We compare rates of gain and loss of cognate words for basic vocabulary in Polynesian languages, an ideal test case with a well-defined history. We demonstrate that larger populations have higher rates of gain of new words whereas smaller populations have higher rates of word loss. These results show that demographic factors can influence rates of language evolution and that rates of gain and loss are affected differently. These findings are strikingly consistent with general predictions of evolutionary models.

Detecting Macroevolutionary Self-Destruction from Phylogenies.

Phylogenetic analyses have lent support to the concept of lineage selection: that biological lineages can have heritable traits that influence their capacity to persist and diversify, and thereby affect their representation in biodiversity. While many discussions have focused on "positive" lineage selection, where stably heritable properties of lineages enhance their diversification rate, there are also intriguing examples that seem to represent "negative" lineage selection, where traits reduce the likelihood that a lineage will persist or speciate. In this article, we test whether a particular pattern of negative lineage selection is detectable from the distributions of the trait on a phylogeny. "Self-destructive" traits are those that arise often but then disappear again because they confer either a raised extinction rate or they are prone to a high rate of trait loss. For such a trait, the reconstructed origins will tend to be dispersed across the tips of the phylogeny, rather than defining large clades of related lineages that all share the trait. We examine the utility of four possible measures of "tippiness" as potential indicators of macroevolutionary self-destruction, applying them to phylogenies on which trait evolution has been simulated under different combinations of parameters for speciation, extinction, trait gain, and trait loss. We use an efficient simulation approach that starts with the required number of tips with and without the trait and uses a model to work "backwards" to construct different possible trees that result in that set of tips. We then apply these methods to a number of case studies: salt tolerance in grasses, color polymorphism in birds of prey, and selfing in nightshades. We find that the relative age of species, measured from tip length, can indicate a reduced speciation rate but does not identify traits that increase the extinction rate or the trait loss rate. We show that it is possible to detect cases of macroevolutionary self-destruction by considering the number of tips with the trait that arise from each inferred origin, and the degree to which the trait is scattered across the phylogeny. These metrics, and the methods we present, may be useful for testing macroevolutionary hypotheses from phylogenetic patterns.

Longevity Is Linked to Mitochondrial Mutation Rates in Rockfish: A Test Using Poisson Regression.

The mitochondrial theory of ageing proposes that the cumulative effect of biochemical damage in mitochondria causes mitochondrial mutations and plays a key role in ageing. Numerous studies have applied comparative approaches to test one of the predictions of the theory: That the rate of mitochondrial mutations is negatively correlated with longevity. Comparative studies face three challenges in detecting correlates of mutation rate: Covariation of mutation rates between species due to ancestry, covariation between life-history traits, and difficulty obtaining accurate estimates of mutation rate. We address these challenges using a novel Poisson regression method to examine the link between mutation rate and lifespan in rockfish (Sebastes). This method has better performance than traditional sister-species comparisons when sister species are too recently diverged to give reliable estimates of mutation rate. Rockfish are an ideal model system: They have long life spans with indeterminate growth and little evidence of senescence, which minimizes the confounding tradeoffs between lifespan and fecundity. We show that lifespan in rockfish is negatively correlated to rate of mitochondrial mutation, but not the rate of nuclear mutation. The life history of rockfish allows us to conclude that this relationship is unlikely to be driven by the tradeoffs between longevity and fecundity, or by the frequency of DNA replications in the germline. Instead, the relationship is compatible with the hypothesis that mutation rates are reduced by selection in long-lived taxa to reduce the chance of mitochondrial damage over its lifespan, consistent with the mitochondrial theory of ageing.

Testing hypotheses in macroevolution.

Experimental manipulation of microevolution (changes in frequency of heritable traits in populations) has shed much light on evolutionary processes. But many evolutionary processes occur on scales that are not amenable to experimental manipulation. Indeed, one of the reasons that macroevolution (changes in biodiversity over time, space and lineages) has sometimes been a controversial topic is that processes underlying the generation of biological diversity generally operate at scales that are not open to direct observation or manipulation. Macroevolutionary hypotheses can be tested by using them to generate predictions then asking whether observations from the biological world match those predictions. Each study that identifies significant correlations between evolutionary events, processes or outcomes can generate new predictions that can be further tested with different datasets, allowing a cumulative process that may narrow down on plausible explanations, or lead to rejection of other explanations as inconsistent or unsupported. A similar approach can be taken even for unique events, for example by comparing patterns in different regions, lineages, or time periods. I will illustrate the promise and pitfalls of these approaches using a range of examples, and discuss the problems of inferring causality from significant evolutionary associations.

Salt tolerance is evolutionarily labile in a diverse set of angiosperm families.

BACKGROUND: Salt tolerance in plants is rare, yet it is found across a diverse set of taxonomic groups. This suggests that, although salt tolerance often involves a set of complex traits, it has evolved many times independently in different angiosperm lineages. However, the pattern of evolution of salt tolerance can vary dramatically between families. A recent phylogenetic study of the Chenopodiaceae (goosefoot family) concluded that salt tolerance has a conserved evolutionary pattern, being gained early in the evolution of the lineage then retained by most species in the family. Conversely, a phylogenetic study of the Poaceae (grass family) suggested over 70 independent gains of salt tolerance, most giving rise to only one or a few salt tolerant species. Here, we use a phylogenetic approach to explore the macroevolutionary patterns of salt tolerance in a sample of angiosperm families, in order to ask whether either of these two patterns - deep and conserved or shallow and labile - represents a common mode of salt tolerance evolution. We analyze the distribution of halophyte species across the angiosperms and identify families with more or less halophytes than expected under a random model. Then, we explore the phylogenetic distribution of halophytes in 22 families using phylogenetic comparative methods. RESULTS: We find that salt tolerance species have been reported from over one-third of angiosperm families, but that salt tolerant species are not distributed evenly across angiosperm families. We find that salt tolerance has been gained hundreds of times over the history of the angiosperms. In a few families, we find deep and conserved gains of salt tolerance, but in the majority of families analyzed, we find that the pattern of salt tolerant species is best explained by multiple independent gains that occur near the tips of the phylogeny and often give rise to only one or a few halophytes. CONCLUSIONS: Our results suggest that the pattern of many independent gains of salt tolerance near the tips of the phylogeny is found in many angiosperm families. This suggests that the pattern reported in the grasses of high evolutionary lability may be a common feature of salt tolerance evolution in angiosperms.

Exploring the Relationships between Mutation Rates, Life History, Genome Size, Environment, and Species Richness in Flowering Plants.

A new view is emerging of the interplay between mutation at the genomic level, substitution at the population level, and diversification at the lineage level. Many studies have suggested that rate of molecular evolution is linked to rate of diversification, but few have evaluated competing hypotheses. By analyzing sequences from 130 families of angiosperms, we show that variation in the synonymous substitution rate is correlated among genes from the mitochondrial, chloroplast, and nuclear genomes and linked to differences in traits among families (average height and genome size). Within each genome, synonymous rates are correlated to nonsynonymous substitution rates, suggesting that increasing the mutation rate results in a faster rate of genome evolution. Substitution rates are correlated with species richness in protein-coding sequences from the chloroplast and nuclear genomes. These data suggest that species traits contribute to lineage-specific differences in the mutation rate that drive both synonymous and nonsynonymous rates of change across all three genomes, which in turn contribute to greater rates of divergence between populations, generating higher rates of diversification. These observations link mutation in individuals to population-level processes and to patterns of lineage divergence.

Macroevolutionary patterns of salt tolerance in angiosperms.

BACKGROUND: Halophytes are rare, with only 0·25% of angiosperm species able to complete their life cycle in saline conditions. This could be interpreted as evidence that salt tolerance is difficult to evolve. However, consideration of the phylogenetic distribution of halophytes paints a different picture: salt tolerance has evolved independently in many different lineages, and halophytes are widely distributed across angiosperm families. In this Viewpoint, I will consider what phylogenetic analysis of halophytes can tell us about the macroevolution of salt tolerance. HYPOTHESIS: Phylogenetic analyses of salt tolerance have shown contrasting patterns in different families. In some families, such as chenopods, salt tolerance evolved early in the lineage and has been retained in many lineages. But in other families, including grasses, there have been a surprisingly large number of independent origins of salt tolerance, most of which are relatively recent and result in only one or a few salt-tolerant species. This pattern of many recent origins implies either a high transition rate (salt tolerance is gained and lost often) or a high extinction rate (salt-tolerant lineages do not tend to persist over macroevolutionary timescales). While salt tolerance can evolve in a wide range of genetic backgrounds, some lineages are more likely to produce halophytes than others. This may be due to enabling traits that act as stepping stones to developing salt tolerance. The ability to tolerate environmental salt may increase tolerance of other stresses or vice versa. CONCLUSIONS: Phylogenetic analyses suggest that enabling traits and cross-tolerances may make some lineages more likely to adapt to increasing salinization, a finding that may prove useful in assessing the probable impact of rapid environmental change on vegetation communities, and in selecting taxa to develop for use in landscape rehabilitation and agriculture.

Predicting species' tolerance to salinity and alkalinity using distribution data and geochemical modelling: a case study using Australian grasses.

BACKGROUND AND AIMS: Salt tolerance has evolved many times independently in different plant groups. One possible explanation for this pattern is that it builds upon a general suite of stress-tolerance traits. If this is the case, then we might expect a correlation between salt tolerance and other tolerances to different environmental stresses. This association has been hypothesized for salt and alkalinity tolerance. However, a major limitation in investigating large-scale patterns of these tolerances is that lists of known tolerant species are incomplete. This study explores whether species' salt and alkalinity tolerance can be predicted using geochemical modelling for Australian grasses. The correlation between taxa found in conditions of high predicted salinity and alkalinity is then assessed. METHODS: Extensive occurrence data for Australian grasses is used together with geochemical modelling to predict values of pH and electrical conductivity to which species are exposed in their natural distributions. Using parametric and phylogeny-corrected tests, the geochemical predictions are evaluated using a list of known halophytes as a control, and it is determined whether taxa that occur in conditions of high predicted salinity are also found in conditions of high predicted alkalinity. KEY RESULTS: It is shown that genera containing known halophytes have higher predicted salinity conditions than those not containing known halophytes. Additionally, taxa occurring in high predicted salinity tend to also occur in high predicted alkalinity. CONCLUSIONS: Geochemical modelling using species' occurrence data is a potentially useful approach to predict species' relative natural tolerance to challenging environmental conditions. The findings also demonstrate a correlation between salinity tolerance and alkalinity tolerance. Further investigations can consider the phylogenetic distribution of specific traits involved in these ecophysiological strategies, ideally by incorporating more complete, finer-scale geochemical information, as well as laboratory experiments.

Combining Molecular, Macroevolutionary, and Macroecological Perspectives on the Generation of Diversity.

Charles Darwin presented a unified process of diversification driven by the gradual accumulation of heritable variation. The growth in DNA databases and the increase in genomic sequencing, combined with advances in molecular phylogenetic analyses, gives us an opportunity to realize Darwin's vision, connecting the generation of variation to the diversification of lineages. The rate of molecular evolution is correlated with the rate of diversification across animals and plants, but the relationship between genome change and speciation is complex: Mutation rates evolve in response to life history and niche; substitution rates are influenced by mutation, selection, and population size; rates of acquisition of reproductive isolation vary between populations; and traits, niches, and distribution can influence diversification rates. The connection between mutation rate and diversification rate is one part of the complex and varied story of speciation, which has theoretical importance for understanding the generation of biodiversity and also practical impacts on the use of DNA to understand the dynamics of speciation over macroevolutionary timescales.

Rates of molecular evolution and diversification in plants: chloroplast substitution rates correlate with species-richness in the Proteaceae.

BACKGROUND: Many factors have been identified as correlates of the rate of molecular evolution, such as body size and generation length. Analysis of many molecular phylogenies has also revealed correlations between substitution rates and clade size, suggesting a link between rates of molecular evolution and the process of diversification. However, it is not known whether this relationship applies to all lineages and all sequences. Here, in order to investigate how widespread this phenomenon is, we investigate patterns of substitution in chloroplast genomes of the diverse angiosperm family Proteaceae. We used DNA sequences from six chloroplast genes (6278bp alignment with 62 taxa) to test for a correlation between diversification and the rate of substitutions. RESULTS: Using phylogenetically-independent sister pairs, we show that species-rich lineages of Proteaceae tend to have significantly higher chloroplast substitution rates, for both synonymous and non-synonymous substitutions. CONCLUSIONS: We show that the rate of molecular evolution in chloroplast genomes is correlated with net diversification rates in this large plant family. We discuss the possible causes of this relationship, including molecular evolution driving diversification, speciation increasing the rate of substitutions, or a third factor causing an indirect link between molecular and diversification rates. The link between the synonymous substitution rate and clade size is consistent with a role for the mutation rate of chloroplasts driving the speed of reproductive isolation. We find no significant differences in the ratio of non-synonymous to synonymous substitutions between lineages differing in net diversification rate, therefore we detect no signal of population size changes or alteration in selection pressures that might be causing this relationship.

Parasitic plants have increased rates of molecular evolution across all three genomes.

BACKGROUND: Theoretical models and experimental evidence suggest that rates of molecular evolution could be raised in parasitic organisms compared to non-parasitic taxa. Parasitic plants provide an ideal test for these predictions, as there are at least a dozen independent origins of the parasitic lifestyle in angiosperms. Studies of a number of parasitic plant lineages have suggested faster rates of molecular evolution, but the results of some studies have been mixed. Comparative analysis of all parasitic plant lineages, including sequences from all three genomes, is needed to examine the generality of the relationship between rates of molecular evolution and parasitism in plants. RESULTS: We analysed DNA sequence data from the mitochondrial, nuclear and chloroplast genomes for 12 independent evolutionary origins of parasitism in angiosperms. We demonstrated that parasitic lineages have a faster rate of molecular evolution than their non-parasitic relatives in sequences for all three genomes, for both synonymous and nonsynonymous substitutions. CONCLUSIONS: Our results prove that raised rates of molecular evolution are a general feature of parasitic plants, not confined to a few taxa or specific genes. We discuss possible causes for this relationship, including increased positive selection associated with host-parasite arms races, relaxed selection, reduced population size or repeated bottlenecks, increased mutation rates, and indirect causal links with generation time and body size. We find no evidence that faster rates are due to smaller effective populations sizes or changes in selection pressure. Instead, our results suggest that parasitic plants have a higher mutation rate than their close non-parasitic relatives. This may be due to a direct connection, where some aspect of the parasitic lifestyle drives the evolution of raised mutation rates. Alternatively, this pattern may be driven by an indirect connection between rates and parasitism: for example, parasitic plants tend to be smaller than their non-parasitic relatives, which may result in more cell generations per year, thus a higher rate of mutations arising from DNA copy errors per unit time. Demonstration that adoption of a parasitic lifestyle influences the rate of genomic evolution is relevant to attempts to infer molecular phylogenies of parasitic plants and to estimate their evolutionary divergence times using sequence data.

Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales).

Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.

Mutation rate is linked to diversification in birds.

How does genome evolution affect the rate of diversification of biological lineages? Recent studies have suggested that the overall rate of genome evolution is correlated with the rate of diversification. If true, this claim has important consequences for understanding the process of diversification, and implications for the use of DNA sequence data to reconstruct evolutionary history. However, the generality and cause of this relationship have not been established. Here, we test the relationship between the rate of molecular evolution and net diversification with a 19-gene, 17-kb DNA sequence dataset from 64 families of birds. We show that rates of molecular evolution are positively correlated to net diversification in birds. Using a 7.6-kb dataset of protein-coding DNA, we show that the synonymous substitution rate, and therefore the mutation rate, is correlated to net diversification. Further analysis shows that the link between mutation rates and net diversification is unlikely to be the indirect result of correlations with life-history variables that may influence both quantities, suggesting that there might be a causal link between mutation rates and net diversification.

Neighbours and relatives: accounting for spatial distribution when testing causal hypotheses in cultural evolution.

Many important and interesting hypotheses about cultural evolution are evaluated using cross-cultural correlations: if knowing one particular feature of a culture (e.g. environmental conditions such as temperature, humidity or parasite load) allows you to predict other features (e.g. language features, religious beliefs, cuisine), it is often interpreted as indicating a causal link between the two (e.g. hotter climates carry greater disease risk, which encourages belief in supernatural forces and favours the use of antimicrobial ingredients in food preparation; dry climates make the production of distinct tones more difficult). However, testing such hypotheses from cross-cultural comparisons requires us to take proximity of cultures into account: nearby cultures share many aspects of their environment and are more likely to be similar in many culturally inherited traits. This can generate indirect associations between environment and culture which could be misinterpreted as signals of a direct causal link. Evaluating examples of cross-cultural correlations from the literature, we show that significant correlations interpreted as causal relationships can often be explained as a result of similarity between neighbouring cultures. We discuss some strategies for sorting the explanatory wheat from the co-varying chaff, distinguishing incidental correlations from causal relationships.

Reconstructing past species assemblages reveals the changing patterns and drivers of extinction through time.

Predicting future species extinctions from patterns of past extinctions or current threat status relies on the assumption that the taxonomic and biological selectivity of extinction is consistent through time. If the driving forces of extinction change through time, this assumption may be unrealistic. Testing the consistency of extinction patterns between the past and the present has been difficult, because the phylogenetically explicit methods used to model present-day extinction risk typically cannot be applied to the data from the fossil record. However, the detailed historical and fossil records of the New Zealand avifauna provide a unique opportunity to reconstruct a complete, large faunal assemblage for different periods in the past. Using the first complete phylogeny of all known native New Zealand bird species, both extant and extinct, we show how the taxonomic and phylogenetic selectivity of extinction, and biological correlates of extinction, change from the pre-human period through Polynesian and European occupation, to the present. These changes can be explained both by changes in primary threatening processes, and by the operation of extinction filter effects. The variable patterns of extinction through time may confound attempts to identify risk factors that apply across time periods, and to infer future species declines from past extinction patterns and current threat status.

Investigating the reliability of molecular estimates of evolutionary time when substitution rates and speciation rates vary.

BACKGROUND: An accurate timescale of evolutionary history is essential to testing hypotheses about the influence of historical events and processes, and the timescale for evolution is increasingly derived from analysis of DNA sequences. But variation in the rate of molecular evolution complicates the inference of time from DNA. Evidence is growing for numerous factors, such as life history and habitat, that are linked both to the molecular processes of mutation and fixation and to rates of macroevolutionary diversification. However, the most widely used methods rely on idealised models of rate variation, such as the uncorrelated and autocorrelated clocks, and molecular dating methods are rarely tested against complex models of rate change. One relationship that is not accounted for in molecular dating is the potential for interaction between molecular substitution rates and speciation, a relationship that has been supported by empirical studies in a growing number of taxa. If these relationships are as widespread as current evidence suggests, they may have a significant influence on molecular dates. RESULTS: We simulate phylogenies and molecular sequences under three different realistic rate variation models-one in which speciation rates and substitution rates both vary but are unlinked, one in which they covary continuously and one punctuated model in which molecular change is concentrated in speciation events, using empirical case studies to parameterise realistic simulations. We test three commonly used "relaxed clock" molecular dating methods against these realistic simulations to explore the degree of error in molecular dates under each model. We find average divergence time inference errors ranging from 12% of node age for the unlinked model when reconstructed under an uncorrelated rate prior using BEAST 2, to up to 91% when sequences evolved under the punctuated model are reconstructed under an autocorrelated prior using PAML. CONCLUSIONS: We demonstrate the potential for substantial errors in molecular dates when both speciation rates and substitution rates vary between lineages. This study highlights the need for tests of molecular dating methods against realistic models of rate variation generated from empirical parameters and known relationships.