Comparative transcriptomics of the garden dormouse hypothalamus during hibernation.

Torpor or heterothermy is an energy-saving mechanism used by endotherms to overcome harsh environmental conditions. During winter, the garden dormouse (Eliomys quercinus) hibernates with multiday torpor bouts and body temperatures of a few degrees Celsius, interrupted by brief euthermic phases. This study investigates gene expression within the hypothalamus, the key brain area controlling energy balance, adding information on differential gene expression potentially relevant to orchestrate torpor. A de novo assembled transcriptome of the hypothalamus was generated from garden dormice hibernating under constant darkness without food and water at 5 °C. Samples were collected during early torpor, late torpor, and interbout arousal. During early torpor, 765 genes were differentially expressed as compared with interbout arousal. Twenty-seven pathways were over-represented, including pathways related to hemostasis, extracellular matrix organization, and signaling of small molecules. Only 82 genes were found to be differentially expressed between early and late torpor, and no pathways were over-represented. During late torpor, 924 genes were differentially expressed relative to interbout arousal. Despite the high number of differentially expressed genes, only 10 pathways were over-represented. Of these, eight were also observed to be over-represented when comparing early torpor and interbout arousal. Our results are largely consistent with previous findings in other heterotherms. The addition of a transcriptome of a novel species may help to identify species-specific and overarching torpor mechanisms through future species comparisons.

Comparative transcriptomics of the Djungarian hamster hypothalamus during short photoperiod acclimation and spontaneous torpor.

The energy-saving strategy of Djungarian hamsters (Phodopus sungorus, Cricetidae) to overcome harsh environmental conditions comprises of behavioral, morphological, and physiological adjustments, including spontaneous daily torpor, a metabolic downstate. These acclimatizations are triggered by short photoperiod and orchestrated by the hypothalamus. Key mechanisms of long-term photoperiodic acclimatizations have partly been described, but specific mechanisms that acutely control torpor remain incomplete. Here, we performed comparative transcriptome analysis on hypothalamus of normometabolic hamsters in their summer- and winter-like state to enable us to identify changes in gene expression during photoperiodic acclimations. Comparing nontorpid and torpid hamsters may also be able to pin down mechanisms relevant for torpor control. A de novo assembled transcriptome of the hypothalamus was generated from hamsters acclimated to long photoperiod or to short photoperiod. The hamsters were sampled either during long photoperiod normothermia, short photoperiod normothermia, or short photoperiod-induced spontaneous torpor with a body temperature of 24.6 ± 1.0 °C, or. The mRNA-seq analysis revealed that 32 and 759 genes were differentially expressed during photoperiod or torpor, respectively. Biological processes were not enriched during photoperiodic acclimatization but were during torpor, where transcriptional and metabolic processes were reinforced. Most extremely regulated genes (those genes with |log2(FC)| > 2.0 and padj < 0.05 of a pairwise group comparison) underpinned the role of known key players in photoperiodic comparison, but these genes exhibit adaptive and protective adjustments during torpor. Targeted analyses of genes from potentially involved hypothalamic systems identified gene regulation of previously described torpor-relevant systems and a potential involvement of glucose transport.

Phylogenomics from transcriptomic "bycatch" clarify the origins and diversity of avian trypanosomes in North America.

The eukaryotic blood parasite genus Trypanosoma includes several important pathogens of humans and livestock, but has been understudied in wildlife broadly. The trypanosomes that infect birds are in particular need of increased attention, as these parasites are abundant and globally distributed, yet few studies have addressed their evolutionary origins and diversity using modern molecular and analytical approaches. Of specific interest are the deep evolutionary relationships of the avian trypanosomes relative to the trypanosome species that are pathogenic in humans, as well as their species level diversity in regions where they have been understudied such as North America. Here, we address these unresolved areas of study using phylogenomic data for two species of avian trypanosomes that were isolated as "bycatch" from host transcriptome assemblies, as well as a large 18S DNA barcode sequence dataset that includes 143 novel avian Trypanosoma 18S sequences from North America. Using a phylogenomic approach, we find that the avian trypanosomes are nested within a clade of primarily mammalian trypanosomes that includes the human pathogen Trypanosoma cruzi, and are paraphyletic with respect to the ruminant trypanosome Trypanosoma theileri. DNA barcode sequences showed that T. avium and an unidentified small, non-striated trypanosome that was morphologically similar to T. everetti are each represented by highly abundant and divergent 18S haplotypes in North America. Community-level sampling revealed that additional species-level Trypanosoma lineages exist in this region. We compared the newly sequenced DNA barcodes from North America to a global database, and found that avian Trypanosoma 18S haplotypes generally exhibited a marked lack of host specificity with at least one T. avium haplotype having an intercontinental distribution. This highly abundant T. avium haplotype appears to have a remarkably high dispersal ability and cosmopolitan capacity to evade avian host immune defenses, which warrant further study.

Evolution of Host Specificity by Malaria Parasites through Altered Mechanisms Controlling Genome Maintenance.

The protozoan parasites that cause malaria infect a wide variety of vertebrate hosts, including birds, reptiles, and mammals, and the evolutionary pressures inherent to the host-parasite relationship have profoundly shaped the genomes of both host and parasite. Here, we report that these selective pressures have resulted in unexpected alterations to one of the most basic aspects of eukaryotic biology, the maintenance of genome integrity through DNA repair. Malaria parasites that infect humans continuously generate genetic diversity within their antigen-encoding gene families through frequent ectopic recombination between gene family members, a process that is a crucial feature of the persistence of malaria globally. The continuous generation of antigen diversity ensures that different parasite isolates are antigenically distinct, thus preventing extensive cross-reactive immunity and enabling parasites to maintain stable transmission within human populations. However, the molecular basis of the recombination between gene family members is not well understood. Through computational analyses of the antigen-encoding, multicopy gene families of different Plasmodium species, we report the unexpected observation that malaria parasites that infect rodents do not display the same degree of antigen diversity as observed in Plasmodium falciparum and appear to undergo significantly less ectopic recombination. Using comparative genomics, we also identify key molecular components of the diversification process, thus shedding new light on how malaria parasites balance the maintenance of genome integrity with the requirement for continuous genetic diversification.IMPORTANCE Malaria remains one of the most prevalent and deadly infectious diseases of the developing world, causing approximately 228 million clinical cases and nearly half a million deaths annually. The disease is caused by protozoan parasites of the genus Plasmodium, and of the five species capable of infecting humans, infections with P. falciparum are the most severe. In addition to the parasites that infect people, there are hundreds of additional species that infect birds, reptiles, and other mammals, each exquisitely evolved to meet the specific challenges inherent to survival within their respective hosts. By comparing the unique strategies that each species has evolved, key insights into host-parasite interactions can be gained, including discoveries regarding the pathogenesis of human disease. Here, we describe the surprising observation that closely related parasites with different hosts have evolved remarkably different methods for repairing their genomes. This observation has important implications for the ability of parasites to maintain chronic infections and for the development of host immunity.

Metatranscriptomics yields new genomic resources and sensitive detection of infections for diverse blood parasites.

Metatranscriptomics is a powerful method for studying the composition and function of complex microbial communities. The application of metatranscriptomics to multispecies parasite infections is of particular interest, as research on parasite evolution and diversification has been hampered by technical challenges to genome-scale DNA sequencing. In particular, blood parasites of vertebrates are abundant and diverse although they often occur at low infection intensities and exist as multispecies infections, rendering the isolation of genomic sequence data challenging. Here, we use birds and their diverse haemosporidian parasites to illustrate the potential for metatranscriptome sequencing to generate large quantities of genome-wide sequence data from multiple blood parasite species simultaneously. We used RNA-sequencing of 24 blood samples from songbirds in North America to show that metatranscriptomes can yield large proportions of haemosporidian protein-coding gene repertoires even when infections are of low intensity (<0.1% red blood cells infected) and consist of multiple parasite taxa. By bioinformatically separating host and parasite transcripts and assigning them to the haemosporidian genus of origin, we found that transcriptomes detected ~23% more total parasite infections across all samples than were identified using microscopy and DNA barcoding. For single-species infections, we obtained data for >1,300 loci from samples with as low as 0.03% parasitaemia, with the number of loci increasing with infection intensity. In total, we provide data for 1,502 single-copy orthologous loci from a phylogenetically diverse set of 33 haemosporidian mitochondrial lineages. The metatranscriptomic approach described here has the potential to accelerate ecological and evolutionary research on haemosporidians and other diverse parasites.

The polyphyly of Plasmodium: comprehensive phylogenetic analyses of the malaria parasites (order Haemosporida) reveal widespread taxonomic conflict.

The evolutionary relationships among the apicomplexan blood pathogens known as the malaria parasites (order Haemosporida), some of which infect nearly 200 million humans each year, has remained a vexing phylogenetic problem due to limitations in taxon sampling, character sampling and the extreme nucleotide base composition biases that are characteristic of this clade. Previous phylogenetic work on the malaria parasites has often lacked sufficient representation of the broad taxonomic diversity within the Haemosporida or the multi-locus sequence data needed to resolve deep evolutionary relationships, rendering our understanding of haemosporidian life-history evolution and the origin of the human malaria parasites incomplete. Here we present the most comprehensive phylogenetic analysis of the malaria parasites conducted to date, using samples from a broad diversity of vertebrate hosts that includes numerous enigmatic and poorly known haemosporidian lineages in addition to genome-wide multi-locus sequence data. We find that if base composition differences were corrected for during phylogenetic analysis, we recovered a well-supported topology indicating that the evolutionary history of the malaria parasites was characterized by a complex series of transitions in life-history strategies and host usage. Notably we find that Plasmodium, the malaria parasite genus that includes the species of human medical concern, is polyphyletic with the life-history traits characteristic of this genus having evolved in a dynamic manner across the phylogeny. We find support for multiple instances of gain and loss of asexual proliferation in host blood cells and production of haemozoin pigment, two traits that have been used for taxonomic classification as well as considered to be important factors for parasite virulence and used as drug targets. Lastly, our analysis illustrates the need for a widespread reassessment of malaria parasite taxonomy.

Parasite infection of public databases: a data mining approach to identify apicomplexan contaminations in animal genome and transcriptome assemblies.

BACKGROUND: Contaminations from various exogenous sources are a common problem in next-generation sequencing. Another possible source of contaminating DNA are endogenous parasites. On the one hand, undiscovered contaminations of animal sequence assemblies may lead to erroneous interpretation of data; on the other hand, when identified, parasite-derived sequences may provide a valuable source of information. RESULTS: Here we show that sequences deriving from apicomplexan parasites can be found in many animal genome and transcriptome projects, which in most cases derived from an infection of the sequenced host specimen. The apicomplexan sequences were extracted from the sequence assemblies using a newly developed bioinformatic pipeline (ContamFinder) and tentatively assigned to distinct taxa employing phylogenetic methods. We analysed 920 assemblies and found 20,907 contigs of apicomplexan origin in 51 of the datasets. The contaminating species were identified as members of the apicomplexan taxa Gregarinasina, Coccidia, Piroplasmida, and Haemosporida. For example, in the platypus genome assembly, we found a high number of contigs derived from a piroplasmid parasite (presumably Theileria ornithorhynchi). For most of the infecting parasite species, no molecular data had been available previously, and some of the datasets contain sequences representing large amounts of the parasite's gene repertoire. CONCLUSION: Our study suggests that parasite-derived contaminations represent a valuable source of information that can help to discover and identify new parasites, and provide information on previously unknown host-parasite interactions. We, therefore, argue that uncurated assembly data should routinely be made available in addition to the final assemblies.

Phylogeny of haemosporidian blood parasites revealed by a multi-gene approach.

The apicomplexan order Haemosporida is a clade of unicellular blood parasites that infect a variety of reptilian, avian and mammalian hosts. Among them are the agents of human malaria, parasites of the genus Plasmodium, which pose a major threat to human health. Illuminating the evolutionary history of Haemosporida may help us in understanding their enormous biological diversity, as well as tracing the multiple host switches and associated acquisitions of novel life-history traits. However, the deep-level phylogenetic relationships among major haemosporidian clades have remained enigmatic because the datasets employed in phylogenetic analyses were severely limited in either gene coverage or taxon sampling. Using a PCR-based approach that employs a novel set of primers, we sequenced fragments of 21 nuclear genes from seven haemosporidian parasites of the genera Leucocytozoon, Haemoproteus, Parahaemoproteus, Polychromophilus and Plasmodium. After addition of genomic data from 25 apicomplexan species, the unreduced alignment comprised 20,580 bp from 32 species. Phylogenetic analyses were performed based on nucleotide, codon and amino acid data employing Bayesian inference, maximum likelihood and maximum parsimony. All analyses resulted in highly congruent topologies. We found consistent support for a basal position of Leucocytozoon within Haemosporida. In contrast to all previous studies, we recovered a sister group relationship between the genera Polychromophilus and Plasmodium. Within Plasmodium, the sauropsid and mammal-infecting lineages were recovered as sister clades. Support for these relationships was high in nearly all trees, revealing a novel phylogeny of Haemosporida, which is robust to the choice of the outgroup and the method of tree inference.

Evaluating the Hypoxia Response of Ruffe and Flounder Gills by a Combined Proteome and Transcriptome Approach.

Hypoxia has gained ecological importance during the last decades, and it is the most dramatically increasing environmental factor in coastal areas and estuaries. The gills of fish are the prime target of hypoxia and other stresses. Here we have studied the impact of the exposure to hypoxia (1.5 mg O2/l for 48 h) on the protein expression of the gills of two estuarine fish species, the ruffe (Gymnocephalus cernua) and the European flounder (Platichthys flesus). First, we obtained the transcriptomes of mixed tissues (gills, heart and brain) from both species by Illumina next-generation sequencing. Then, the gill proteomes were investigated using two-dimensional gel electrophoresis and mass spectrometry. Quantification of the normalized proteome maps resulted in a total of 148 spots in the ruffe, of which 28 (18.8%) were significantly regulated (> 1.5-fold). In the flounder, 121 spots were found, of which 27 (22.3%) proteins were significantly regulated. The transcriptomes were used for the identification of these proteins, which was successful for 15 proteins of the ruffe and 14 of the flounder. The ruffe transcriptome dataset comprised 87,169,850 reads, resulting in an assembly of 72,108 contigs (N50 = 1,828 bp). 20,860 contigs (26.93%) had blastx hits with E < 1e-5 in the human sequences in the RefSeq database, representing 14,771 unique accession numbers. The flounder transcriptome with 78,943,030 reads assembled into 49,241 contigs (N50 = 2,106 bp). 20,127 contigs (40.87%) had a hit with human proteins, corresponding to 14,455 unique accession numbers. The regulation of selected genes was confirmed by quantitative real-time RT-PCR. Most of the regulated proteins that were identified by this approach function in the energy metabolism, while others are involved in the immune response, cell signalling and the cytoskeleton.

The Plasmodium falciparum exportome contains non-canonical PEXEL/HT proteins.

The pathogenicity of Plasmodium falciparum is partly due to parasite-induced host cell modifications. These modifications are facilitated by exported P. falciparum proteins, collectively referred to as the exportome. Export of several hundred proteins is mediated by the PEXEL/HT, a protease cleavage site. The PEXEL/HT is usually comprised of five amino acids, of which R at position 1, L at position 3 and E, D or Q at position 5 are conserved and important for export. Non-canonical PEXEL/HTs with K or H at position 1 and/or I at position 3 are presently considered non-functional. Here, we show that non-canonical PEXEL/HT proteins are overrepresented in P. falciparum and other Plasmodium species. Furthermore, we show that non-canonical PEXEL/HTs can be cleaved and can promote export in both a REX3 and a GBP reporter, but not in a KAHRP reporter, indicating that non-canonical PEXEL/HTs are functional in concert with a supportive sequence environment. We then selected P. falciparum proteins with a non-canonical PEXEL/HT and show that some of these proteins are exported and that their export depends on non-canonical PEXEL/HTs. We conclude that PEXEL/HT plasticity is higher than appreciated and that non-canonical PEXEL/HT proteins cannot categorically be excluded from Plasmodium exportome predictions.

A transcriptome approach to ecdysozoan phylogeny.

The monophyly of Ecdysozoa, which comprise molting phyla, has received strong support from several lines of evidence. However, the internal relationships of Ecdysozoa are still contended. We generated expressed sequence tags from a priapulid (penis worm), a kinorhynch (mud dragon), a tardigrade (water bear) and five chelicerate taxa by 454 transcriptome sequencing. A multigene alignment was assembled from 63 taxa, which comprised after matrix optimization 24,249 amino acid positions with high data density (2.6% gaps, 19.1% missing data). Phylogenetic analyses employing various models support the monophyly of Ecdysozoa. A clade combining Priapulida and Kinorhyncha (i.e. Scalidophora) was recovered as the earliest branch among Ecdysozoa. We conclude that Cycloneuralia, a taxon erected to combine Priapulida, Kinorhyncha and Nematoda (and others), are paraphyletic. Rather Arthropoda (including Onychophora) are allied with Nematoda and Tardigrada. Within Arthropoda, we found strong support for most clades, including monophyletic Mandibulata and Pancrustacea. The phylogeny within the Euchelicerata remained largely unresolved. There is conflicting evidence on the position of tardigrades: While Bayesian and maximum likelihood analyses of only slowly evolving genes recovered Tardigrada as a sister group to Arthropoda, analyses of the full data set, and of subsets containing genes evolving at fast and intermediate rates identified a clade of Tardigrada and Nematoda. Notably, the latter topology is also supported by the analyses of indel patterns.

Phylogenetic position of Myriapoda revealed by 454 transcriptome sequencing.

Myriapods had been considered closely allied to hexapods (insects and relatives). However, analyses of molecular sequence data have consistently placed Myriapoda either as a sister group of Pancrustacea, comprising crustaceans and hexapods, and thereby supporting the monophyly of Mandibulata, or retrieved Myriapoda as a sister group of Chelicerata (spiders, ticks, mites and allies). In addition, the relationships among the four myriapod groups (Pauropoda, Symphyla, Diplopoda, Chilopoda) are unclear. To resolve the phylogeny of myriapods and their relationship to other main arthropod groups, we collected transcriptome data from the symphylan Symphylella vulgaris, the centipedes Lithobius forficatus and Scolopendra dehaani, and the millipedes Polyxenus lagurus, Glomeris pustulata and Polydesmus angustus by 454 sequencing. We concatenated a multiple sequence alignment that contained 1550 orthologous single copy genes (1,109,847 amino acid positions) from 55 euarthropod and 14 outgroup taxa. The final selected alignment included 181 genes and 37,425 amino acid positions from 55 taxa, with eight myriapods and 33 other euarthropods. Bayesian analyses robustly recovered monophyletic Mandibulata, Pancrustacea and Myriapoda. Most analyses support a sister group relationship of Symphyla in respect to a clade comprising Chilopoda and Diplopoda. Inclusion of additional sequence data from nine myriapod species resulted in an alignment with poor data density, but broader taxon average. With this dataset we inferred Diplopoda+Pauropoda as closest relatives (i.e., Dignatha) and recovered monophyletic Helminthomorpha. Molecular clock calculations suggest an early Cambrian emergence of Myriapoda ∼513 million years ago and a late Cambrian divergence of myriapod classes. This implies a marine origin of the myriapods and independent terrestrialization events during myriapod evolution.

Decisive data sets in phylogenomics: lessons from studies on the phylogenetic relationships of primarily wingless insects.

Phylogenetic relationships of the primarily wingless insects are still considered unresolved. Even the most comprehensive phylogenomic studies that addressed this question did not yield congruent results. To get a grip on these problems, we here analyzed the sources of incongruence in these phylogenomic studies by using an extended transcriptome data set. Our analyses showed that unevenly distributed missing data can be severely misleading by inflating node support despite the absence of phylogenetic signal. In consequence, only decisive data sets should be used which exclusively comprise data blocks containing all taxa whose relationships are addressed. Additionally, we used Four-cluster Likelihood Mapping (FcLM) to measure the degree of congruence among genes of a data set, as a measure of support alternative to bootstrap. FcLM showed incongruent signal among genes, which in our case is correlated neither with functional class assignment of these genes nor with model misspecification due to unpartitioned analyses. The herein analyzed data set is the currently largest data set covering primarily wingless insects, but failed to elucidate their interordinal phylogenetic relationships. Although this is unsatisfying from a phylogenetic perspective, we try to show that the analyses of structure and signal within phylogenomic data can protect us from biased phylogenetic inferences due to analytical artifacts.

Oligonucleotide primers for targeted amplification of single-copy nuclear genes in apocritan Hymenoptera.

BACKGROUND: Published nucleotide sequence data from the mega-diverse insect order Hymenoptera (sawflies, bees, wasps, and ants) are taxonomically scattered and still inadequate for reconstructing a well-supported phylogenetic tree for the order. The analysis of comprehensive multiple gene data sets obtained via targeted PCR could provide a cost-effective solution to this problem. However, oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce. FINDINGS: Here we present a suite of degenerate oligonucleotide primer pairs for PCR amplification of 154 single-copy nuclear protein-coding genes from Hymenoptera. These primers were inferred from genome sequence data from nine Hymenoptera (seven species of ants, the honeybee, and the parasitoid wasp Nasonia vitripennis). We empirically tested a randomly chosen subset of these primer pairs for amplifying target genes from six Hymenoptera, representing the families Chrysididae, Crabronidae, Gasteruptiidae, Leucospidae, Pompilidae, and Stephanidae. Based on our results, we estimate that these primers are suitable for studying a large number of nuclear genes across a wide range of apocritan Hymenoptera (i.e., all hymenopterans with a wasp-waist) and of aculeate Hymenoptera in particular (i.e., apocritan wasps with stingers). CONCLUSIONS: The amplified nucleotide sequences are (a) with high probability from single-copy genes, (b) easily generated at low financial costs, especially when compared to phylogenomic approaches, (c) easily sequenced by means of an additionally provided set of sequencing primers, and (d) suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides.

The diversity and evolution of chelicerate hemocyanins.

BACKGROUND: Oxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as hemocyanins. Arthropod hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O2 transport capacity and a high cooperativity, thereby enhancing O2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of hemocyanins and the emergence of the large hemocyanin oligomers, hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes. RESULTS: Hemocyanin subunits from a sea spider, a scorpion, a whip scorpion and a whip spider were sequenced. Hemocyanin has been lost in Opiliones, Pseudoscorpiones, Solifugae and Acari, which may be explained by the evolution of trachea (i.e., taxon Apulmonata). Bayesian phylogenetic analysis was used to reconstruct the evolution of hemocyanin subunits and a relaxed molecular clock approach was applied to date the major events. While the sea spider has a simple hexameric hemocyanin, four distinct subunit types evolved before Xiphosura and Arachnida diverged around 470 Ma ago, suggesting the existence of a 4 × 6mer at that time. Subsequently, independent gene duplication events gave rise to the other distinct subunits in each of the 8 × 6mer hemocyanin of Xiphosura and the 4 × 6mer of Arachnida. The hemocyanin sequences were used to infer the evolutionary history of chelicerates. The phylogenetic trees support a basal position of Pycnogonida, a sister group relationship of Xiphosura and Arachnida, and a sister group relationship of the whip scorpions and the whip spiders. CONCLUSION: Formation of a complex hemocyanin oligomer commenced early in the evolution of euchelicerates. A 4 × 6mer hemocyanin consisting of seven subunit types is conserved in most arachnids since more than 400 Ma, although some entelegyne spiders display selective subunit loss and independent oligomerization. Hemocyanins also turned out to be a good marker to trace chelicerate evolution, which is, however, limited by the loss of hemocyanin in some taxa. The molecular clock calculations were in excellent agreement with the fossil record, also demonstrating the applicability of hemocyanins for such approach.

Dating the arthropod tree based on large-scale transcriptome data.

Molecular sequences do not only allow the reconstruction of phylogenetic relationships among species, but also provide information on the approximate divergence times. Whereas the fossil record dates the origin of most multicellular animal phyla during the Cambrian explosion less than 540 million years ago(mya), molecular clock calculations usually suggest much older dates. Here we used a large multiple sequence alignment derived from Expressed Sequence Tags and genomes comprising 129genes (37,476 amino acid positions) and 117 taxa, including 101 arthropods. We obtained consistent divergence time estimates applying relaxed Bayesian clock models with different priors and multiple calibration points. While the influence of substitution rates, missing data, and model priors were negligible, the clock model had significant effect. A log-normal autocorrelated model was selected on basis of cross-validation. We calculated that arthropods emerged ~600 mya. Onychophorans (velvet worms) and euarthropods split ~590 mya, Pancrustacea and Myriochelata ~560 mya, Myriapoda and Chelicerata ~555 mya, and 'Crustacea' and Hexapoda ~510 mya. Endopterygote insects appeared ~390 mya. These dates are considerably younger than most previous molecular clock estimates and in better agreement with the fossil record. Nevertheless, a Precambrian origin of arthropods and other metazoan phyla is still supported. Our results also demonstrate the applicability of large datasets of random nuclear sequences for approximating the timing of multicellular animal evolution.

The taming of an impossible child: a standardized all-in approach to the phylogeny of Hymenoptera using public database sequences.

BACKGROUND: Enormous molecular sequence data have been accumulated over the past several years and are still exponentially growing with the use of faster and cheaper sequencing techniques. There is high and widespread interest in using these data for phylogenetic analyses. However, the amount of data that one can retrieve from public sequence repositories is virtually impossible to tame without dedicated software that automates processes. Here we present a novel bioinformatics pipeline for downloading, formatting, filtering and analyzing public sequence data deposited in GenBank. It combines some well-established programs with numerous newly developed software tools (available at http://software.zfmk.de/). RESULTS: We used the bioinformatics pipeline to investigate the phylogeny of the megadiverse insect order Hymenoptera (sawflies, bees, wasps and ants) by retrieving and processing more than 120,000 sequences and by selecting subsets under the criteria of compositional homogeneity and defined levels of density and overlap. Tree reconstruction was done with a partitioned maximum likelihood analysis from a supermatrix with more than 80,000 sites and more than 1,100 species. In the inferred tree, consistent with previous studies, "Symphyta" is paraphyletic. Within Apocrita, our analysis suggests a topology of Stephanoidea + (Ichneumonoidea + (Proctotrupomorpha + (Evanioidea + Aculeata))). Despite the huge amount of data, we identified several persistent problems in the Hymenoptera tree. Data coverage is still extremely low, and additional data have to be collected to reliably infer the phylogeny of Hymenoptera. CONCLUSIONS: While we applied our bioinformatics pipeline to Hymenoptera, we designed the approach to be as general as possible. With this pipeline, it is possible to produce phylogenetic trees for any taxonomic group and to monitor new data and tree robustness in a taxon of interest. It therefore has great potential to meet the challenges of the phylogenomic era and to deepen our understanding of the tree of life.

A 454 sequencing approach for large scale phylogenomic analysis of the common emperor scorpion (Pandinus imperator).

In recent years, phylogenetic tree reconstructions that rely on multiple gene alignments that had been deduced from expressed sequence tags (ESTs) have become a popular method in molecular systematics. Here, we present a 454 pyrosequencing approach to infer the transcriptome of the Emperor scorpion Pandinus imperator. We obtained 428,844 high-quality reads (mean length=223+/-50 b) from total cDNA, which were assembled into 8334 contigs (mean length 422+/-313 bp) and 26,147 singletons. About 1200 contigs were successfully annotated by BLAST and orthology search. Specific analyses of eight distinct hemocyanin sequences provided further proof for the quality of the 454 reads and the assembly process. The P. imperator sequences were included in a concatenated alignment of 149 orthologous genes of 67 metazoan taxa that covers 39,842 amino acids. After removal of low-quality regions, 11,168 positions were employed for phylogenetic reconstructions. Using Bayesian and maximum likelihood methods, we obtained strongly supported monophyletic Ecdysozoa, Arthropoda (excluding Tardigrada), Euarthropoda, Pancrustacea and Hexapoda. We also recovered the Myriochelata (Chelicerata+Myriapoda). Within the chelicerates, Pycnogonida form the sister group of Euchelicerata. However, Arachnida were found paraphyletic because the Acari (mites and ticks) were recovered as sister group of a clade comprising Xiphosura, Scorpiones and Araneae. In summary, we have shown that 454 pyrosequencing is a cost-effective method that provides sufficient data and coverage depth for gene detection and multigene-based phylogenetic analyses.