Two C++ libraries for counting trees on a phylogenetic terrace.

Motivation: The presence of terraces in phylogenetic tree space, i.e. a potentially large number of distinct tree topologies that have exactly the same analytical likelihood score, was first described by Sanderson et al. However, popular software tools for maximum likelihood and Bayesian phylogenetic inference do not yet routinely report, if inferred phylogenies reside on a terrace, or not. We believe, this is due to the lack of an efficient library to (i) determine if a tree resides on a terrace, (ii) calculate how many trees reside on a terrace and (iii) enumerate all trees on a terrace. Results: In our bioinformatics practical that is set up as a programming contest we developed two efficient and independent C++ implementations of the SUPERB algorithm by Constantinescu and Sankoff (1995) for counting and enumerating trees on a terrace. Both implementations yield exactly the same results, are more than one order of magnitude faster, and require one order of magnitude less memory than a previous thirrd party python implementation. Availability and implementation: The source codes are available under GNU GPL at https://github.com/terraphast. Supplementary information: Supplementary data are available at Bioinformatics online.

Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo.

MOTIVATION: In recent years, molecular species delimitation has become a routine approach for quantifying and classifying biodiversity. Barcoding methods are of particular importance in large-scale surveys as they promote fast species discovery and biodiversity estimates. Among those, distance-based methods are the most common choice as they scale well with large datasets; however, they are sensitive to similarity threshold parameters and they ignore evolutionary relationships. The recently introduced "Poisson Tree Processes" (PTP) method is a phylogeny-aware approach that does not rely on such thresholds. Yet, two weaknesses of PTP impact its accuracy and practicality when applied to large datasets; it does not account for divergent intraspecific variation and is slow for a large number of sequences. RESULTS: We introduce the multi-rate PTP (mPTP), an improved method that alleviates the theoretical and technical shortcomings of PTP. It incorporates different levels of intraspecific genetic diversity deriving from differences in either the evolutionary history or sampling of each species. Results on empirical data suggest that mPTP is superior to PTP and popular distance-based methods as it, consistently yields more accurate delimitations with respect to the taxonomy (i.e., identifies more taxonomic species, infers species numbers closer to the taxonomy). Moreover, mPTP does not require any similarity threshold as input. The novel dynamic programming algorithm attains a speedup of at least five orders of magnitude compared to PTP, allowing it to delimit species in large (meta-) barcoding data. In addition, Markov Chain Monte Carlo sampling provides a comprehensive evaluation of the inferred delimitation in just a few seconds for millions of steps, independently of tree size. AVAILABILITY AND IMPLEMENTATION: mPTP is implemented in C and is available for download at http://github.com/Pas-Kapli/mptp under the GNU Affero 3 license. A web-service is available at http://mptp.h-its.org . CONTACT: : paschalia.kapli@h-its.org or alexandros.stamatakis@h-its.org or tomas.flouri@h-its.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Efficient Detection of Repeating Sites to Accelerate Phylogenetic Likelihood Calculations.

The phylogenetic likelihood function (PLF) is the major computational bottleneck in several applications of evolutionary biology such as phylogenetic inference, species delimitation, model selection, and divergence times estimation. Given the alignment, a tree and the evolutionary model parameters, the likelihood function computes the conditional likelihood vectors for every node of the tree. Vector entries for which all input data are identical result in redundant likelihood operations which, in turn, yield identical conditional values. Such operations can be omitted for improving run-time and, using appropriate data structures, reducing memory usage. We present a fast, novel method for identifying and omitting such redundant operations in phylogenetic likelihood calculations, and assess the performance improvement and memory savings attained by our method. Using empirical and simulated data sets, we show that a prototype implementation of our method yields up to 12-fold speedups and uses up to 78% less memory than one of the fastest and most highly tuned implementations of the PLF currently available. Our method is generic and can seamlessly be integrated into any phylogenetic likelihood implementation. [Algorithms; maximum likelihood; phylogenetic likelihood function; phylogenetics].

Effects of pre- and postnatal exposure to 1880-1900MHz DECT base radiation on development in the rat.

In the present study, to evaluate the effects of wireless 1880-1900MHz Digital Enhanced Communication Telephony (DECT) base radiation on fetal and postnatal development, Wistar rats were exposed at an average electric field intensity of 3.7V/m, 12h/day, during pregnancy. After parturition, a group of dams and offspring were similarly exposed for another 22days. Controls were sham-exposed. The data showed that DECT base radiation exposure caused heart rate increase in the embryos on the 17th day of pregnancy. Moreover, significant changes on the newborns' somatometric characteristics were noticed. Pyramidal cell loss and glia fibrilliary acidic protein (GFAP) over-expression were detected in the CA4 region of the hippocampus of the 22-day old pups that were irradiated either during prenatal life or both pre- and postnatally. Changes in the integrity of the brain in the 22-day old pups could potentially be related to developmental behavioral changes during the fetal period.

The levels of the GluN2A NMDA receptor subunit are modified in both the neonatal and adult rat brain by an early experience involving denial of maternal contact.

The composition of the N-methyl-d-aspartate receptor receptor in GluN2A/GluN2B subunits is important in determining its characteristics and its role in plasticity, a property of the brain which is known to be critically affected by early experiences. In the present work we employed an early experience model involving either receipt (RER) or denial (DER) of the expected reward of maternal contact within the context of learning by the pups of a T-maze on postnatal days (PND) 10-13. We investigated the effects of the RER and DER early experiences on GluN1, GluN2A and GluN2B levels in the prefrontal cortex (PFC), hippocampus and amygdala of the rat. We show that on PND13 the DER animals had lower GluN2A levels in the PFC. In adulthood DER males had higher GluN2A levels in the hippocampus, both under basal conditions and after exposure to a novel environment. The early experiences did not affect the response to the novelty. After exposure to a novel environment animals of all three groups (DER, RER, Control) responded with an increase in GluN2A levels in the brain areas examined. We did not detect any effects on GluN1 or GluN2B levels. The alterations in GluN2A levels observed in the DER animals could in part be responsible for their behavioral phenotype, described previously, which includes an increased susceptibility for the expression of depressive-like behavior.

INSECT PHYLOGENOMICS. Response to Comment on "Phylogenomics resolves the timing and pattern of insect evolution".

Tong et al. comment on the accuracy of the dating analysis presented in our work on the phylogeny of insects and provide a reanalysis of our data. They replace log-normal priors with uniform priors and add a "roachoid" fossil as a calibration point. Although the reanalysis provides an interesting alternative viewpoint, we maintain that our choices were appropriate.

The phylogenetic likelihood library.

We introduce the Phylogenetic Likelihood Library (PLL), a highly optimized application programming interface for developing likelihood-based phylogenetic inference and postanalysis software. The PLL implements appropriate data structures and functions that allow users to quickly implement common, error-prone, and labor-intensive tasks, such as likelihood calculations, model parameter as well as branch length optimization, and tree space exploration. The highly optimized and parallelized implementation of the phylogenetic likelihood function and a thorough documentation provide a framework for rapid development of scalable parallel phylogenetic software. By example of two likelihood-based phylogenetic codes we show that the PLL improves the sequential performance of current software by a factor of 2-10 while requiring only 1 month of programming time for integration. We show that, when numerical scaling for preventing floating point underflow is enabled, the double precision likelihood calculations in the PLL are up to 1.9 times faster than those in BEAGLE. On an empirical DNA dataset with 2000 taxa the AVX version of PLL is 4 times faster than BEAGLE (scaling enabled and required). The PLL is available at http://www.libpll.org under the GNU General Public License (GPL).

An optimal algorithm for computing all subtree repeats in trees.

Given a labelled tree T, our goal is to group repeating subtrees of T into equivalence classes with respect to their topologies and the node labels. We present an explicit, simple and time-optimal algorithm for solving this problem for unrooted unordered labelled trees and show that the running time of our method is linear with respect to the size of T. By unordered, we mean that the order of the adjacent nodes (children/neighbours) of any node of T is irrelevant. An unrooted tree T does not have a node that is designated as root and can also be referred to as an undirected tree. We show how the presented algorithm can easily be modified to operate on trees that do not satisfy some or any of the aforementioned assumptions on the tree structure; for instance, how it can be applied to rooted, ordered or unlabelled trees.

Effects of an early experience of reward through maternal contact or its denial on the dopaminergic system of the rat brain.

The mesolimbic/mesocortical dopaminergic pathway plays a pivotal role in the reward system. During the neonatal period the mother is the main source of rewarding stimuli. We have developed an experimental model in which rat pups learn a T-maze during the neonatal period (postnatal day (PND) 10-13) using contact with the mother as the reward. One group of animals is allowed contact with the mother (receipt of expected reward, RER) while the other was denied (denial of expected reward, DER). We determined the effects of these two early experiences in the prefrontal cortex (PFC) and the nucleus accumbens (nAc), the levels of dopamine (DA) and its metabolites [3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] by high-performance liquid chromatography and those of D1 and D2 receptors by autoradiographic in vitro binding both on PND 13 and in adulthood. On PND13, 2h after the end of training, the RER experience resulted in higher DA, HVA and D1 receptor levels in the nAc, while the DER in lower DA and its metabolites (DOPAC and HVA) in the PFC. These results could be related to the reward the RER pups received through the contact with their mother. The RER and DER early experience had long-term sex-dependent effects: The RER-induced activation of the dopaminergic system in the nAc was also evident in adult female rats. In contrast, adult DER males, similar to PND13 animals, had reduced dopamine in the PFC. Our results document that early experiences, a key determinant of adult brain function, affect the dopaminergic system which is disturbed in many psychiatric diseases.

Sexually dimorphic long-term effects of an early life experience on AMPA receptor subunit expression in rat brain.

Neonatal handling, an experimental model of early life experiences, is known to affect hypothalamic-pituitary-adrenal (HPA) axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. AMPA receptors (AMPARs) mediate fast synaptic transmission at excitatory glutamatergic synapses in the CNS and are crucial during neuronal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as AMPA glutamate receptor subunits (GluA1, GluA2, GluA3 and GluA4), which combine to form tetramers. The present study addressed the question of whether neonatal handling (15min daily maternal separation from postnatal day 1 (PND1) to PND21) might have an effect on GluA1-4 mRNA levels in adult rat male and female brain using in situ hybridization. We have identified selective sexually dimorphic effects of neonatal handling on the mRNA expression levels of AMPAR subunits in adult rat hippocampus and nuclei of the amygdaloid complex. In the dorsal hippocampus GluA1 mRNA levels were increased in handled males, while they were decreased in handled female animals. In the ventral hippocampus and the amygdaloid complex GluA2 mRNA was lower in handled females, while no effect was observed in handled males. Furthermore, we observed that neonatal handling induced in both sexes decreases of GluA2 mRNA in the dorsal hippocampus, as well as in the somatosensory and occipital cortex, of GluA3 mRNA in most hippocampal areas, amygdaloid complex and cortical regions studied, and of GluA4 mRNA in the ventral hippocampus. These results show that glutamatergic transmission is markedly affected by an early experience. The neonatal handling-induced alterations in AMPAR subunit composition are in line with the increased brain plasticity, the more effective HPA axis function, and in general the more adaptive behavioral phenotype known to characterize the handled animals.

OmegaPlus: a scalable tool for rapid detection of selective sweeps in whole-genome datasets.

UNLABELLED: Recent advances in sequencing technologies have led to the rapid accumulation of molecular sequence data. Analyzing whole-genome data (as obtained from next-generation sequencers) from intra-species samples allows to detect signatures of positive selection along the genome and therefore identify potentially advantageous genes in the course of the evolution of a population. We introduce OmegaPlus, an open-source tool for rapid detection of selective sweeps in whole-genome data based on linkage disequilibrium. The tool is up to two orders of magnitude faster than existing programs for this purpose and also exhibits up to two orders of magnitude smaller memory requirements. AVAILABILITY: OmegaPlus is available under GNU GPL at http://www.exelixis-lab.org/software.html.

RAxML-Light: a tool for computing terabyte phylogenies.

MOTIVATION: Due to advances in molecular sequencing and the increasingly rapid collection of molecular data, the field of phyloinformatics is transforming into a computational science. Therefore, new tools are required that can be deployed in supercomputing environments and that scale to hundreds or thousands of cores. RESULTS: We describe RAxML-Light, a tool for large-scale phylogenetic inference on supercomputers under maximum likelihood. It implements a light-weight checkpointing mechanism, deploys 128-bit (SSE3) and 256-bit (AVX) vector intrinsics, offers two orthogonal memory saving techniques and provides a fine-grain production-level message passing interface parallelization of the likelihood function. To demonstrate scalability and robustness of the code, we inferred a phylogeny on a simulated DNA alignment (1481 taxa, 20 000 000 bp) using 672 cores. This dataset requires one terabyte of RAM to compute the likelihood score on a single tree. CODE AVAILABILITY: https://github.com/stamatak/RAxML-Light-1.0.5 DATA AVAILABILITY: http://www.exelixis-lab.org/onLineMaterial.tar.bz2 CONTACT: alexandros.stamatakis@h-its.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effects of interaction of an early experience of reward through maternal contact or its denial with social stress during adolescence on the serotonergic system and the stress responsiveness of adult female rats.

Experiences during critical periods, such as the neonatal and adolescence, play a critical role in determining adult stress-coping behavior. Based on the aforementioned we developed an experimental protocol, which included a neonatal experience and a social stress during adolescence. The serotonergic system is known as an important modulator of coping ability and, in general, emotional balance in both normal and pathological states, such as depression and anxiety, for which females are more vulnerable. Thus in the present work we used female rats and determined 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), and 5-hydroxytryptamine receptor type 1A (5-HT(1A)) receptor levels in the prefrontal cortex (PFC) and the amygdala (AMY). During postnatal days 10-13 (PND 10-13) rat pups were exposed to a T-maze, one arm of which lead to the mother. One group of animals was allowed contact with the mother (rewarded-receiving expected reward (RER)), whereas the other was denied the expected reward (DER). High performance liquid chromatography (HPLC) analysis revealed that in both the PFC and in AMY, adult RER animals had higher basal 5-HT levels. Furthermore, in the AMY of this group of animals, higher levels of 5-HT(1A) receptors were detected by Western blot analysis. In adulthood rats were exposed to the Forced Swimming Test/Stress (FST/S). RER animals not exposed to the adolescent stress exhibited longer immobility time during both the first and second day of FST. Corticosterone levels following the FST fell faster in the DER animals. Adolescent stress affected the responses to the adult FSS only in the DER animals, which had decreased 5-HT in the AMY and increased immobility time on both days of the FST, compared with the DER, not stressed in adolescence. The phenotype of the DER animals is in line with the "match-mismatch" hypothesis, which states that if two events during critical periods of life "match" in being mildly stressful, their interaction can be adaptive.

Reward or its denial during the neonatal period affects adult spatial memory and hippocampal phosphorylated cAMP response element-binding protein levels of both the neonatal and adult rat.

Early life experiences, particularly mother-infant interactions, have been shown to influence adult coping and learning abilities via gene-environment interactions. We have developed a paradigm, in which mother contact is used as either a positive or a negative reinforcer in a T-maze, during postnatal days 10-13. In both neonates receiving (RER) or denied (DER) the expected reward, exposure to the memory test in the absence of the mother resulted in a remarkable increase in the number of pCREB immunopositive cells, when compared to their corresponding levels 2 h after the completion of the training process, but also to the levels of naïve animals. In the CA3 area, the pattern of pCREB immunoreactivity, when evaluated 2 h after the completion of the training on postnatal day 13 seemed to distinguish between the two different neonatal experiences in the T-maze, with the DER pups showing higher levels of pCREB immunopositive cells than the RER. Exposure to the Morris Water Maze (MWM) during adulthood revealed a memory advantage of the DER animals compared to the RER and the animals not exposed to the neonatal experience. Relevantly, in the DER animals an increased number of pCREB immunopositive cells was observed in the CA3 area even 24 h after the end of MWM training. When also measured after exposure to the probe trial, the number of pCREB immunopositive cells was again higher in the DER compared to the RER animals. In conclusion, we show that a learning experience involving discrepancy during the particularly plastic neonatal period is able to induce long-term effects, which result in enhanced adult hippocampal dependent spatial memory. Furthermore, our data document a role of plasticity molecules like pCREB in mediating hippocampal dependent learning and detection of novelty not only in adulthood, but also more importantly in the neonatal period of the rat.

Short-term memory in mice is affected by mobile phone radiation.

The effects of mobile phone electromagnetic fields (EMFs) were studied on a non-spatial memory task (Object Recognition Task - ORT) that requires entorhinal cortex function. The task was applied to three groups of mice Mus musculus C57BL/6 (exposed, sham-exposed and control) combined with 3 different radiation exposure protocols. In the first protocol designated "acute exposure", mice 45 days old (PND45 - postnatal day 45) were exposed to mobile phone (MP) radiation (SAR value 0.22W/kg) during the habituation, the training and the test sessions of the ORT, but not during the 10min inter-trial interval (ITI) where consolidation of stored object information takes place. On the second protocol designated "chronic exposure-I", the same mice were exposed for 17 days for 90min/per day starting at PND55 to the same MP radiation. ORT recognition memory was performed at PND72 with radiation present only during the ITI phase. In the third protocol designated "chronic exposure-II", mice continued to be exposed daily under the same conditions up to PND86 having received radiation for 31 days. One day later the ORT test was performed without irradiation present in any of the sessions. The ORT-derived discrimination indices in all three exposure protocols revealed a major effect on the "chronic exposure-I" suggesting a possible severe interaction of EMF with the consolidation phase of recognition memory processes. This may imply that the primary EMF target may be the information transfer pathway connecting the entorhinal-parahippocampal regions which participate in the ORT memory task.

Whole body exposure with GSM 900MHz affects spatial memory in mice.

Extended work has been performed worldwide on the effects of mobile phone radiation upon rats' cognitive functions, however there is great controversy to the existence or not of deficits. The present work has been designed in order to test the effects of mobile phone radiation on spatial learning and memory in mice Mus musculus Balb/c using the Morris water maze (a hippocampal-dependent spatial memory task), since there is just one other study on mice with very low SAR level (0.05W/kg) showing no effects. We have applied a 2h daily dose of pulsed GSM 900MHz radiation from commercially available mobile phone for 4 days at SAR values ranging from 0.41 to 0.98W/kg. Statistical analysis revealed that during learning, exposed animals showed a deficit in transferring the acquired spatial information across training days (increased escape latency and distance swam, compared to the sham-exposed animals, on the first trial of training days 2-4). Moreover, during the memory probe-trial sham-exposed animals showed the expected preference for the target quadrant, while the exposed animals showed no preference, indicating that the exposed mice had deficits in consolidation and/or retrieval of the learned spatial information. Our results provide a basis for more thorough investigations considering reports on non-thermal effects of electromagnetic fields (EMFs).

Selective effects of neonatal handling on rat brain N-methyl-D-aspartate receptors.

Neonatal handling, an experimental model of early life experiences, is known to affect the hypothalamic-pituitary-adrenal axis function thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. A molecule that plays a most critical role in such processes is the N-methyl-D-aspartate (NMDA) receptor, a tetramer consisting of two obligatory, channel forming NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. Since the subunit composition of the NMDA receptor affects brain plasticity, in the present study we investigated the effect of neonatal handling on NR1, NR2A and NR2B mRNA levels using in situ hybridization, and on NR2B binding sites, using autoradiography of in vitro binding of [(3)H]-ifenprodil, in adult rat limbic brain areas. We found that neonatal handling specifically increased NR2B mRNA and binding sites, while it had no effect on the NR1 and NR2A subunits. More specifically, neonatally handled animals, both males and females, had higher NR2B mRNA and binding sites in the dorsal CA1 hippocampal area, as well as the prelimbic, the anterior cingulate and the somatosensory cortex, compared to the non-handled. Moreover NR2B binding sites were increased in the dorsal CA3 area of handled animals of both sexes. Furthermore, neonatal handling had a sexually dimorphic effect, increasing NR2B mRNA and binding sites in the central and medial amygdaloid nuclei only of the females. The neonatal handling-induced increase in the NR2B subunit of the NMDA receptor could underlie the higher brain plasticity, which neonatally handled animals exhibit.

Effect of neonatal handling on adult rat spatial learning and memory following acute stress.

Brief neonatal handling permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress. Since stress is known to affect cognitive abilities, in the present study we investigated the effect of brief (15 min) handling on learning and memory in the Morris water maze, following exposure to an acute restraint stress either before training or recall. Exposure of non-handled rats to the acute stress prior to training resulted in quicker learning of the task, than in the absence of the stressor. When acute stress preceded acquisition, male handled rats showed an overall better learning performance, and both sexes of handled animals were less impaired in the subsequent memory trial, compared to the respective non-handled. In addition, the number of neurons immunoreactive for GR was higher in all areas of Ammon's horn of the handled rats during the recall. In contrast, the number of neurons immunoreactive for MR was higher in the CA1 and CA2 areas of the non-handled males. When the acute restraint stress was applied prior to the memory test, neonatal handling was not effective in preventing mnemonic impairment, as all animal groups showed a similar deficit in recall. In this case, no difference between handled and non-handled rats was observed in the number of GR positive neurons in the CA2 and CA3 hippocampal areas during the memory test. These results indicate that early experience interacts with sex and acute stress exposure in adulthood to affect performance in the water maze. Hippocampal corticosterone receptors may play a role in determining the final outcome.

Cellular mechanisms underlying the effect of a single exposure to neonatal handling on neurotrophin-3 in the brain of 1-day-old rats.

Neurotrophin-3 (NT-3) has an important role in brain development and is thus a good candidate molecule to be involved in the cellular mechanisms mediating the effects of early experiences on the brain. In the present work we employed the model of neonatal handling, which is known to affect the ability of the adult organism to respond to stressful stimuli, and determined its effects on NT-3 levels in the rat hippocampus and cortex 2, 4 and 8 h after handling on postnatal day 1. We also recorded maternal behavior during the 8 h following handling. At both the 4 and 8 h time-points there was an increase in NT-3 positive cells in field 1 of Ammon's horn (CA1 area of the hippocampus) and parietal cortex of the handled animals. In the parietal cortex NT-3 levels increased with time following handling: at 8 h there were more NT-3 positive cells than at 4 h. During the 4 h following the end of handling, handled pups were subject to more maternal licking, indicating that the more intense maternal care could underlie the handling-induced increase in NT-3. In the hippocampus, the handling induced increase in NT-3 was cancelled by inhibition of N-methyl-D-aspartate (NMDA), AMPA/kainate, or GABA-A receptors, as well as L-type voltage-gated Ca(2+) channels. It thus appears that neonatal handling activates these neurotransmitter receptors and channels, leading to increased intracellular Ca(2+) and increased NT-3 expression. NT-3 can then activate downstream effectors and exert its morphogenetic actions and thus imprint the effects of handling on the brain.

Effects of neonatal handling on the basal forebrain cholinergic system of adult male and female rats.

Neonatal handling is an early experience which results in improved function of the hypothalamic-pituitary-adrenal axis, increased adaptability and coping as a response to stress, as well as better cognitive abilities. In the present study, we investigated the effect of neonatal handling on the basal forebrain cholinergic system, since this system is known to play an important role in cognitive processes. We report that neonatal handling results in increased number of choline-acetyl transferase immunopositive cells in the septum/diagonal band, in both sexes, while no such effect was observed in the other cholinergic nuclei, such as the magnocellular preoptic nucleus and the nucleus basalis of Meynert. In addition, neonatal handling resulted in increased M1 and M2 muscarinic receptor binding sites in the cingulate and piriform cortex of both male and female rats. A handling-induced increase in M1 muscarinic receptor binding sites was also observed in the CA3 and CA4 (fields 3 and 4 of Ammon's horn) areas of the hippocampus. Furthermore, a handling-induced increase in acetylcholinesterase staining was found only in the hippocampus of females. Our results thus show that neonatal handling acts in a sexually dimorphic manner on one of the cholinergic parameters, and has a beneficial effect on BFCS function, which could be related to the more efficient and adaptive stress response and the superior cognitive abilities of handled animals.