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Analyses of genome sequence data have revealed pervasive interspecific gene flow and enriched our understanding of the role of gene flow in speciation and adaptation. Inference of gene flow using genomic data requires powerful statistical methods. Yet current likelihood-based methods involve heavy computation and are feasible for small datasets only. Here, we implement the multispecies-coalescent-with-migration model in the Bayesian program bpp, which can be used to test for gene flow and estimate migration rates, as well as species divergence times and population sizes. We develop Markov chain Monte Carlo algorithms for efficient sampling from the posterior, enabling the analysis of genome-scale datasets with thousands of loci. Implementation of both introgression and migration models in the same program allows us to test whether gene flow occurred continuously over time or in pulses. Analyses of genomic data from Anopheles mosquitoes demonstrate rich information in typical genomic datasets about the mode and rate of gene flow.

Close-kin mark-recapture (CKMR) methods have recently been used to infer demographic parameters such as census population size and survival for fish of interest to fisheries and conservation. These methods have advantages over traditional mark-recapture methods as the mark is genetic, removing the need for physical marking and recapturing that may interfere with parameter estimation. For mosquitoes, the spatial distribution of close-kin pairs has been used to estimate mean dispersal distance, of relevance to vector-borne disease transmission and novel biocontrol strategies. Here, we extend CKMR methods to the life history of mosquitoes and comparable insects. We derive kinship probabilities for mother-offspring, father-offspring, full-sibling and half-sibling pairs, where an individual in each pair may be a larva, pupa or adult. A pseudo-likelihood approach is used to combine the marginal probabilities of all kinship pairs. To test the effectiveness of this approach at estimating mosquito demographic parameters, we develop an individual-based model of mosquito life history incorporating egg, larva, pupa and adult life stages. The simulation labels each individual with a unique identification number, enabling close-kin relationships to be inferred for sampled individuals. Using the dengue vector Aedes aegypti as a case study, we find the CKMR approach provides unbiased estimates of adult census population size, adult and larval mortality rates, and larval life stage duration for logistically feasible sampling schemes. Considering a simulated population of 3,000 adult mosquitoes, estimation of adult parameters is accurate when ca. 40 adult females are sampled biweekly over a three month period. Estimation of larval parameters is accurate when adult sampling is supplemented with ca. 120 larvae sampled biweekly over the same period. The methods are also effective at detecting intervention-induced increases in adult mortality and decreases in population size. As the cost of genome sequencing declines, CKMR holds great promise for characterizing the demography of mosquitoes and comparable insects of epidemiological and agricultural significance.

Modeling human diseases as networks simplify complex multi-cellular processes, helps understand patterns in noisy data that humans cannot find, and thereby improves precision in prediction. Using Inflammatory Bowel Disease (IBD) as an example, here we outline an unbiased AI-assisted approach for target identification and validation. A network was built in which clusters of genes are connected by directed edges that highlight asymmetric Boolean relationships. Using machine-learning, a path of continuum states was pinpointed, which most effectively predicted disease outcome. This path was enriched in gene-clusters that maintain the integrity of the gut epithelial barrier. We exploit this insight to prioritize one target, choose appropriate pre-clinical murine models for target validation and design patient-derived organoid models. Potential for treatment efficacy is confirmed in patient-derived organoids using multivariate analyses. This AI-assisted approach identifies a first-in-class gut barrier-protective agent in IBD and predicted Phase-III success of candidate agents.

The effectiveness of immune responses depends on the precision of stimulus-responsive gene expression programs. Cells specify which genes to express by activating stimulus-specific combinations of stimulus-induced transcription factors (TFs). Their activities are decoded by a gene regulatory strategy (GRS) associated with each response gene. Here, we examined whether the GRSs of target genes may be inferred from stimulus-response (input-output) datasets, which remains an unresolved model-identifiability challenge. We developed a mechanistic modeling framework and computational workflow to determine the identifiability of all possible combinations of synergistic (AND) or non-synergistic (OR) GRSs involving three transcription factors. Considering different sets of perturbations for stimulus-response studies, we found that two thirds of GRSs are easily distinguishable but that substantially more quantitative data is required to distinguish the remaining third. To enhance the accuracy of the inference with timecourse experimental data, we developed an advanced error model that avoids error overestimates by distinguishing between value and temporal error. Incorporating this error model into a Bayesian framework, we show that GRS models can be identified for individual genes by considering multiple datasets. Our analysis rationalizes the allocation of experimental resources by identifying most informative TF stimulation conditions. Applying this computational workflow to experimental data of immune response genes in macrophages, we found that a much greater fraction of genes are combinatorially controlled than previously reported by considering compensation among transcription factors. Specifically, we revealed that a group of known NFκB target genes may also be regulated by IRF3, which is supported by chromatin immuno-precipitation analysis. Our study provides a computational workflow for designing and interpreting stimulus-response gene expression studies to identify underlying gene regulatory strategies and further a mechanistic understanding.

The rooting of the SARS-CoV-2 phylogeny is important for understanding the origin and early spread of the virus. Previously published phylogenies have used different rootings that do not always provide consistent results. We investigate several different strategies for rooting the SARS-CoV-2 tree and provide measures of statistical uncertainty for all methods. We show that methods based on the molecular clock tend to place the root in the B clade, whereas methods based on outgroup rooting tend to place the root in the A clade. The results from the two approaches are statistically incompatible, possibly as a consequence of deviations from a molecular clock or excess back-mutations. We also show that none of the methods provide strong statistical support for the placement of the root in any particular edge of the tree. These results suggest that phylogenetic evidence alone is unlikely to identify the origin of the SARS-CoV-2 virus and we caution against strong inferences regarding the early spread of the virus based solely on such evidence.

Despite the substantial role that chickens have played in human societies across the world, both the geographic and temporal origins of their domestication remain controversial. To address this issue, we analyzed 863 genomes from a worldwide sampling of chickens and representatives of all four species of wild jungle fowl and each of the five subspecies of red jungle fowl (RJF). Our study suggests that domestic chickens were initially derived from the RJF subspecies Gallus gallus spadiceus whose present-day distribution is predominantly in southwestern China, northern Thailand and Myanmar. Following their domestication, chickens were translocated across Southeast and South Asia where they interbred locally with both RJF subspecies and other jungle fowl species. In addition, our results show that the White Leghorn chicken breed possesses a mosaic of divergent ancestries inherited from other subspecies of RJF. Despite the strong episodic gene flow from geographically divergent lineages of jungle fowls, our analyses show that domestic chickens undergo genetic adaptations that underlie their unique behavioral, morphological and reproductive traits. Our study provides novel insights into the evolutionary history of domestic chickens and a valuable resource to facilitate ongoing genetic and functional investigations of the world's most numerous domestic animal.

Maximum likelihood estimators are used extensively to estimate unknown parameters of stochastic trait evolution models on phylogenetic trees. Although the MLE has been proven to converge to the true value in the independent-sample case, we cannot appeal to this result because trait values of different species are correlated due to shared evolutionary history. In this paper, we consider a 2-state symmetric model for a single binary trait and investigate the theoretical properties of the MLE for the transition rate in the large-tree limit. Here, the large-tree limit is a theoretical scenario where the number of taxa increases to infinity and we can observe the trait values for all species. Specifically, we prove that the MLE converges to the true value under some regularity conditions. These conditions ensure that the tree shape is not too irregular, and holds for many practical scenarios such as trees with bounded edges, trees generated from the Yule (pure birth) process, and trees generated from the coalescent point process. Our result also provides an upper bound for the distance between the MLE and the true value.

Marburg virus (MARV) causes sporadic outbreaks of severe Marburg virus disease (MVD). Most MVD outbreaks originated in East Africa and field studies in East Africa, South Africa, Zambia, and Gabon identified the Egyptian rousette bat (ERB; Rousettus aegyptiacus) as a natural reservoir. However, the largest recorded MVD outbreak with the highest case-fatality ratio happened in 2005 in Angola, where direct spillover from bats was not  shown. Here, collaborative studies by the Centers for Disease Control and Prevention, Njala University, University of California, Davis USAID-PREDICT, and the University of Makeni identify MARV circulating in ERBs in Sierra Leone. PCR, antibody and virus isolation data from 1755 bats of 42 species shows active MARV infection in approximately 2.5% of ERBs. Phylogenetic analysis identifies MARVs that are similar to the Angola strain. These results provide evidence of MARV circulation in West Africa and demonstrate the value of pathogen surveillance to identify previously undetected threats.

Single-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. We observe that snRNA-seq is commonly subject to contamination by high amounts of ambient RNA, which can lead to biased downstream analyses, such as identification of spurious cell types if overlooked. We present a novel approach to quantify contamination and filter droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: (1) human differentiating preadipocytes in vitro, (2) fresh mouse brain tissue, and (3) human frozen adipose tissue (AT) from six individuals. All three data sets showed evidence of extranuclear RNA contamination, and we observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq, our clustering strategy also successfully filtered single-cell RNA-seq data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

Previous genome-scale phylogenetic analyses of Fungi have under sampled taxa from Zoopagales; this order contains many predacious or parasitic genera, and most have never been grown in pure culture. We sequenced the genomes of 4 zoopagalean taxa that are predators of amoebae, nematodes, or rotifers and the genome of one taxon that is a parasite of amoebae using single cell sequencing methods with whole genome amplification. Each genome was a metagenome, which was assembled and binned using multiple techniques to identify the target genomes. We inferred phylogenies with both super matrix and coalescent approaches using 192 conserved proteins mined from the target genomes and performed ancestral state reconstructions to determine the ancestral trophic lifestyle of the clade. Our results indicate that Zoopagales is monophyletic. Ancestral state reconstructions provide moderate support for mycoparasitism being the ancestral state of the clade.

Leeches and oligochaetes comprise a monophyletic group of annelids, the Clitellata, whose reproduction is characterized by simultaneous hermaphroditism. While most clitellate species reproduce by cross-fertilization, self-fertilization has been described within the speciose genus Helobdella. Here we document the reproductive life histories and reproductive capacities for three other Helobdella species. Under laboratory conditions, both H. robusta and H. octatestisaca exhibit uniparental reproduction, apparently reflecting self-fertility, and suggesting that this trait is ancestral for the genus. However, the third species, H. austinensis, seems incapable of reproduction by self-fertilization, so we inferred its reproductive life history by analyzing reproduction in breeding cohorts. Comparing the reproductive parameters for H. robusta reproducing in isolation and in cohorts revealed that reproduction in cohorts is dramatically delayed with respect to that of isolated individuals, and that cohorts of leeches coordinate their cocoon deposition in a manner that is not predicted from the reproductive parameters of individuals reproducing in isolation. Finally, our comparisons of reproductive capacity for individuals versus cohorts for H. robusta, and between different sizes of cohorts for H. austinensis, reveal differences in resource allocation between male and female reproductive roles that are consistent with evolutionary theory.

BackgroundThe synchronous ionic currents that give rise to neural oscillations have complex influences on neuronal spiking activity that are challenging to characterize.New methodHere we present a method to estimate probabilistic relationships between neural spiking activity and the phase of field oscillations using a generalized linear model (GLM) with an overcomplete basis of circular functions. We first use an L1-regularized maximum likelihood procedure to select an active set of regressors from the overcomplete set and perform model fitting using standard maximum likelihood estimation. An information theoretic model selection procedure is then used to identify an optimal subset of regressors and associated coefficients that minimize overfitting. To assess goodness of fit, we apply the time-rescaling theorem and compare model predictions to original data using quantile-quantile plots.ResultsSpike-phase relationships in synthetic data were robustly characterized. When applied to in vivo hippocampal data from an awake behaving rat, our method captured a multimodal relationship between the spiking activity of a CA1 interneuron, a theta (5-10 Hz) rhythm, and a nested high gamma (65-135 Hz) rhythm.Comparison with existing methodsPrevious methods for characterizing spike-phase relationships are often only suitable for unimodal relationships, impose specific relationship shapes, or have limited ability to assess the accuracy or fit of their characterizations.ConclusionsThis method advances the way spike-phase relationships are visualized and quantified, and captures multimodal spike-phase relationships, including relationships with multiple nested rhythms. Overall, our method is a powerful tool for revealing a wide range of neural circuit interactions.

Austropurcellia, a genus of dispersal-limited arachnids endemic to isolated patches of coastal rainforest in Queensland, Australia, has a remarkable biogeographic history. The genus is a member of the family Pettalidae, which has a classical temperate Gondwanan distribution; previous work has suggested that Austropurcellia is an ancient lineage, with an origin that predates Gondwanan rifting. Subsequently, this lineage has persisted through major climatic fluctuations, such as major aridification during the Miocene and contraction and fragmentation of forest habitats during the Last Glacial Maximum (LGM). In order to understand Austropurcellia's evolutionary and biogeographic history, we generated DNA sequences from both mitochondrial and nuclear loci and combined this information with previously published datasets for the globally-distributed suborder Cyphophthalmi (i.e., all mite harvestmen). We generated phylogenetic trees using maximum likelihood and Bayesian approaches to date divergences using a relaxed molecular clock. According to our estimates, the family Pettalidae diversified in the late Jurassic, in accordance with Gondwanan vicariance. Within Pettalidae, Austropurcellia split from its sister group in the early Cretaceous and began to diversify some 15 Ma later. Therefore, its presence in Australia predates continental rifting-making it one of very few hypothesized examples of Gondwanan vicariance that have withstood rigorous testing. We found a steady rate of diversification within the genus, with no evidence for a shift in rate associated with Miocene aridification. Ages of splits between species predate the Pleistocene, consistent with a "museum" model in which forest refugia acted to preserve existing lineages rather than drive speciation within the group.

Birth-death processes track the size of a univariate population, but many biological systems involve interaction between populations, necessitating models for two or more populations simultaneously. A lack of efficient methods for evaluating finite-time transition probabilities of bivariate processes, however, has restricted statistical inference in these models. Researchers rely on computationally expensive methods such as matrix exponentiation or Monte Carlo approximation, restricting likelihood-based inference to small systems, or indirect methods such as approximate Bayesian computation. In this paper, we introduce the birth/birth-death process, a tractable bivariate extension of the birth-death process, where rates are allowed to be nonlinear. We develop an efficient algorithm to calculate its transition probabilities using a continued fraction representation of their Laplace transforms. Next, we identify several exemplary models arising in molecular epidemiology, macro-parasite evolution, and infectious disease modeling that fall within this class, and demonstrate advantages of our proposed method over existing approaches to inference in these models. Notably, the ubiquitous stochastic susceptible-infectious-removed (SIR) model falls within this class, and we emphasize that computable transition probabilities newly enable direct inference of parameters in the SIR model. We also propose a very fast method for approximating the transition probabilities under the SIR model via a novel branching process simplification, and compare it to the continued fraction representation method with application to the 17th century plague in Eyam. Although the two methods produce similar maximum a posteriori estimates, the branching process approximation fails to capture the correlation structure in the joint posterior distribution.

The wide and rapid spread of Chikungunya (CHIKV) and Zika (ZIKV) viruses represent a global public health problem, especially for tropical and subtropical environments. The early detection of CHIKV and ZIKV in mosquitoes may help to understand the dynamics of the diseases in high-risk areas, and to design data based epidemiological surveillance to activate the preparedness and response of the public health system and vector control programs. This study was done to detect ZIKV and CHIKV viruses in naturally infected fed female Aedes aegypti (L.) mosquitoes from active epidemic urban areas in Ecuador. Pools (n=193; 22 pools) and individuals (n=22) of field collected Ae. aegypti mosquitoes from high-risk arboviruses infection sites in Ecuador were analyzed for the presence of CHIKV and ZIKV using RT-PCR. Phylogenetic analysis demonstrated that both ZIKV and CHIKV viruses circulating in Ecuador correspond to the Asian lineages. Minimum infection rate (MIR) of CHIKV for Esmeraldas city was 2.3% and the maximum likelihood estimation (MLE) was 3.3%. The minimum infection rate (MIR) of ZIKV for Portoviejo city was 5.3% and for Manta city was 2.1%. Maximum likelihood estimation (MLE) for Portoviejo city was 6.9% and 2.6% for Manta city. Detection of arboviruses and infection rates in the arthropod vectors may help to predict an outbreak and serve as a warning tool in surveillance programs.

Phylogenetics is undergoing a revolution as large-scale molecular datasets reveal unexpected but repeatable rearrangements of clades that were previously thought to be disparate lineages. One of the most unusual clades of fishes that has been found using large-scale molecular datasets is an expanded Syngnathiformes including traditional long-snouted syngnathiform lineages (Aulostomidae, Centriscidae, Fistulariidae, Solenostomidae, Syngnathidae), as well as a diverse set of largely benthic-associated fishes (Callionymoidei, Dactylopteridae, Mullidae, Pegasidae) that were previously dispersed across three orders. The monophyly of this surprising clade of fishes has been upheld by recent studies utilizing both nuclear and mitogenomic data, but the relationships among major lineages within Syngnathiformes remain ambiguous; previous analyses have inconsistent topologies and are plagued by low support at deep divergences between the major lineages. In this study, we use a dataset of ultraconserved elements (UCEs) to conduct the first phylogenomic study of Syngnathiformes. UCEs have been effective markers for resolving deep phylogenetic relationships in fishes and, combined with increased taxon sampling, we expected UCEs to resolve problematic syngnathiform relationships. Overall, UCEs were effective at resolving relationships within Syngnathiformes at a range of evolutionary timescales. We find consistent support for the monophyly of traditional long-snouted syngnathiform lineages (Aulostomidae, Centriscidae, Fistulariidae, Solenostomidae, Syngnathidae), which better agrees with morphological hypotheses than previously published topologies from molecular data. This result was supported by all Bayesian and maximum likelihood analyses, was robust to differences in matrix completeness and potential sources of bias, and was highly supported in coalescent-based analyses in ASTRAL when matrices were filtered to contain the most phylogenetically informative loci. While Bayesian and maximum likelihood analyses found support for a benthic-associated clade (Callionymidae, Dactylopteridae, Mullidae, and Pegasidae) as sister to the long-snouted clade, this result was not replicated in the ASTRAL analyses. The base of our phylogeny is characterized by short internodes separating major syngnathiform lineages and is consistent with the hypothesis of an ancient rapid radiation at the base of Syngnathiformes. Syngnathiformes therefore present an exciting opportunity to study patterns of morphological variation and functional innovation arising from rapid but ancient radiation.

Gene transfer among reproductively isolated organisms can lead to novel phenotypes and increased fitness. Among the Sternorrhyncha, a suborder of plant sap-feeding hemipteran insects, both aphids and adelgids acquired carotenoid biosynthesis genes from a fungal donor that result in ecologically relevant pigmentation. Phylloxerids form another family that are closely related to aphids and adelgids and share similar pigmentation, but are largely uncharacterized for their presence and number of pigment genes that have duplicated among aphids. Here, we examined the transcriptomes of nine phylloxerid species, and performed PCR to amplify carotenoid genes from their genomic DNA. We identified carotenoid cyclase/synthase and desaturase genes in each species and demonstrated that they share the common fungal origin as those of aphids and adelgids based on their exon-intron gene structures and phylogenetic relationships. The phylogenetic analyses also indicated that carotenoid genes evolved following the differentiation of aphids, adelgids, and phylloxerids at the levels of family, genus, and species. Unlike aphids that duplicated these genes in their genomes, phylloxerids maintained only single copies, and some species may lack expression of certain genes. These results suggest that the phylloxerid lifestyle undergoes reduced selection pressure to expand carotenoid synthesis genes, and provides insight into these gene functions in insects.

Ascaridomorph nematodes threaten the health of humans and other animals worldwide. Despite their medical, veterinary and economic importance, the identification of species lineages and establishing their phylogenetic relationships have proved difficult in some cases. Many working hypotheses regarding the phylogeny of ascaridomorphs have been based on single-locus data, most typically nuclear ribosomal RNA. Such single-locus hypotheses lack independent corroboration, and for nuclear rRNA typically lack resolution for deep relationships. As an alternative approach, we analyzed the mitochondrial (mt) genomes of anisakids (~14 kb) from different fish hosts in multiple countries, in combination with those of other ascaridomorphs available in the GenBank database. The circular mt genomes range from 13,948-14,019 bp in size and encode 12 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNA genes. Our analysis showed that the Pseudoterranova decipiens complex consists of at least six cryptic species. In contrast, the hypothesis that Contracaecum ogmorhini represents a complex of cryptic species is not supported by mt genome data. Our analysis recovered several fundamental and uncontroversial ascaridomorph clades, including the monophyly of superfamilies and families, except for Ascaridiidae, which was consistent with the results based on nuclear rRNA analysis. In conclusion, mt genome analysis provided new insights into the phylogeny and taxonomy of ascaridomorph nematodes.

BackgroundDespite considerable effort, progress in spider molecular systematics has lagged behind many other comparable arthropod groups, thereby hindering family-level resolution, classification, and testing of important macroevolutionary hypotheses. Recently, alternative targeted sequence capture techniques have provided molecular systematics a powerful tool for resolving relationships across the Tree of Life. One of these approaches, Anchored Hybrid Enrichment (AHE), is designed to recover hundreds of unique orthologous loci from across the genome, for resolving both shallow and deep-scale evolutionary relationships within non-model systems. Herein we present a modification of the AHE approach that expands its use for application in spiders, with a particular emphasis on the infraorder Mygalomorphae.ResultsOur aim was to design a set of probes that effectively capture loci informative at a diversity of phylogenetic timescales. Following identification of putative arthropod-wide loci, we utilized homologous transcriptome sequences from 17 species across all spiders to identify exon boundaries. Conserved regions with variable flanking regions were then sought across the tick genome, three published araneomorph spider genomes, and raw genomic reads of two mygalomorph taxa. Following development of the 585 target loci in the Spider Probe Kit, we applied AHE across three taxonomic depths to evaluate performance: deep-level spider family relationships (33 taxa, 327 loci); family and generic relationships within the mygalomorph family Euctenizidae (25 taxa, 403 loci); and species relationships in the North American tarantula genus Aphonopelma (83 taxa, 581 loci). At the deepest level, all three major spider lineages (the Mesothelae, Mygalomorphae, and Araneomorphae) were supported with high bootstrap support. Strong support was also found throughout the Euctenizidae, including generic relationships within the family and species relationships within the genus Aptostichus. As in the Euctenizidae, virtually identical topologies were inferred with high support throughout Aphonopelma.ConclusionsThe Spider Probe Kit, the first implementation of AHE methodology in Class Arachnida, holds great promise for gathering the types and quantities of molecular data needed to accelerate an understanding of the spider Tree of Life by providing a mechanism whereby different researchers can confidently and effectively use the same loci for independent projects, yet allowing synthesis of data across independent research groups.

BackgroundThe sperm gene bindin encodes a gamete recognition protein, which plays an important role in conspecific fertilization and reproductive isolation of sea urchins. Molecular evolution of the gene has been extensively investigated with the attention focused on the protein coding regions. Intron evolution has been investigated to a much lesser extent. We have studied nucleotide variability in the complete bindin locus, including two exons and one intron, in the sea urchin Strongylocentrotus intermedius represented by two morphological forms. We have also analyzed all available bindin sequences for two other sea urchin species, S. pallidus and S. droebachiensis.ResultsThe results show that the bindin sequences from the two forms of S. intermedius are intermingled with no evidence of genetic divergence; however, the forms exhibit slightly different patterns in bindin variability. The level of the bindin nucleotide diversity is close for S. intermedius and S. droebachiensis, but noticeably higher for S. pallidus. The distribution of variability is non-uniform along the gene; however there are striking similarities among the species, indicating similar evolutionary trends in this gene engaged in reproductive function. The patterns of nucleotide variability and divergence are radically different in the bindin coding and intron regions. Positive selection is detected in the bindin coding region. The neutrality tests as well as the maximum likelihood approaches suggest the action of diversifying selection in the bindin intron.ConclusionsSignificant deviation from neutrality has been detected in the bindin coding region and suggested in the intron, indicating the possible functional importance of the bindin intron variability. To clarify the question concerning possible involvement of diversifying selection in the bindin intron evolution more data combining population genetic and functional approaches are necessary.

Bayesian analysis of macroevolutionary mixtures (BAMM) has recently taken the study of lineage diversification by storm. BAMM estimates the diversification-rate parameters (speciation and extinction) for every branch of a study phylogeny and infers the number and location of diversification-rate shifts across branches of a tree. Our evaluation of BAMM reveals two major theoretical errors: (i) the likelihood function (which estimates the model parameters from the data) is incorrect, and (ii) the compound Poisson process prior model (which describes the prior distribution of diversification-rate shifts across branches) is incoherent. Using simulation, we demonstrate that these theoretical issues cause statistical pathologies; posterior estimates of the number of diversification-rate shifts are strongly influenced by the assumed prior, and estimates of diversification-rate parameters are unreliable. Moreover, the inability to correctly compute the likelihood or to correctly specify the prior for rate-variable trees precludes the use of Bayesian approaches for testing hypotheses regarding the number and location of diversification-rate shifts using BAMM.

Naturally occurring admixture has now been documented in every major primate lineage, suggesting its key role in primate evolutionary history. Active primate hybrid zones can provide valuable insight into this process. Here, we investigate the history of admixture in one of the best-studied natural primate hybrid zones, between yellow baboons (Papio cynocephalus) and anubis baboons (Papio anubis) in the Amboseli ecosystem of Kenya. We generated a new genome assembly for yellow baboon and low-coverage genomewide resequencing data from yellow baboons, anubis baboons and known hybrids (n = 44). Using a novel composite likelihood method for estimating local ancestry from low-coverage data, we found high levels of genetic diversity and genetic differentiation between the parent taxa, and excellent agreement between genome-scale ancestry estimates and a priori pedigree, life history and morphology-based estimates (r(2)  = 0.899). However, even putatively unadmixed Amboseli yellow individuals carried a substantial proportion of anubis ancestry, presumably due to historical admixture. Further, the distribution of shared vs. fixed differences between a putatively unadmixed Amboseli yellow baboon and an unadmixed anubis baboon, both sequenced at high coverage, is inconsistent with simple isolation-migration or equilibrium migration models. Our findings suggest a complex process of intermittent contact that has occurred multiple times in baboon evolutionary history, despite no obvious fitness costs to hybrids or major geographic or behavioural barriers. In combination with the extensive phenotypic data available for baboon hybrids, our results provide valuable context for understanding the history of admixture in primates, including in our own lineage.

Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

The genes of the Major Histocompatibility Complex (MHC) play an important role in the vertebrate immune response and are among the most polymorphic genes known in vertebrates. In some marine mammals, MHC genes have been shown to be characterized by low levels of polymorphism compared to terrestrial taxa; this reduction in variation is often explained as a result of lower pathogen pressures in marine habitats. To determine if this same reduction in variation applies to the migratory population of blue whales (Balaenoptera musculus) that occurs in the Gulf of California, we genotyped a 172 bp fragment of exon 2 of the MHC Class II DQB locus for 80 members of this population. Twenty-two putatively functional DQB allotypes were identified, all of which were homologous with DQB sequences from other cetacean species. Up to 5 putative alleles per individual were identified, suggesting that gene duplication has occurred at this locus. Rates of non-synonymous to synonymous substitutions (ω) and maximum likelihood analyses of models of nucleotide variation provided potential evidence of ongoing positive selection at this exon. Phylogenetic analyses of DQB alleles from B. musculus and 16 other species of cetaceans revealed trans-specific conservation of MHC variants, suggesting that selection has acted on this locus over prolonged periods of time. Collectively our findings reveal that immunogenic variation in blue whales is comparable to that in terrestrial mammals, thereby providing no evidence that marine taxa are subject to reduced pathogen-induced selective pressures.

Continuous-time birth-death-shift (BDS) processes are frequently used in stochastic modeling, with many applications in ecology and epidemiology. In particular, such processes can model evolutionary dynamics of transposable elements-important genetic markers in molecular epidemiology. Estimation of the effects of individual covariates on the birth, death, and shift rates of the process can be accomplished by analyzing patient data, but inferring these rates in a discretely and unevenly observed setting presents computational challenges. We propose a multi-type branching process approximation to BDS processes and develop a corresponding expectation maximization algorithm, where we use spectral techniques to reduce calculation of expected sufficient statistics to low-dimensional integration. These techniques yield an efficient and robust optimization routine for inferring the rates of the BDS process, and apply broadly to multi-type branching processes whose rates can depend on many covariates. After rigorously testing our methodology in simulation studies, we apply our method to study intrapatient time evolution of IS6110 transposable element, a genetic marker frequently used during estimation of epidemiological clusters of Mycobacterium tuberculosis infections.

BackgroundAntarctic notothenioids are an impressive adaptive radiation. While they share recent common ancestry with several species-depauperate lineages that exhibit a relictual distribution in areas peripheral to the Southern Ocean, an understanding of their evolutionary origins and biogeographic history is limited as the sister lineage of notothenioids remains unidentified. The phylogenetic placement of notothenioids among major lineages of perciform fishes, which include sculpins, rockfishes, sticklebacks, eelpouts, scorpionfishes, perches, groupers and soapfishes, remains unresolved. We investigate the phylogenetic position of notothenioids using DNA sequences of 10 protein coding nuclear genes sampled from more than 650 percomorph species. The biogeographic history of notothenioids is reconstructed using a maximum likelihood method that integrates phylogenetic relationships, estimated divergence times, geographic distributions and paleogeographic history.ResultsPercophis brasiliensis is resolved, with strong node support, as the notothenioid sister lineage. The species is endemic to the subtropical and temperate Atlantic coast of southern South America. Biogeographic reconstructions imply the initial diversification of notothenioids involved the western portion of the East Gondwanan Weddellian Province. The geographic disjunctions among the major lineages of notothenioids show biogeographic and temporal correspondence with the fragmentation of East Gondwana.ConclusionsThe phylogenetic resolution of Percophis requires a change in the classification of percomorph fishes and provides evidence for a western Weddellian origin of notothenioids. The biogeographic reconstruction highlights the importance of the geographic and climatic isolation of Antarctica in driving the radiation of cold-adapted notothenioids.

UnlabelledWhile a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (~92 days) before they were detected in gp120 (~182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains.ImportanceThe SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at 3 months postinfection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. The results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration, resulting in HIV neuropathology.

The number of genes controlling mimetic traits has been a topic of much research and discussion. In this paper, we examine a mimetic, dendrobatid frog Ranitomeya imitator, which harbours extensive phenotypic variation with multiple mimetic morphs, not unlike the celebrated Heliconius system. However, the genetic basis for this polymorphism is unknown, and not easy to determine using standard experimental approaches, for this hard-to-breed species. To circumvent this problem, we first develop a new protocol for automatic quantification of complex colour pattern phenotypes from images. Using this method, which has the potential to be applied in many other systems, we define a phenotype associated with differences in colour pattern between different mimetic morphs. We then proceed to develop a maximum-likelihood method for estimating the number of genes affecting a quantitative trait segregating in a hybrid zone. This method takes advantage of estimates of admixture proportions obtained using genetic data, such as microsatellite markers, and is applicable to any other system where a phenotype has been quantified in an admixture/introgression zone. We evaluate the method using extensive simulations and apply it to the R. imitator system. We show that probably one or two, or at most three genes, control the mimetic phenotype segregating in a R. imitator hybrid zone identified using image analyses.

Repeated low-dose (RLD) challenge designs are important in HIV vaccine research. Current methods for RLD designs rely heavily on an assumption of homogeneous risk of infection among animals, which, upon violation, can lead to invalid inferences and underpowered study designs. We propose to fit a discrete-time survival model with random effects that allows for heterogeneity in the risk of infection among animals and allows for predetermined challenge dose changes over time. Based on this model, we derive likelihood ratio tests and estimators for vaccine efficacy. A two-stage approach is proposed for optimizing the RLD design under cost constraints. Simulation studies demonstrate good finite sample properties of the proposed method and its superior performance compared to existing methods. We illustrate the application of the heterogeneous infection risk model on data from a real simian immunodeficiency virus vaccine study using Rhesus Macaques. The results of our study provide useful guidance for future RLD experimental design.

Manta and devil rays are an iconic group of globally distributed pelagic filter feeders, yet their evolutionary history remains enigmatic. We employed next generation sequencing of mitogenomes for nine of the 11 recognized species and two outgroups; as well as additional Sanger sequencing of two mitochondrial and two nuclear genes in an extended taxon sampling set. Analysis of the mitogenome coding regions in a Maximum Likelihood and Bayesian framework provided a well-resolved phylogeny. The deepest divergences distinguished three clades with high support, one containing Manta birostris, Manta alfredi, Mobula tarapacana, Mobula japanica and Mobula mobular; one containing Mobula kuhlii, Mobula eregoodootenkee and Mobula thurstoni; and one containing Mobula munkiana, Mobula hypostoma and Mobula rochebrunei. Mobula remains paraphyletic with the inclusion of Manta, a result that is in agreement with previous studies based on molecular and morphological data. A fossil-calibrated Bayesian random local clock analysis suggests that mobulids diverged from Rhinoptera around 30 Mya. Subsequent divergences are characterized by long internodes followed by short bursts of speciation extending from an initial episode of divergence in the Early and Middle Miocene (19-17 Mya) to a second episode during the Pliocene and Pleistocene (3.6 Mya - recent). Estimates of divergence dates overlap significantly with periods of global warming, during which upwelling intensity - and related high primary productivity in upwelling regions - decreased markedly. These periods are hypothesized to have led to fragmentation and isolation of feeding regions leading to possible regional extinctions, as well as the promotion of allopatric speciation. The closely shared evolutionary history of mobulids in combination with ongoing threats from fisheries and climate change effects on upwelling and food supply, reinforces the case for greater protection of this charismatic family of pelagic filter feeders.

BackgroundRecent genomic information has revealed that neuroglobin and cytoglobin are the two principal lineages of vertebrate hemoglobins, with the latter encompassing the familiar myoglobin and α-globin/β-globin tetramer hemoglobin, and several minor groups. In contrast, very little is known about hemoglobins in echinoderms, a phylum of exclusively marine organisms closely related to vertebrates, beyond the presence of coelomic hemoglobins in sea cucumbers and brittle stars. We identified about 50 hemoglobins in sea urchin, starfish and sea cucumber genomes and transcriptomes, and used Bayesian inference to carry out a molecular phylogenetic analysis of their relationship to vertebrate sequences, specifically, to assess the hypothesis that the neuroglobin and cytoglobin lineages are also present in echinoderms.ResultsThe genome of the sea urchin Strongylocentrotus purpuratus encodes several hemoglobins, including a unique chimeric 14-domain globin, 2 androglobin isoforms and a unique single androglobin domain protein. Other strongylocentrotid genomes appear to have similar repertoires of globin genes. We carried out molecular phylogenetic analyses of 52 hemoglobins identified in sea urchin, brittle star and sea cucumber genomes and transcriptomes, using different multiple sequence alignment methods coupled with Bayesian and maximum likelihood approaches. The results demonstrate that there are two major globin lineages in echinoderms, which are related to the vertebrate neuroglobin and cytoglobin lineages. Furthermore, the brittle star and sea cucumber coelomic hemoglobins appear to have evolved independently from the cytoglobin lineage, similar to the evolution of erythroid oxygen binding globins in cyclostomes and vertebrates.ConclusionThe presence of echinoderm globins related to the vertebrate neuroglobin and cytoglobin lineages suggests that the split between neuroglobins and cytoglobins occurred in the deuterostome ancestor shared by echinoderms and vertebrates.

Tandem duplications are an essential source of genetic novelty, and their variation in natural populations is expected to influence adaptive walks. Here, we describe evolutionary impacts of recently-derived, segregating tandem duplications in Drosophila yakuba and Drosophila simulans. We observe an excess of duplicated genes involved in defense against pathogens, insecticide resistance, chorion development, cuticular peptides, and lipases or endopeptidases associated with the accessory glands across both species. The observed agreement is greater than expectations on chance alone, suggesting large amounts of convergence across functional categories. We document evidence of widespread selection on the D. simulans X, suggesting adaptation through duplication is common on the X. Despite the evidence for positive selection, duplicates display an excess of low frequency variants consistent with largely detrimental impacts, limiting the variation that can effectively facilitate adaptation. Standing variation for tandem duplications spans less than 25% of the genome in D. yakuba and D. simulans, indicating that evolution will be strictly limited by mutation, even in organisms with large population sizes. Effective whole gene duplication rates are low at 1.17 × 10-9 per gene per generation in D. yakuba and 6.03 × 10-10 per gene per generation in D. simulans, suggesting long wait times for new mutations on the order of thousands of years for the establishment of sweeps. Hence, in cases where adaptation depends on individual tandem duplications, evolution will be severely limited by mutation. We observe low levels of parallel recruitment of the same duplicated gene in different species, suggesting that the span of standing variation will define evolutionary outcomes in spite of convergence across gene ontologies consistent with rapidly evolving phenotypes.

The POU genes represent a diverse class of animal-specific transcription factors that play important roles in neurogenesis, pluripotency, and cell-type specification. Although previous attempts have been made to reconstruct the evolution of the POU class, these studies have been limited by a small number of representative taxa, and a lack of sequences from basally branching organisms. In this study, we performed comparative analyses on available genomes and sequences recovered through "gene fishing" to better resolve the topology of the POU gene tree. We then used ancestral state reconstruction to map the most likely changes in amino acid evolution for the conserved domains. Our work suggests that four of the six POU families evolved before the last common ancestor of living animals-doubling previous estimates-and were followed by extensive clade-specific gene loss. Amino acid changes are distributed unequally across the gene tree, consistent with a neofunctionalization model of protein evolution. We consider our results in the context of early animal evolution, and the role of POU5 genes in maintaining stem cell pluripotency.

BackgroundTools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families.ResultsWe generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository ( http://bitbucket.org/osiris_phylogenetics/pia/ ) and we demonstrate PIA on a publicly-accessible web server ( http://galaxy-dev.cnsi.ucsb.edu/pia/ ).ConclusionsOur new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.

Understanding the factors that influence larval dispersal and connectivity among marine populations is critical to the conservation and sustainable management of marine resources. We assessed genetic subdivision among ten populations of copper rockfish (Sebastes caurinus) representing paired samples of outer coast and the heads of inlets in five replicate sounds on the west coast of Vancouver Island, British Columbia, using 17 microsatellite DNA loci. Overall, subdivision (F(ST)) was low (F(ST) = 0.031, P < 0.001), but consistently higher between paired coast and head of inlet sites (mean FST = 0.047, P < 0.001) compared to among the five coast sites (mean F(ST) = -0.001, P > 0.5) or among the five head of inlet sites (mean F(ST) = 0.026, P < 0.001). Heterozygosity, allelic richness and estimates of effective population size were also lower in head of inlet sites than in coast sites. Bayesian analysis identified two genetic groups across all samples, a single genetic group among only coast samples, two genetic groups among head of inlet samples and two genetic groups within each sound analysed separately. Head of inlet copper rockfish tended to be shorter with lower condition factors and grew more slowly than coast sites fish. Reduced physical connectivity and selection against immigrants in contrasting outer coast-head of inlet environments likely contribute to the evolution of population structure of copper rockfish. Based on genetic connectivity, coast sites appear to be better served by existing marine protected areas than are head of inlet sites.

The M and S forms of Anopheles gambiae have been the focus of intense study by malaria researchers and evolutionary biologists interested in ecological speciation. Divergence occurs at three discrete islands in genomes that are otherwise nearly identical. An "islands of speciation" model proposes that diverged regions contain genes that are maintained by selection in the face of gene flow. An alternative "incidental island" model maintains that gene flow between M and S is effectively zero and that divergence islands are unrelated to speciation. A "divergence island SNP" assay was used to explore the spatial and temporal distributions of hybrid genotypes. Results revealed that hybrid individuals occur at frequencies ranging between 5% and 97% in every population examined. A temporal analysis revealed that assortative mating is unstable and periodically breaks down, resulting in extensive hybridization. Results suggest that hybrids suffer a fitness disadvantage, but at least some hybrid genotypes are viable. Stable introgression of the 2L speciation island occurred at one site following a hybridization event.

Over the past few years, new high-throughput DNA sequencing technologies have dramatically increased speed and reduced sequencing costs. However, the use of these sequencing technologies is often challenged by errors and biases associated with the bioinformatical methods used for analyzing the data. In particular, the use of naïve methods to identify polymorphic sites and infer genotypes can inflate downstream analyses. Recently, explicit modeling of genotype probability distributions has been proposed as a method for taking genotype call uncertainty into account. Based on this idea, we propose a novel method for quantifying population genetic differentiation from next-generation sequencing data. In addition, we present a strategy for investigating population structure via principal components analysis. Through extensive simulations, we compare the new method herein proposed to approaches based on genotype calling and demonstrate a marked improvement in estimation accuracy for a wide range of conditions. We apply the method to a large-scale genomic data set of domesticated and wild silkworms sequenced at low coverage. We find that we can infer the fine-scale genetic structure of the sampled individuals, suggesting that employing this new method is useful for investigating the genetic relationships of populations sampled at low coverage.

We analyzed 83 fully sequenced great ape genomes for mobile element insertions, predicting a total of 49,452 fixed and polymorphic Alu and long interspersed element 1 (L1) insertions not present in the human reference assembly and assigning each retrotransposition event to a different time point during great ape evolution. We used these homoplasy-free markers to construct a mobile element insertions-based phylogeny of humans and great apes and demonstrate their differential power to discern ape subspecies and populations. Within this context, we find a good correlation between L1 diversity and single-nucleotide polymorphism heterozygosity (r(2) = 0.65) in contrast to Alu repeats, which show little correlation (r(2) = 0.07). We estimate that the "rate" of Alu retrotransposition has differed by a factor of 15-fold in these lineages. Humans, chimpanzees, and bonobos show the highest rates of Alu accumulation--the latter two since divergence 1.5 Mya. The L1 insertion rate, in contrast, has remained relatively constant, with rates differing by less than a factor of three. We conclude that Alu retrotransposition has been the most variable form of genetic variation during recent human-great ape evolution, with increases and decreases occurring over very short periods of evolutionary time.

Spider silk fibers have impressive mechanical properties and are primarily composed of highly repetitive structural proteins (termed spidroins) encoded by a single gene family. Most characterized spidroin genes are incompletely known because of their extreme size (typically >9 kb) and repetitiveness, limiting understanding of the evolutionary processes that gave rise to their unusual gene architectures. The only complete spidroin genes characterized thus far form the dragline in the Western black widow, Latrodectus hesperus. Here, we describe the first complete gene sequence encoding the aciniform spidroin AcSp1, the primary component of spider prey-wrapping fibers. L. hesperus AcSp1 contains a single enormous (∼19 kb) exon. The AcSp1 repeat sequence is exceptionally conserved between two widow species (∼94% identity) and between widows and distantly related orb-weavers (∼30% identity), consistent with a history of strong purifying selection on its amino acid sequence. Furthermore, the 16 repeats (each 371-375 amino acids long) found in black widow AcSp1 are, on average, >99% identical at the nucleotide level. A combination of stabilizing selection on amino acid sequence, selection on silent sites, and intragenic recombination likely explains the extreme homogenization of AcSp1 repeats. In addition, phylogenetic analyses of spidroin paralogs support a gene duplication event occurring concomitantly with specialization of the aciniform glands and the tubuliform glands, which synthesize egg-case silk. With repeats that are dramatically different in length and amino acid composition from dragline spidroins, our L. hesperus AcSp1 expands the knowledge base for developing silk-based biomimetic technologies.

BackgroundWe describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.ResultsOur phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.ConclusionsOur comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle's extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.

The hypothesis that patterns of sex-biased dispersal are related to social mating system in mammals and birds has gained widespread acceptance over the past 30 years. However, two major complications have obscured the relationship between these two behaviors: 1) dispersal frequency and dispersal distance, which measure different aspects of the dispersal process, have often been confounded, and 2) the relationship between mating system and sex-biased dispersal in these vertebrate groups has not been examined using modern phylogenetic comparative methods. Here, we present a phylogenetic analysis of the relationship between mating system and sex-biased dispersal in mammals and birds. Results indicate that the evolution of female-biased dispersal in mammals may be more likely on monogamous branches of the phylogeny, and that females may disperse farther than males in socially monogamous mammalian species. However, we found no support for a relationship between social mating system and sex-biased dispersal in birds when the effects of phylogeny are taken into consideration. We caution that although there are larger-scale behavioral differences in mating system and sex-biased dispersal between mammals and birds, mating system and sex-biased dispersal are far from perfectly associated within these taxa.

Ray-finned fishes constitute the dominant radiation of vertebrates with over 32,000 species. Although molecular phylogenetics has begun to disentangle major evolutionary relationships within this vast section of the Tree of Life, there is no widely available approach for efficiently collecting phylogenomic data within fishes, leaving much of the enormous potential of massively parallel sequencing technologies for resolving major radiations in ray-finned fishes unrealized. Here, we provide a genomic perspective on longstanding questions regarding the diversification of major groups of ray-finned fishes through targeted enrichment of ultraconserved nuclear DNA elements (UCEs) and their flanking sequence. Our workflow efficiently and economically generates data sets that are orders of magnitude larger than those produced by traditional approaches and is well-suited to working with museum specimens. Analysis of the UCE data set recovers a well-supported phylogeny at both shallow and deep time-scales that supports a monophyletic relationship between Amia and Lepisosteus (Holostei) and reveals elopomorphs and then osteoglossomorphs to be the earliest diverging teleost lineages. Our approach additionally reveals that sequence capture of UCE regions and their flanking sequence offers enormous potential for resolving phylogenetic relationships within ray-finned fishes.

In a variety of organisms, adulthood is divided into aging and late life, where aging is a period of exponentially increasing mortality rates and late life is a period of roughly plateaued mortality rates. In this study we used ∼57,600 Drosophila melanogaster from six replicate populations to examine the physiological transitions from aging to late life in four functional characters that decline during aging: desiccation resistance, starvation resistance, time spent in motion, and negative geotaxis. Time spent in motion and desiccation resistance declined less quickly in late life compared to their patterns of decline during aging. Negative geotaxis declined at a faster rate in late life compared to its rate of decline during aging. These results yield two key findings: (1) Late-life physiology is distinct from the physiology of aging, in that there is not simply a continuation of the physiological trends which characterize aging; and (2) late life physiology is complex, in that physiological characters vary with respect to their stabilization, deceleration, or acceleration in the transition from aging to late life. These findings imply that a correct understanding of adulthood requires identifying and appropriately characterizing physiology during properly delimited late-life periods as well as aging periods.

Evolutionary and biogeographic studies increasingly rely on calibrated molecular clocks to date key events. Although there has been significant recent progress in development of the techniques used for molecular dating, many issues remain. In particular, controversies abound over the appropriate use and placement of fossils for calibrating molecular clocks. Several methods have been proposed for evaluating candidate fossils; however, few studies have compared the results obtained by different approaches. Moreover, no previous study has incorporated the effects of nucleotide saturation from different data types in the evaluation of candidate fossils. In order to address these issues, we compared three approaches for evaluating fossil calibrations: the single-fossil cross-validation method of Near, Meylan, and Shaffer (2005. Assessing concordance of fossil calibration points in molecular clock studies: an example using turtles. Am. Nat. 165:137-146), the empirical fossil coverage method of Marshall (2008. A simple method for bracketing absolute divergence times on molecular phylogenies using multiple fossil calibration points. Am. Nat. 171:726-742), and the Bayesian multicalibration method of Sanders and Lee (2007. Evaluating molecular clock calibrations using Bayesian analyses with soft and hard bounds. Biol. Lett. 3:275-279) and explicitly incorporate the effects of data type (nuclear vs. mitochondrial DNA) for identifying the most reliable or congruent fossil calibrations. We used advanced (Caenophidian) snakes as a case study; however, our results are applicable to any taxonomic group with multiple candidate fossils, provided appropriate taxon sampling and sufficient molecular sequence data are available. We found that data type strongly influenced which fossil calibrations were identified as outliers, regardless of which method was used. Despite the use of complex partitioned models of sequence evolution and multiple calibrations throughout the tree, saturation severely compressed basal branch lengths obtained from mitochondrial DNA compared with nuclear DNA. The effects of mitochondrial saturation were not ameliorated by analyzing a combined nuclear and mitochondrial data set. Although removing the third codon positions from the mitochondrial coding regions did not ameliorate saturation effects in the single-fossil cross-validations, it did in the Bayesian multicalibration analyses. Saturation significantly influenced the fossils that were selected as most reliable for all three methods evaluated. Our findings highlight the need to critically evaluate the fossils selected by data with different rates of nucleotide substitution and how data with different evolutionary rates affect the results of each method for evaluating fossils. Our empirical evaluation demonstrates that the advantages of using multiple independent fossil calibrations significantly outweigh any disadvantages.

BackgroundThe fish family Haemulidae is divided in two subfamilies, Haemulinae and Plectorhynchinae (sweetlips), including approximately 17 genera and 145 species. The family has a broad geographic distribution that encompasses contrasting ecological habitats resulting in a unique potential for evolutionary hypotheses testing. In the present work we have examined the phylogenetic relationships of the family using selected representatives of additional Percomorpha based on Bayesian and Maximum likelihood methods by means of three mitochondrial genes. We also developed a phylogenetic hypothesis of the New World species based on five molecular markers (three mitochondrial and two nuclear) as a framework to evaluate the evolutionary history, the ecological diversification and speciation patterns of this group.ResultsMitochondrial genes and different reconstruction methods consistently recovered a monophyletic Haemulidae with the Sillaginidae as its sister clade (although with low support values). Previous studies proposed different relationships that were not recovered in this analysis. We also present a robust molecular phylogeny of Haemulinae based on the combined data of two nuclear and three mitochondrial genes. All topologies support the monophyly of both sub-families (Haemulinae, Plectorhinchinae). The genus Pomadasys was shown to be polyphyletic and Haemulon, Anisotremus, and Plectorhinchus were found to be paraphyletic. Four of seven presumed geminate pairs were indeed found to be sister species, however our data did not support a contemporaneous divergence. Analyses also revealed that differential use of habitat might have played an important role in the speciation dynamics of this group of fishes, in particular among New World species where extensive sample coverage was available.ConclusionsThis study provides a new hypothesis for the sister clade of Hamulidae and a robust phylogeny of the latter. The presence of para- and polyphyletic genera underscores the need for a taxonomic reassessment within the family. A scarce sampling of the Old World Pomadasys species prevents us to definitively point to a New World origin of the sub-familiy Hamulinae, however our data suggest that this is likely to be the case. This study also illustrates how life history habitat influences speciation and evolutionary trajectories.

Recent molecular exploration of the Plasmodium species circulating in great apes in Africa has revealed the existence of a large and previously unknown diversity of Plasmodium. For instance, gorillas were found to be infected by parasites closely related to Plasmodium falciparum, suggesting that the human malignant malaria agent may have arisen after a transfer from gorillas. Although this scenario is likely in light of the data collected in great apes, it remained to be ascertained whether P. falciparum-related parasites may infect other nonhuman primates in Africa. Using molecular tools, we here explore the diversity of Plasmodium species infecting monkeys in Central Africa. In addition to previously described Hepatocystis and Plasmodium species (Plasmodium gonderi and Plasmodium sp DAJ-2004), we have found one African monkey to be infected by a P. falciparum-related parasite. Examination of the nuclear and mitochondrial genomes of this parasite reveals that it is specific of nonhuman primates, indicating that P. falciparum-related pathogens can naturally circulate in some monkey populations in Africa. We also show that at least two distinct genetic entities of P. falciparum infect nonhuman primates and humans, respectively. Our discoveries bring into question the proposed gorilla origin of human P. falciparum.

One of the goals of systems genetics is the reconstruction of gene networks that underlie key processes in development and disease. To identify cartilage gene networks that play an important role in bone development, we used a systems genetics approach that integrated microarray gene expression profiles from cartilage and bone phenotypic data from two sets of recombinant inbred strains. Microarray profiles generated from isolated chondrocytes were used to generate weighted gene coexpression networks. This analysis resulted in the identification of subnetworks (modules) of coexpressed genes that then were examined for relationships with bone geometry and density. One module exhibited significant correlation with femur length (r = 0.416), anteroposterior diameter (r = 0.418), mediolateral diameter (r = 0.576), and bone mineral density (r = 0.475). Highly connected genes (n = 28) from this and other modules were tested in vitro using prechondrocyte ATDC5 cells and RNA interference. Five of the 28 genes were found to play a role in chondrocyte differentiation. Two of these, Hspd1 and Cdkn1a, were known previously to function in chondrocyte development, whereas the other three, Bhlhb9, Cugbp1, and Spcs3, are novel genes. Our integrative analysis provided a systems-level view of cartilage development and identified genes that may be involved in bone development.

Mammals are serially infected with a variety of microorganisms, including bacteria and parasites. Each infection reprograms the immune system's responses to re-exposure and potentially alters responses to first-time infection by different microorganisms. To examine whether infection with a metazoan parasite modulates host responses to subsequent bacterial infection, mice were infected with the hookworm-like intestinal nematode Nippostrongylus brasiliensis, followed in 2-4 weeks by peritoneal injection of the pathogenic bacterium Klebsiella pneumoniae. Survival from Klebsiella peritonitis two weeks after parasite infection was better in Nippostrongylus-infected animals than in unparasitized mice, with Nippostrongylus-infected mice having fewer peritoneal bacteria, more neutrophils, and higher levels of protective interleukin 6. The improved survival of Nippostrongylus-infected mice depends on IL-4 because the survival benefit is lost in mice lacking IL-4. Because mast cells protect mice from Klebsiella peritonitis, we examined responses in mast cell-deficient Kit(W-sh)/Kit(W-sh) mice, in which parasitosis failed to improve survival from Klebsiella peritonitis. However, adoptive transfer of cultured mast cells to Kit(W-sh)/Kit(W-sh) mice restored survival benefits of parasitosis. These results show that recent infection with Nippostrongylus brasiliensis protects mice from Klebsiella peritonitis by modulating mast cell contributions to host defense, and suggest more generally that parasitosis can yield survival advantages to a bacterially infected host.

BackgroundNematodes represent the most abundant benthic metazoa in one of the largest habitats on earth, the deep sea. Characterizing major patterns of biodiversity within this dominant group is a critical step towards understanding evolutionary patterns across this vast ecosystem. The present study has aimed to place deep-sea nematode species into a phylogenetic framework, investigate relationships between shallow water and deep-sea taxa, and elucidate phylogeographic patterns amongst the deep-sea fauna.ResultsMolecular data (18 S and 28 S rRNA) confirms a high diversity amongst deep-sea Enoplids. There is no evidence for endemic deep-sea lineages in Maximum Likelihood or Bayesian phylogenies, and Enoplids do not cluster according to depth or geographic location. Tree topologies suggest frequent interchanges between deep-sea and shallow water habitats, as well as a mixture of early radiations and more recently derived lineages amongst deep-sea taxa. This study also provides convincing evidence of cosmopolitan marine species, recovering a subset of Oncholaimid nematodes with identical gene sequences (18 S, 28 S and cox1) at trans-Atlantic sample sites.ConclusionsThe complex clade structures recovered within the Enoplida support a high global species richness for marine nematodes, with phylogeographic patterns suggesting the existence of closely related, globally distributed species complexes in the deep sea. True cosmopolitan species may additionally exist within this group, potentially driven by specific life history traits of Enoplids. Although this investigation aimed to intensively sample nematodes from the order Enoplida, specimens were only identified down to genus (at best) and our sampling regime focused on an infinitesimal small fraction of the deep-sea floor. Future nematode studies should incorporate an extended sample set covering a wide depth range (shelf, bathyal, and abyssal sites), utilize additional genetic loci (e.g. mtDNA) that are informative at the species level, and apply high-throughput sequencing methods to fully assay community diversity. Finally, further molecular studies are needed to determine whether phylogeographic patterns observed in Enoplids are common across other ubiquitous marine groups (e.g. Chromadorida, Monhysterida).