Dynamics and timekeeping in biological systems.

This article introduces three reviews on the theme of circadian rhythms.

Methods for Assessing Population Relationships and History Using Genomic Data.

Genetic data contain a record of our evolutionary history. The availability of large-scale datasets of human populations from various geographic areas and timescales, coupled with advances in the computational methods to analyze these data, has transformed our ability to use genetic data to learn about our evolutionary past. Here, we review some of the widely used statistical methods to explore and characterize population relationships and history using genomic data. We describe the intuition behind commonly used approaches, their interpretation, and important limitations. For illustration, we apply some of these techniques to genome-wide autosomal data from 929 individuals representing 53 worldwide populations that are part of the Human Genome Diversity Project. Finally, we discuss the new frontiers in genomic methods to learn about population history. In sum, this review highlights the power (and limitations) of DNA to infer features of human evolutionary history, complementing the knowledge gleaned from other disciplines, such as archaeology, anthropology, and linguistics.

Time-of-day effects on ex vivo muscle contractility following short-term satellite cell ablation.

Muscle isometric torque fluctuates according to time-of-day with such variation owed to the influence of circadian molecular clock genes. Satellite cells (SCs), the muscle stem cell population, also express molecular clock genes with several contractile-related genes oscillating in a diurnal pattern. Currently, limited evidence exists regarding the relationship between SCs and contractility, although long-term SC ablation alters muscle contractile function. Whether there are acute alterations in contractility following SC ablation and with respect to the time-of-day is unknown. We investigated whether short-term SC ablation affected contractile function at two times of day and whether any such alterations led to different extents of eccentric contraction-induced injury. Using an established mouse model to deplete SCs, we characterized muscle clock gene expression and ex vivo contractility at two times-of-day (morning: 0700 and afternoon: 1500). Morning-SC+ animals demonstrated ∼25%-30% reductions in tetanic/eccentric specific forces and, after eccentric injury, exhibited ∼30% less force-loss and ∼50% less dystrophinnegative fibers versus SC- counterparts; no differences were noted between Afternoon groups (Morning-SC+: -5.63 ± 0.61, Morning-SC-: -7.93 ± 0.61; N/cm2; P < 0.05) (Morning-SC+: 32 ± 2.1, Morning-SC-: 64 ± 10.2; dystrophinnegative fibers; P < 0.05). As Ca++ kinetics underpin force generation, we also evaluated caffeine-induced contracture force as an indirect marker of Ca++ availability and found similar force reductions in Morning-SC+ vs. SC- mice. We conclude that force production is reduced in the presence of SCs in the morning but not in the afternoon, suggesting that SCs may have a time-of-day influence over contractile function.NEW & NOTEWORTHY Muscle isometric torque fluctuates according to time-of-day with such variation owed to molecular clock regulation. Satellite cells (SCs) have recently demonstrated diurnal characteristics related to muscle physiology. In our work, force production was reduced in the presence versus absence of SCs in the morning but, not in the afternoon. Morning-SC+ animals, producing lower force, sustained lesser degrees of injury versus SC- counterparts. One potential mechanism underpinning lower forces produced appears to be lower calcium availability.

Two Notorious Nodes: a Critical Examination of Relaxed Molecular Clock Age Estimates of the Bilaterian Animals and Placental Mammals.

The popularity of relaxed clock Bayesian inference of clade origin timings has generated several recent publications with focal results considerably older than the fossils of the clades in question. Here we critically examine two such clades: the animals (with focus on the bilaterians); and the mammals (with focus on the placentals). Each example displays a set of characteristic pathologies which, although much commented on, are rarely corrected for. We conclude that in neither case does the molecular clock analysis provide any evidence for an origin of the clade deeper than what is suggested by the fossil record. In addition, both these clades have other features (including, in the case of the placental mammals, proximity to a large mass extinction) that allow us to generate precise expectations of the timings of their origins. Thus, in these instances the fossil record can provide a powerful test of molecular clock methodology, and why it goes astray; and we have every reason to think these problems are general.

The Circadian Clocks, Oscillations of Pain-Related Mediators, and Pain.

The circadian clock is a biochemical oscillator that is synchronized with solar time. Normal circadian rhythms are necessary for many physiological functions. Circadian rhythms have also been linked with many physiological functions, several clinical symptoms, and diseases. Accumulating evidence suggests that the circadian clock appears to modulate the processing of nociceptive information. Many pain conditions display a circadian fluctuation pattern clinically. Thus, the aim of this review is to summarize the existing knowledge about the circadian clocks involved in diurnal rhythms of pain. Possible cellular and molecular mechanisms regarding the connection between the circadian clocks and pain are discussed.

Immunological and inflammatory effects of infectious diseases in circadian rhythm disruption and future therapeutic directions.

BACKGROUND: Circadian rhythm is characterised by daily variations in biological activity to align with the light and dark cycle. These diurnal variations, in turn, influence physiological functions such as blood pressure, temperature, and sleep-wake cycle. Though it is well established that the circadian pathway is linked to pro-inflammatory responses and circulating immune cells, its association with infectious diseases is widely unknown. OBJECTIVE: This comprehensive review aims to describe the association between circadian rhythm and host immune response to various kinds of infection. METHODS: We conducted a literature search in databases Pubmed/Medline and Science direct. Our paper includes a comprehensive analysis of findings from articles in English which was related to our hypothesis. FINDINGS: Molecular clocks determine circadian rhythm disruption in response to infection, influencing the host's response toward infection. Moreover, there is a complex interplay with intrinsic oscillators of pathogens and the influence of specific infectious processes on the CLOCK: BMAL1 pathway. Such mechanisms vary for bacterial and viral infections, both well studied in the literature. However, less is known about the association of parasitic infections and fungal pathogens with circadian rhythm modulation. CONCLUSION: It is shown that bidirectional relationships exist between circadian rhythm disruption and infectious process, which contains interplay between the host's and pathogens' circadian oscillator, immune response, and the influence of specific infectious. Further studies exploring the modulations of circadian rhythm and immunity can offer novel explanations of different susceptibilities to infection and can lead to therapeutic avenues in circadian immune modulation of infectious diseases.

Biogeography and genome size evolution of the oldest extant vascular plant genus, Equisetum (Equisetaceae).

BACKGROUND AND AIMS: Extant plant groups with a long fossil history are key elements in understanding vascular plant evolution. Horsetails (Equisetum, Equisetaceae) have a nearly continuous fossil record dating back to the Carboniferous, but their phylogenetic and biogeographic patterns are still poorly understood. We use here the most extensive phylogenetic analysis to date as a framework to evaluate their age, biogeography and genome size evolution. METHODS: DNA sequences of four plastid loci were used to estimate divergence times and investigate the biogeographic history of all extant species of Equisetum. Flow cytometry was used to study genome size evolution against the framework of phylogenetic relationships in Equisetum. KEY RESULTS: On a well-supported phylogenetic tree including all extant Equisetum species, a molecular clock calibrated with multiple fossils places the node at which the outgroup and Equisetum diverged at 343 Mya (Early Carboniferous), with the first major split among extant species occurring 170 Mya (Middle Jurassic). These dates are older than those reported in some other recent molecular clock studies but are largely in agreement with a timeline established by fossil appearance in the geological record. Representatives of evergreen subgenus Hippochaete have much larger genome sizes than those of deciduous subgenus Equisetum, despite their shared conserved chromosome number. Subgenus Paramochaete has an intermediate genome size and maintains the same number of chromosomes. CONCLUSIONS: The first divergences among extant members of the genus coincided with the break-up of Pangaea and the resulting more humid, warmer climate. Subsequent tectonic activity most likely involved vicariance events that led to species divergences combined with some more recent, long-distance dispersal events. We hypothesize that differences in genome size between subgenera may be related to the number of sperm flagellae.

Molecular clocks in ancient proteins: Do they reflect the age at death even after millennia?

Age at death estimation in cases of human skeletal finds is an important task in forensic medicine as well as in anthropology. In forensic medicine, methods based on "molecular clocks" in dental tissues and bone play an increasing role. The question, whether these methods are applicable also in cases with post-depositional intervals far beyond the forensically relevant period, was investigated for two "protein clocks", the accumulation of D-aspartic acid (D-Asp) and the accumulation of pentosidine (Pen) in dentine. Eight teeth of skeletons from different burial sites in Austria and with post-depositional intervals between c. 1216 and c. 8775 years were analysed. The results of age at death estimation based on D-Asp and Pen in dentine were compared to that derived from a classical morphological examination. Age at death estimation based on D-Asp resulted consistently in false high values. This finding can be explained by a post-mortem accumulation of D-Asp that may be enhanced by protein degradation. In contrast, the Pen-based age estimates fitted well with the morphological age diagnoses. The described effect of post-mortem protein degradation is negligible in forensically relevant time horizons, but not for post-depositional intervals of thousands of years. That means that the "D-Asp clock" loses its functionality with increasing post-depositional intervals, whereas Pen seems to be very stable. The "Pen-clock" may have the potential to become an interesting supplement to the existing repertoire of methods even in cases with extremely long post-depositional intervals. Further investigations have to test this hypothesis.

MondoA:MLX complex regulates glucose-dependent gene expression and links to circadian rhythm in liver and brain of the freeze-tolerant wood frog, Rana sylvatica.

The wood frog, Rana sylvatica, is one of only a few vertebrate species that display natural freeze tolerance. Frogs survive the freezing of about two-thirds of their body water as extracellular ice over the winter months. Multiple adaptations support freeze tolerance including metabolic rate depression and the production of huge amounts of glucose (often 200 mM or more) as a cryoprotectant that protects cells from freeze damage. To understand how high glucose levels affect gene expression, we studied MondoA, a glucose sensing transcription factor, and its partner MLX (Max-like protein) to assess their ability to modulate the expression of genes involved in glucose metabolism and circadian rhythm. Wood frog liver and brain tissues were analyzed, assessing protein levels, nuclear distribution, and DNA binding activity of MondoA:MLX during freezing (24 h at - 2.5 °C) and subsequent thawing (8 h returned to 5 °C), as compared with 5 °C controls. Downstream targets of MondoA:MLX were also evaluated: TXNIP (thioredoxin interacting protein), ARRDC4 (arrestin domain containing 4), HK-2 (hexokinase-2), PFKFB-3 (6-phosphofructo-2-kinase isozyme 3) and KLF-10 (Kruppel-like factor-10). Both KLF-10 and PFKFB-3 are also involved in circadian dependant regulation which was also explored in the current study via analysis of BMAL-1 (aryl hydrocarbon receptor nuclear translocator-like protein 1) and CLOCK (circadian locomotor output cycles kaput) proteins. Our data establish the MondoA-MLX complex as active under the hyperglycemic conditions in liver to regulate glucose metabolism and may also link to circadian rhythm in liver via KLF-10 and PFKFB-3 but not in brain.

Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

Several molecular modifications accumulate in the human organism with increasing age. Some of these "molecular clocks" in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of D-aspartic acid (D-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5-11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, D-Asp) and expand our set of tissues.

Uplift-driven diversification in the Hengduan Mountains, a temperate biodiversity hotspot.

A common hypothesis for the rich biodiversity found in mountains is uplift-driven diversification-that orogeny creates conditions favoring rapid in situ speciation of resident lineages. We tested this hypothesis in the context of the Qinghai-Tibetan Plateau (QTP) and adjoining mountain ranges, using the phylogenetic and geographic histories of multiple groups of plants to infer the tempo (rate) and mode (colonization versus in situ diversification) of biotic assembly through time and across regions. We focused on the Hengduan Mountains region, which in comparison with the QTP and Himalayas was uplifted more recently (since the late Miocene) and is smaller in area and richer in species. Time-calibrated phylogenetic analyses show that about 8 million y ago the rate of in situ diversification increased in the Hengduan Mountains, significantly exceeding that in the geologically older QTP and Himalayas. By contrast, in the QTP and Himalayas during the same period the rate of in situ diversification remained relatively flat, with colonization dominating lineage accumulation. The Hengduan Mountains flora was thus assembled disproportionately by recent in situ diversification, temporally congruent with independent estimates of orogeny. This study shows quantitative evidence for uplift-driven diversification in this region, and more generally, tests the hypothesis by comparing the rate and mode of biotic assembly jointly across time and space. It thus complements the more prevalent method of examining endemic radiations individually and could be used as a template to augment such studies in other biodiversity hotspots.

Genomic Signature of an Avian Lilliput Effect across the K-Pg Extinction.

Survivorship following major mass extinctions may be associated with a decrease in body size-a phenomenon called the Lilliput Effect. Body size is a strong predictor of many life history traits (LHTs), and is known to influence demography and intrinsic biological processes. Pronounced changes in organismal size throughout Earth history are therefore likely to be associated with concomitant genome-wide changes in evolutionary rates. Here, we report pronounced heterogeneity in rates of molecular evolution (varying up to $\sim$20-fold) across a large-scale avian phylogenomic data set and show that nucleotide substitution rates are strongly correlated with body size and metabolic rate. We also identify potential body size reductions associated with the Cretaceous-Paleogene (K-Pg) transition, consistent with a Lilliput Effect in the wake of that mass extinction event. We posit that selection for reduced body size across the K-Pg extinction horizon may have resulted in transient increases in substitution rate along the deepest branches of the extant avian tree of life. This "hidden" rate acceleration may result in both strict and relaxed molecular clocks over-estimating the age of the avian crown group through the relationship between life history and demographic parameters that scale with molecular substitution rate. If reductions in body size (and/or selection for related demographic parameters like short generation times) are a common property of lineages surviving mass extinctions, this phenomenon may help resolve persistent divergence time debates across the tree of life. Furthermore, our results suggest that selection for certain LHTs may be associated with deterministic molecular evolutionary outcomes.

The origin of animals: Can molecular clocks and the fossil record be reconciled?

The evolutionary emergence of animals is one of the most significant episodes in the history of life, but its timing remains poorly constrained. Molecular clocks estimate that animals originated and began diversifying over 100 million years before the first definitive metazoan fossil evidence in the Cambrian. However, closer inspection reveals that clock estimates and the fossil record are less divergent than is often claimed. Modern clock analyses do not predict the presence of the crown-representatives of most animal phyla in the Neoproterozoic. Furthermore, despite challenges provided by incomplete preservation, a paucity of phylogenetically informative characters, and uncertain expectations of the anatomy of early animals, a number of Neoproterozoic fossils can reasonably be interpreted as metazoans. A considerable discrepancy remains, but much of this can be explained by the limited preservation potential of early metazoans and the difficulties associated with their identification in the fossil record. Critical assessment of both records may permit better resolution of the tempo and mode of early animal evolution.

Fast and Accurate Estimates of Divergence Times from Big Data.

Ongoing advances in sequencing technology have led to an explosive expansion in the molecular data available for building increasingly larger and more comprehensive timetrees. However, Bayesian relaxed-clock approaches frequently used to infer these timetrees impose a large computational burden and discourage critical assessment of the robustness of inferred times to model assumptions, influence of calibrations, and selection of optimal data subsets. We analyzed eight large, recently published, empirical datasets to compare time estimates produced by RelTime (a non-Bayesian method) with those reported by using Bayesian approaches. We find that RelTime estimates are very similar to Bayesian approaches, yet RelTime requires orders of magnitude less computational time. This means that the use of RelTime will enable greater rigor in molecular dating, because faster computational speeds encourage more extensive testing of the robustness of inferred timetrees to prior assumptions (models and calibrations) and data subsets. Thus, RelTime provides a reliable and computationally thrifty approach for dating the tree of life using large-scale molecular datasets.

Molecular clocks indicate turnover and diversification of modern coleoid cephalopods during the Mesozoic Marine Revolution.

Coleoid cephalopod molluscs comprise squid, cuttlefish and octopuses, and represent nearly the entire diversity of modern cephalopods. Sophisticated adaptations such as the use of colour for camouflage and communication, jet propulsion and the ink sac highlight the unique nature of the group. Despite these striking adaptations, there are clear parallels in ecology between coleoids and bony fishes. The coleoid fossil record is limited, however, hindering confident analysis of the tempo and pattern of their evolution. Here we use a molecular dataset (180 genes, approx. 36 000 amino acids) of 26 cephalopod species to explore the phylogeny and timing of cephalopod evolution. We show that crown cephalopods diverged in the Silurian-Devonian, while crown coleoids had origins in the latest Palaeozoic. While the deep-sea vampire squid and dumbo octopuses have ancient origins extending to the Early Mesozoic Era, 242 ± 38 Ma, incirrate octopuses and the decabrachian coleoids (10-armed squid) diversified in the Jurassic Period. These divergence estimates highlight the modern diversity of coleoid cephalopods emerging in the Mesozoic Marine Revolution, a period that also witnessed the radiation of most ray-finned fish groups in addition to several other marine vertebrates. This suggests that that the origin of modern cephalopod biodiversity was contingent on ecological competition with marine vertebrates.

Recent origin and rapid speciation of Neotropical orchids in the world's richest plant biodiversity hotspot.

The Andean mountains of South America are the most species-rich biodiversity hotspot worldwide with c. 15% of the world's plant species, in only 1% of the world's land surface. Orchids are a key element of the Andean flora, and one of the most prominent components of the Neotropical epiphyte diversity, yet very little is known about their origin and diversification. We address this knowledge gap by inferring the biogeographical history and diversification dynamics of the two largest Neotropical orchid groups (Cymbidieae and Pleurothallidinae), using two unparalleled, densely sampled orchid phylogenies (including more than 400 newly generated DNA sequences), comparative phylogenetic methods, geological and biological datasets. We find that the majority of Andean orchid lineages only originated in the last 20-15 million yr. Andean lineages are derived from lowland Amazonian ancestors, with additional contributions from Central America and the Antilles. Species diversification is correlated with Andean orogeny, and multiple migrations and recolonizations across the Andes indicate that mountains do not constrain orchid dispersal over long timescales. Our study sheds new light on the timing and geography of a major Neotropical diversification, and suggests that mountain uplift promotes species diversification across all elevational zones.

Coevolution with pollinating resin midges led to resin-filled nurseries in the androecia, gynoecia and tepals of Kadsura (Schisandraceae).

Background and Aims: Resin is a defence against herbivores and a floral reward in a few African and South American species whose bee pollinators collect it for nest construction. Here we describe a new role for floral resin from the Asian genus Kadsura (Schisandraceae). Kadsura tepals tightly cover a globe formed by carpels (in females) or near-fused stamens with fleshy connectives (in male flowers of most, but not all species). Methods: We carried out field observations at four sites in China and used pollinator behavioural assays, chemical analyses and time-calibrated insect and plant phylogenies to investigate the specificity of the interactions and their relationship to floral structure. Key Results: Nocturnal resin midges ( Resseliella , Cecidomyiidae) walk around on the flowers' sexual organs to oviposit, thereby transferring pollen and wounding tissues. The larvae then develop in resin-filled chambers. Male and female floral scents are dominated by α-pinene, while the resinous exudate is dominated by caryophyllene. As revealed by barcoding of multiple midge larvae per flower species, the mutualisms are species specific and appear to have evolved over the past 6-9 million years. Conclusions: Resin feeding, not pollen or ovule feeding, by midge larvae explains the abundant Kadsura exudates, highlighting the poorly known world of nocturnal flower-fly interactions.

A Protocol for Diagnosing the Effect of Calibration Priors on Posterior Time Estimates: A Case Study for the Cambrian Explosion of Animal Phyla.

We present a procedure to test the effect of calibration priors on estimated times, which applies a recently developed calibration-free approach (RelTime) method that produces relative divergence times for all nodes in the tree. We illustrate this protocol by applying it to a timetree of metazoan diversification (Erwin DH, Laflamme M, Tweedt SM, Sperling EA, Pisani D, Peterson KJ. 2011. The Cambrian conundrum: early divergence and later ecological success in the early history of animals. Science 334:1091-1097.), which placed the divergence of animal phyla close to the time of the Cambrian explosion inferred from the fossil record. These analyses revealed that the two maximum-only calibration priors in the pre-Cambrian are the primary determinants of the young divergence times among animal phyla in this study. In fact, these two maximum-only calibrations produce divergence times that severely violate minimum boundaries of almost all of the other 22 calibration constraints. The use of these 22 calibrations produces dates for metazoan divergences that are hundreds of millions of years earlier in the Proterozoic. Our results encourage the use of calibration-free approaches to identify most influential calibration constraints and to evaluate their impact in order to achieve biologically robust interpretations.

Macroevolutionary assembly of ant/plant symbioses: Pseudomyrmex ants and their ant-housing plants in the Neotropics.

Symbioses include some of the clearest cases of coevolution, but their origin, loss or reassembly with different partners can rarely be inferred. Here we use ant/plant symbioses involving three plant clades to investigate the evolution of symbioses. We generated phylogenies for the big-eyed arboreal ants (Pseudomyrmecinae), including 72% of their 286 species, as well as for five of their plant host groups, in each case sampling more than 61% of the species. We show that the ant-housing Vachellia (Mimosoideae) clade and its ants co-diversified for the past 5 Ma, with some species additionally colonized by younger plant-nesting ant species, some parasitic. An apparent co-radiation of ants and Tachigali (Caesalpinioideae) was followed by waves of colonization by the same ant clade, and subsequent occupation by a younger ant group. Wide crown and stem age differences between the ant-housing genus Triplaris (Polygonaceae) and its obligate ant inhabitants, and stochastic trait mapping, indicate that its domatium evolved earlier than the ants now occupying it, suggesting previous symbioses that dissolved. Parasitic ant species evolved from generalists, not from mutualists, and are younger than the mutualistic systems they parasitize. Our study illuminates the macroevolutionary assembly of ant/plant symbioses, which has been highly dynamic, even in very specialized systems.

Green Web or megabiased clock? Plant fossils from Gondwanan Patagonia speak on evolutionary radiations.

Evolutionary divergence-age estimates derived from molecular 'clocks' are frequently correlated with paleogeographic, paleoclimatic and extinction events. One prominent hypothesis based on molecular data states that the dominant pattern of Southern Hemisphere biogeography is post-Gondwanan clade origins and subsequent dispersal across the oceans in a metaphoric 'Green Web'. We tested this idea against well-dated Patagonian fossils of 19 plant lineages, representing organisms that actually lived on Gondwana. Most of these occurrences are substantially older than their respective, often post-Gondwanan molecular dates. The Green Web interpretation probably results from directional bias in molecular results. Gondwanan history remains fundamental to understanding Southern Hemisphere plant radiations, and we urge significantly greater caution when using molecular dating to interpret the biological impacts of geological events.