Salvage cryotherapy for prostate cancer.

BACKGROUND: Most men diagnosed with prostate cancer will be candidates for active treatment and 20 to 50% of patients treated with organ preserving strategies recur within the prostate. Optimal treatment of recurrence is controversial. Prostate cryosurgery has been increasingly used as primary, recurrence and focal treatment for prostate cancer. METHODS: We analysed 55 patients submitted to cryotherapy as salvage treatment after recurrence. RESULTS: Study population presented with a mean age of 70.9 ± 6.2 years, mean initial PSA of 7.6 ng/ml and average prostate volume by ultrasound of 43.2 ± 14.7 grams. Mean follow-up was of 18.0 months. Biochemical free survival at one year of follow-up was of 85%. CONCLUSIONS: Cryotherapy can be an effective and safe treatment for recurrence after primary curative treatment failure.

Impacto de la realización de resonancia magnética multiparamétrica (RMmp) antes de la braquiterapia en pacientes con cáncer de próstata / Prior MRI-imaging impact of patients submitted to brachytherapy for prostate cancer

Introducción La braquiterapia es una opción terapéutica bien establecida para el cáncer de próstata. El uso de la resonancia magnética multiparamétrica (RMmp) para la estadificación y el diagnóstico del cáncer de próstata ha supuesto un cambio en el paradigma actual. En este estudio pretendemos evaluar el impacto, en términos de la recurrencia bioquímica y el tiempo hasta el nadir, de la realización de RMmp para evaluar la presencia de lesiones extracapsulares antes de la braquiterapia en pacientes con cáncer de próstata. Métodos Revisar los datos de 73 pacientes sometidos a braquiterapia. Se evaluaron los siguientes factores: edad, PSA inicial, resultados de la estadificación local por RMmp, ISUP, nadir, tiempo hasta el nadir, PSA a un año, recurrencia bioquímica y tiempo hasta la recurrencia. Resultados La mediana de edad fue de 68años (51-72) y la mediana de seguimiento, de 53meses (30-72). En cuanto a la modalidad de imagen para el diagnóstico, el 30,1% (n=22) de los pacientes se sometieron a RMmp. En el grupo de RMmp, el 90,9% (n=20) tenían al menos una lesión sospechosa en la RMmp. El tiempo hasta el nadir fue de 27meses (3-64) en los pacientes en los que no se realizó RMmp y de 23,5meses (2-48) en los pacientes sometidos a RMmp (p=0,244). La mediana del nadir fue de 0,42ng/ml (<0,001-2) en los pacientes sometidos a RMmp, frente a 0,28ng/ml (<0,001-4) en los pacientes sin RMmp (p=0,062) La recurrencia según los criterios Phoenix fue del 9% (n=2) en los pacientes con RMmp y del 9,2% (n=5) en pacientes sin RMmp (p=0,456), con una mediana de seguimiento de 43meses (12-72) para el grupo con RMmp y de 58meses (30-78) para el grupo sin RMmp. Ambos grupos fueron estadísticamente similares. Conclusión Nuestros resultados nos permiten concluir que en nuestra serie la RMmp no influyó en la recurrencia bioquímica, el tiempo hasta el nadir o el valor del nadir (AU)

Introduction Brachytherapy for the treatment of prostate cancer is a well-established option. Use of Multiparametric Magnetic Resonance Imaging (mpMRI) for staging and diagnosis of prostate cancer has come to change the current paradigm. In this study we aim to assess the impact of performing mpMRI to evaluate the presence of extracapsular lesions before brachytherapy in patients with prostate cancer concerning biochemical recurrence and time to nadir. Methods Review data from 73 patients submitted to brachytherapy. The following factors were evaluated: age, initial PSA, MRI local staging results, ISUP, nadir, time to nadir, PSA at one-year, biochemical recurrence, and time to recurrence. Results Median age was 68years (51-72) and median follow-up 53months (30-72). Concerning imaging modality 30.1% (n=22) patients performed mpMRI. In the mpMRI group, 90.9% (n=20) had at least one suspect lesion on mpMRI. Time to nadir was 27months (3-64) in patients where mpMRI was not performed and 23.5months (2-48) in patients submitted to mpMRI (P=.244). The median value of nadir was 0.42ng/mL (<0.001-2) in patients submitted to mpMRI and vs 0.28ng/mL (<0.001-4) in patients without MRI (P=.062) Recurrence utilizing Phoenix criteria was 9% (n=2) in patients with MRI and 9.2% (n=5) without mpMRI (P=.456), median follow-up of 43months (12-72) for the MRI group with 58months (30-78) for the non-mpMRI group. Both groups were statistically similar. Conclusion Our results allow us to conclude that in our series MRI did not influence biochemical recurrence, time to nadir, or nadir value (AU)

Prior MRI-imaging impact of patients submitted to brachytherapy for prostate cancer.

INTRODUCTION: Brachytherapy for the treatment of prostate cancer is a well-established option. Use of Multiparametric Magnetic Resonance Imaging (mpMRI) for staging and diagnosis of prostate cancer has come to change the current paradigm. In this study we aim to assess the impact of performing mpMRI to evaluate the presence of extracapsular lesions before brachytherapy in patients with prostate cancer concerning biochemical recurrence and time to nadir. METHODS: Review data from 73 patients submitted to brachytherapy. The following factors were evaluated: age, initial PSA, MRI local staging results, ISUP, nadir, time to nadir, PSA at one-year, biochemical recurrence, and time to recurrence. RESULTS: Median age was 68 years (51-72) and median follow-up 53 months (30-72). Concerning imaging modality 30,1% (n = 22) patients performed mpMRI. In the mpMRI group, 90.9% (n = 20) had at least one suspect lesion on mpMRI. Time to nadir was 27 months (3-64) in patients where mpMRI was not performed and 23.5 months (2-48) in patients submitted to mpMRI (P = .244). The median value of nadir was 0.42 ng/mL (<0.001-2) in patients submitted to mpMRI and vs 0.28 ng/mL (<0.001-4) in patients without MRI (P = .062) Recurrence utilizing Phoenix criteria was 9% (n = 2) in patients with MRI and 9.2% (n = 5) without mpMRI (P = .456), median follow-up of 43 months (12-72) for the MRI group with 58 months (30-78) for the non-mpMRI group. Both groups were statistically similar. CONCLUSION: Our results allow us to conclude that in our series MRI did not influence biochemical recurrence, time to nadir, or nadir value.

Dating Microbial Evolution with MCMCtree.

This protocol explains how to use the program MCMCtree to estimate divergence times in microbial phylogenies. The main advantage of MCMCtree is the implementation of an approximation to the molecular data likelihood that dramatically speeds up computation during Bayesian MCMC sampling of divergence times and evolutionary rates. The approximation allows the analysis of large phylogenies with hundreds of taxa and molecular alignments with thousands or millions of sites. Two examples are used to illustrate Bayesian clock dating with MCMCtree. The first is a phylogeny of (mostly) microbial eukaryotes and prokaryotes encompassing the major groups of life on Earth, and for which fossil information, to calibrate the nodes of the phylogeny, is available. The second is a phylogeny of influenza viruses with known sampling times. An overview of Bayesian MCMC sampling is given as well as practical advice on issues such as construction of the time and rate prior and assessment of convergence of MCMC chains. Strategies for estimating times in microbial phylogenies for which neither fossil information nor sampling times are known are discussed.

Receptor deorphanization in an echinoderm reveals kisspeptin evolution and relationship with SALMFamide neuropeptides.

BACKGROUND: Kisspeptins are neuropeptides that regulate reproductive maturation in mammals via G-protein-coupled receptor-mediated stimulation of gonadotropin-releasing hormone secretion from the hypothalamus. Phylogenetic analysis of kisspeptin-type receptors indicates that this neuropeptide signaling system originated in a common ancestor of the Bilateria, but little is known about kisspeptin signaling in invertebrates. RESULTS: Contrasting with the occurrence of a single kisspeptin receptor in mammalian species, here, we report the discovery of an expanded family of eleven kisspeptin-type receptors in a deuterostome invertebrate - the starfish Asterias rubens (phylum Echinodermata). Furthermore, neuropeptides derived from four precursor proteins were identified as ligands for six of these receptors. One or more kisspeptin-like neuropeptides derived from two precursor proteins (ArKPP1, ArKPP2) act as ligands for four A. rubens kisspeptin-type receptors (ArKPR1,3,8,9). Furthermore, a family of neuropeptides that act as muscle relaxants in echinoderms (SALMFamides) are ligands for two A. rubens kisspeptin-type receptors (ArKPR6,7). The SALMFamide neuropeptide S1 (or ArS1.4) and a 'cocktail' of the seven neuropeptides derived from the S1 precursor protein (ArS1.1-ArS1.7) act as ligands for ArKPR7. The SALMFamide neuropeptide S2 (or ArS2.3) and a 'cocktail' of the eight neuropeptides derived from the S2 precursor protein (ArS2.1-ArS2.8) act as ligands for ArKPR6. CONCLUSIONS: Our findings reveal a remarkable diversity of neuropeptides that act as ligands for kisspeptin-type receptors in starfish and provide important new insights into the evolution of kisspeptin signaling. Furthermore, the discovery of the hitherto unknown relationship of kisspeptins with SALMFamides, neuropeptides that were discovered in starfish prior to the identification of kisspeptins in mammals, presents a radical change in perspective for research on kisspeptin signaling.

A species-level timeline of mammal evolution integrating phylogenomic data.

High-throughput sequencing projects generate genome-scale sequence data for species-level phylogenies1-3. However, state-of-the-art Bayesian methods for inferring timetrees are computationally limited to small datasets and cannot exploit the growing number of available genomes4. In the case of mammals, molecular-clock analyses of limited datasets have produced conflicting estimates of clade ages with large uncertainties5,6, and thus the timescale of placental mammal evolution remains contentious7-10. Here we develop a Bayesian molecular-clock dating approach to estimate a timetree of 4,705 mammal species integrating information from 72 mammal genomes. We show that increasingly larger phylogenomic datasets produce diversification time estimates with progressively smaller uncertainties, facilitating precise tests of macroevolutionary hypotheses. For example, we confidently reject an explosive model of placental mammal origination in the Palaeogene8 and show that crown Placentalia originated in the Late Cretaceous with unambiguous ordinal diversification in the Palaeocene/Eocene. Our Bayesian methodology facilitates analysis of complete genomes and thousands of species within an integrated framework, making it possible to address hitherto intractable research questions on species diversifications. This approach can be used to address other contentious cases of animal and plant diversifications that require analysis of species-level phylogenomic datasets.

A Mutation-Selection Model of Protein Evolution under Persistent Positive Selection.

We use first principles of population genetics to model the evolution of proteins under persistent positive selection (PPS). PPS may occur when organisms are subjected to persistent environmental change, during adaptive radiations, or in host-pathogen interactions. Our mutation-selection model indicates protein evolution under PPS is an irreversible Markov process, and thus proteins under PPS show a strongly asymmetrical distribution of selection coefficients among amino acid substitutions. Our model shows the criteria ω>1 (where ω is the ratio of nonsynonymous over synonymous codon substitution rates) to detect positive selection is conservative and indeed arbitrary, because in real proteins many mutations are highly deleterious and are removed by selection even at positively selected sites. We use a penalized-likelihood implementation of the PPS model to successfully detect PPS in plant RuBisCO and influenza HA proteins. By directly estimating selection coefficients at protein sites, our inference procedure bypasses the need for using ω as a surrogate measure of selection and improves our ability to detect molecular adaptation in proteins.

Nectar-feeding bats and birds show parallel molecular adaptations in sugar metabolism enzymes.

In most vertebrates, the demand for glucose as the primary substrate for cellular respiration is met by the breakdown of complex carbohydrates, or energy is obtained by protein and lipid catabolism. In contrast, a few bat and bird species have convergently evolved to subsist on nectar, a sugar-rich mixture of glucose, fructose, and sucrose.1-4 How these nectar-feeders have adapted to cope with life-long high sugar intake while avoiding the onset of metabolic syndrome and diabetes5-7 is not understood. We analyzed gene sequences obtained from 127 taxa, including 22 nectar-feeding bat and bird genera that collectively encompass four independent origins of nectarivory. We show these divergent taxa have undergone pervasive molecular adaptation in sugar catabolism pathways, including parallel selection in key glycolytic and fructolytic enzymes. We also uncover convergent amino acid substitutions in the otherwise evolutionarily conserved aldolase B (ALDOB), which catalyzes rate-limiting steps in fructolysis and glycolysis, and the mitochondrial gatekeeper pyruvate dehydrogenase (PDH), which links glycolysis and the tricarboxylic acid cycle. Metabolomic profile and enzyme functional assays are consistent with increased respiratory flux in nectar-feeding bats and help explain how these taxa can both sustain hovering flight and efficiently clear simple sugars. Taken together, our results indicate that nectar-feeding bats and birds have undergone metabolic adaptations that have enabled them to exploit a unique energy-rich dietary niche among vertebrates.

Understanding the Dependence of Nanoparticles Magnetothermal Properties on Their Size for Hyperthermia Applications: A Case Study for La-Sr Manganites.

Magnetic oxides are promising materials for alternative health diagnoses and treatments. The aim of this work is to understand the dependence of the heating power with the nanoparticle (NP) mean size, for the manganite composition La0.75Sr0.25MnO3 (LSMO)-the one with maximum critical temperature for the whole La/Sr ratio of the series. We have prepared four different samples, each one annealed at different temperatures, in order to produce different mean NP sizes, ranging from 26 nm up to 106 nm. Magnetization measurements revealed a FC-ZFC irreversibility and from the coercive field as function of temperature we determined the blocking temperature. A phase diagram was delivered as a function of the NP mean size and, based on this, the heating mechanism understood. Small NPs (26 nm) is heated up within the paramagnetic range of temperature (T>Tc), and therefore provide low heating efficiency; while bigger NPs are heated up, from room temperature, within the magnetically blocked range of temperature (TT>TB), for intermediate mean diameter size of 37 nm, with maximum efficiency of heat transfer.

Dietary Diversification and Specialization in Neotropical Bats Facilitated by Early Molecular Evolution.

Dietary adaptation is a major feature of phenotypic and ecological diversification, yet the genetic basis of dietary shifts is poorly understood. Among mammals, Neotropical leaf-nosed bats (family Phyllostomidae) show unmatched diversity in diet; from a putative insectivorous ancestor, phyllostomids have radiated to specialize on diverse food sources including blood, nectar, and fruit. To assess whether dietary diversification in this group was accompanied by molecular adaptations for changing metabolic demands, we sequenced 89 transcriptomes across 58 species and combined these with published data to compare ∼13,000 protein coding genes across 66 species. We tested for positive selection on focal lineages, including those inferred to have undergone dietary shifts. Unexpectedly, we found a broad signature of positive selection in the ancestral phyllostomid branch, spanning genes implicated in the metabolism of all major macronutrients, yet few positively selected genes at the inferred switch to plantivory. Branches corresponding to blood- and nectar-based diets showed selection in loci underpinning nitrogenous waste excretion and glycolysis, respectively. Intriguingly, patterns of selection in metabolism genes were mirrored by those in loci implicated in craniofacial remodeling, a trait previously linked to phyllostomid dietary specialization. Finally, we show that the null model of the widely-used branch-site test is likely to be misspecified, with the implication that the test is too conservative and probably under-reports true cases of positive selection. Our findings point to a complex picture of adaptive radiation, in which the evolution of new dietary specializations has been facilitated by early adaptations combined with the generation of new genetic variation.

Pedigree-based and phylogenetic methods support surprising patterns of mutation rate and spectrum in the gray mouse lemur.

Mutations are the raw material on which evolution acts, and knowledge of their frequency and genomic distribution is crucial for understanding how evolution operates at both long and short timescales. At present, the rate and spectrum of de novo mutations have been directly characterized in relatively few lineages. Our study provides the first direct mutation-rate estimate for a strepsirrhine (i.e., the lemurs and lorises), which comprises nearly half of the primate clade. Using high-coverage linked-read sequencing for a focal quartet of gray mouse lemurs (Microcebus murinus), we estimated the mutation rate to be among the highest calculated for a mammal at 1.52 × 10-8 (95% credible interval: 1.28 × 10-8-1.78 × 10-8) mutations/site/generation. Further, we found an unexpectedly low count of paternal mutations, and only a modest overrepresentation of mutations at CpG sites. Despite the surprising nature of these results, we found both the rate and spectrum to be robust to the manipulation of a wide range of computational filtering criteria. We also sequenced a technical replicate to estimate a false-negative and false-positive rate for our data and show that any point estimate of a de novo mutation rate should be considered with a large degree of uncertainty. For validation, we conducted an independent analysis of context-dependent substitution types for gray mouse lemur and five additional primate species for which de novo mutation rates have also been estimated. These comparisons revealed general consistency of the mutation spectrum between the pedigree-based and the substitution-rate analyses for all species compared.

Dire wolves were the last of an ancient New World canid lineage.

Dire wolves are considered to be one of the most common and widespread large carnivores in Pleistocene America1, yet relatively little is known about their evolution or extinction. Here, to reconstruct the evolutionary history of dire wolves, we sequenced five genomes from sub-fossil remains dating from 13,000 to more than 50,000 years ago. Our results indicate that although they were similar morphologically to the extant grey wolf, dire wolves were a highly divergent lineage that split from living canids around 5.7 million years ago. In contrast to numerous examples of hybridization across Canidae2,3, there is no evidence for gene flow between dire wolves and either North American grey wolves or coyotes. This suggests that dire wolves evolved in isolation from the Pleistocene ancestors of these species. Our results also support an early New World origin of dire wolves, while the ancestors of grey wolves, coyotes and dholes evolved in Eurasia and colonized North America only relatively recently.

Radon and thoron exhalation rate, emanation factor and radioactivity risks of building materials of the Iberian Peninsula.

Radon (222Rn) and thoron (220Rn) are radioactive gases emanating from geological materials. Inhalation of these gases is closely related to an increase in the probability of lung cancer if the levels are high. The majority of studies focus on radon, and the thoron is normally ignored because of its short half-life (55.6 s). However, thoron decay products can also cause a significant increase in dose. In buildings with high radon levels, the main mechanism for entry of radon is pressure-driven flow of soil gas through cracks in the floor. Both radon and thoron can also be released from building materials to the indoor atmosphere. In this work, we study the radon and thoron exhalation and emanation properties of an extended variety of common building materials manufactured in the Iberian Peninsula (Portugal and Spain) but exported and used in all countries of the world. Radon and thoron emission from samples collected in the closed chamber was measured by an active method that uses a continuous radon/thoron monitor. The correlations between exhalation rates of these gases and their parent nuclide exhalation (radium/thorium) concentrations were examined. Finally, indoor radon and thoron and the annual effective dose were calculated from radon/thoron concentrations in the closed chamber. Zircon is the material with the highest concentration values of 226Ra and 232Th and the exhalation and emanation rates. Also in the case of zircon and some granites, the annual effective dose was higher than the annual exposure limit for the general public of 1 mSv y-1, recommended by the European regulations.

Molecular Clocks without Rocks: New Solutions for Old Problems.

Molecular data have been used to date species divergences ever since they were described as documents of evolutionary history in the 1960s. Yet, an inadequate fossil record and discordance between gene trees and species trees are persistently problematic. We examine how, by accommodating gene tree discordance and by scaling branch lengths to absolute time using mutation rate and generation time, multispecies coalescent (MSC) methods can potentially overcome these challenges. We find that time estimates can differ - in some cases, substantially - depending on whether MSC methods or traditional phylogenetic methods that apply concatenation are used, and whether the tree is calibrated with pedigree-based mutation rates or with fossils. We discuss the advantages and shortcomings of both approaches and provide practical guidance for data analysis when using these methods.

Prognostic value of p16INK4a overexpression in penile cancer.

INTRODUCTION: Penile cancer is rare, accounting for less than 1% of all male cancers in industrialized countries. It is most common in areas of high prevalence of HPV, being a third of cases attributed to the carcinogenic effect of HPV. Tumour cells infected with HPV overexpress p16INK4a, as such p16INK4a has been used as a surrogate of HPV infections. OBJECTIVE: To evaluate the prognostic factor of p16INK4a overexpression in penile cancer. METHODS: Retrospective analysis of patients diagnosed with penile cancer, submitted to surgery in a Portuguese Oncological Institution in the last 20 years (n = 35). Histological review of surgical pieces and immunohistochemical identification of p16INK4a. Relation between p16INK4a and the following factors were studied: age, histological subtype, tumour dimensions, grade, TNM stage, perineural invasion, perivascular invasion, disease free survival (DFS) and cancer specific survival (CSS). RESULTS: p16INK4a was positive in 8 patients (22.9%). Identification of p16INK4a did not correlate with none of the histopathological factors. In this work we identified a better DFS and CSS in patients positive for p16INK4a (DFS at 36 months was 100.0% vs. 66.7%; CSS at 36 months was 100.0% vs. 70.4%), although without statistical significance (p > 0.05). In multivariate analysis of histopathological factors studied, only N staging correlated with DFS and CSS (p = 0.017 and p = 0.014, respectively). DISCUSSION: the percentage of cases positive for p16INK4a is smaller than the one found in literature, which can suggest a less relevant part of HPV infection in the oncogenesis of penile cancer in the studied population. Identification of p16INK4a did not relate with other clinicopathological factors. Tendency for a more favourable prognosis in patients with p16INK4a agrees with results found in literature. The most relevant factor for prognosis is nodal staging. CONCLUSIONS: penile cancer positive for p16INK4a shows a trend for better survival, although the most relevant factor is nodal staging.

Influence of sociodemographic factors on treatment's choice for localized prostate cancer in Portugal.

INTRODUCTION: Patients with localized prostate cancer (PCa) are active participants in the choice of treatment. OBJECTIVES: To access the effects of social and demographic factors in the choice of treatment in cases of localized PCa, in a Portuguese population. METHODS: Identification of all patients with the diagnosis of localized PCa in the last four years in an oncological centre. Evaluation of the effects of sociodemographic factors (age, profession, literacy, marital status, district and number of inhabitants of the place of residence) in the choice of treatment. RESULTS: 300 patients with localized PCa were evaluated: 17.3% (n = 52) opted for radical prostatectomy (RP); 39,3% had (n = 118) external radiotherapy; brachytherapy in 29.3% (n = 88) and other options (active surveillance, cryotherapy and hormonal therapy) in 14.1% (n = 42). In relation to surgical treatment (RP) the following results were obtained: a) > 70 years: 3.9% (n = 5); ≤ 70 years: 27.5% (n = 47), p < 0.001; b) primary sector: 10.3% (n = 3); secondary sector: 16.2% (n = 27); tertiary sector: 24.1% (n = 21); quaternary sector: 8.3% (n = 1), p = 0.296; c) marital status married: 17.9% (n = 47); single: 0% (n = 0); divorced: 25.0% (n = 5); widow: 0% (n = 0), p = 0.734; d) residency in a city: 14.1% (n = 13); city > 4000 habitants: 22.7% (n = 15); city ≤ 4000 habitants: 16.9% (n = 24), p = 0.701. Using multinomial regression with age (p = 0.001), district (p = 0.035), marital status (p = 0.027) and profession (0.179), this model explained 17.2%-28.4% of therapeutic choices (p < 0.001). CONCLUSIONS: The main socioeconomical factor that influence treatment choice was age. Unmarried patients over 70 years choose less radical prostatectomy. Other sociodemographic factors have minor influence in the choice of the treatment.

Phylogenomic Resolution of the Cetacean Tree of Life Using Target Sequence Capture.

The evolution of cetaceans, from their early transition to an aquatic lifestyle to their subsequent diversification, has been the subject of numerous studies. However, although the higher-level relationships among cetacean families have been largely settled, several aspects of the systematics within these groups remain unresolved. Problematic clades include the oceanic dolphins (37 spp.), which have experienced a recent rapid radiation, and the beaked whales (22 spp.), which have not been investigated in detail using nuclear loci. The combined application of high-throughput sequencing with techniques that target specific genomic sequences provide a powerful means of rapidly generating large volumes of orthologous sequence data for use in phylogenomic studies. To elucidate the phylogenetic relationships within the Cetacea, we combined sequence capture with Illumina sequencing to generate data for $\sim $3200 protein-coding genes for 68 cetacean species and their close relatives including the pygmy hippopotamus. By combining data from $>$38,000 exons with existing sequences from 11 cetaceans and seven outgroup taxa, we produced the first comprehensive comparative genomic data set for cetaceans, spanning 6,527,596 aligned base pairs (bp) and 89 taxa. Phylogenetic trees reconstructed with maximum likelihood and Bayesian inference of concatenated loci, as well as with coalescence analyses of individual gene trees, produced mostly concordant and well-supported trees. Our results completely resolve the relationships among beaked whales as well as the contentious relationships among oceanic dolphins, especially the problematic subfamily Delphinidae. We carried out Bayesian estimation of species divergence times using MCMCTree and compared our complete data set to a subset of clocklike genes. Analyses using the complete data set consistently showed less variance in divergence times than the reduced data set. In addition, integration of new fossils (e.g., Mystacodon selenensis) indicates that the diversification of Crown Cetacea began before the Late Eocene and the divergence of Crown Delphinidae as early as the Middle Miocene. [Cetaceans; phylogenomics; Delphinidae; Ziphiidae; dolphins; whales.].

Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths.

Butterflies and moths (Lepidoptera) are one of the major superradiations of insects, comprising nearly 160,000 described extant species. As herbivores, pollinators, and prey, Lepidoptera play a fundamental role in almost every terrestrial ecosystem. Lepidoptera are also indicators of environmental change and serve as models for research on mimicry and genetics. They have been central to the development of coevolutionary hypotheses, such as butterflies with flowering plants and moths' evolutionary arms race with echolocating bats. However, these hypotheses have not been rigorously tested, because a robust lepidopteran phylogeny and timing of evolutionary novelties are lacking. To address these issues, we inferred a comprehensive phylogeny of Lepidoptera, using the largest dataset assembled for the order (2,098 orthologous protein-coding genes from transcriptomes of 186 species, representing nearly all superfamilies), and dated it with carefully evaluated synapomorphy-based fossils. The oldest members of the Lepidoptera crown group appeared in the Late Carboniferous (∼300 Ma) and fed on nonvascular land plants. Lepidoptera evolved the tube-like proboscis in the Middle Triassic (∼241 Ma), which allowed them to acquire nectar from flowering plants. This morphological innovation, along with other traits, likely promoted the extraordinary diversification of superfamily-level lepidopteran crown groups. The ancestor of butterflies was likely nocturnal, and our results indicate that butterflies became day-flying in the Late Cretaceous (∼98 Ma). Moth hearing organs arose multiple times before the evolutionary arms race between moths and bats, perhaps initially detecting a wide range of sound frequencies before being co-opted to specifically detect bat sonar. Our study provides an essential framework for future comparative studies on butterfly and moth evolution.

Bayesian Molecular Clock Dating Using Genome-Scale Datasets.

Bayesian methods for molecular clock dating of species divergences have been greatly developed during the past decade. Advantages of the methods include the use of relaxed-clock models to describe evolutionary rate variation in the branches of a phylogenetic tree and the use of flexible fossil calibration densities to describe the uncertainty in node ages. The advent of next-generation sequencing technologies has led to a flood of genome-scale datasets for organisms belonging to all domains in the tree of life. Thus, a new era has begun where dating the tree of life using genome-scale data is now within reach. In this protocol, we explain how to use the computer program MCMCTree to perform Bayesian inference of divergence times using genome-scale datasets. We use a ten-species primate phylogeny, with a molecular alignment of over three million base pairs, as an exemplar on how to carry out the analysis. We pay particular attention to how to set up the analysis and the priors and how to diagnose the MCMC algorithm used to obtain the posterior estimates of divergence times and evolutionary rates.