The “Atelocerata” – A vanishing hypothesis? : Molecular phylogeny of arthropods with focus on primary wingless hexapods

Arthropods encompass more than three quarters of all described living species. Among arthropods, hexapods are the most abundant group and show an enormous diversity. To understand evolutionary processes of and within hexapods, it is necessary to resolve phylogenetic relationships, especially early hexapod splits within an arthropod framework. Several contradicting hypotheses have been suggested in the last decade addressing the monophyly of hexapods, and placements and relationships of primary wingless hexapod orders. As a possible sister group of hexapods, traditionally myriapods have been suggested, uniting hexapods and myriapods to a clade "Atelocerata". Alternatively several crustacean taxa (branchiopods or copepods) have been proposed as a possible sister group of hexapods associated with the "Pancrustacea" hypothesis where hexapods and crustaceans are united into a clade. This study concentrates on the analyses of molecular data and aims to resolve deep hexapods relationships within an arthropod context using two different methodological approaches. In the first approach, large phylogenomic (multi-gene and taxon) data sets based on nuclear protein coding genes derived from Expressed Sequence Tags (ESTs) are analyzed. This approach uses raw data sets with more than 100 taxa and more than 700 genes, and optimal data subsets with more than 100 taxa and more than 100 most informative genes to reconstruct phylogenetic trees. This is the first phylogenomic study which takes all entognathous, primary wingless hexapod orders into account. For this study, new EST data have been generated for each entognathous (Protura, Diplura and Collembola) and one ectognathous primary wingless hexapod order (Archaeognatha). A new approach based on a hill climbing algorithm is introduced to select most informative taxa and genes from raw data matrices. The aim is to select an optimized data subset with high information content. Therefore, MARE, (MAtrix REduction, http://www.mare.zfmk.de) has been developed by our work group. Optimized data subsets (SOS) are selected by taking information content of single genes (partitions) and the complete matrix into account without loosing to much taxa. For phylogenomic data sets addressing phylogenetic relationships, such an approach has never previously been applied. Instead, available studies rely on thresholds of available data or on maximal connected groups of data presence. Effects of selecting optimized data subsets towards high information content are examined with respect to phylogenetic reconstructions. Altogether, phylogenomic data can substantially advance our understanding of arthropod evolution and resolve several conflicts among existing hypotheses. Optimized data subsets show strong support for a sister group relationship of onychophorans and euarthropods. Within pancrustaceans, analyses yield paraphyletic crustaceans and monophyletic hexapods and robustly resolved deep hexapod relationships. Within neopteran insects, endopterygote (holometabolous) insects are monophyletic where hymenopterans branch off first with strong support. Analyses show a remarkable sensitivity to methods of analyses for the placement of myriapods. Altogether, results of this thesis show that new heuristics for the selection of optimized submatrices and other applied tools to improve data quality pay off their effort. The second approach to resolve deep hexapod relationships within an arthropod framework relies on two well known nuclear ribosomal RNA genes (large subunit 28S and small subunit 18S rRNA). Both genes are popular markers for studies addressing metazoan, arthropod and hexapod phylogeny. Analyses using an arthropod data set with 148 taxa of all important arthropod groups including both nuclear rRNA genes are improved by employing plausible models of sequence evolution. This rRNA study incorporates background knowledge on the evolution of nuclear ribosomal RNA gene sequences, in particular, into various steps of data processing. Mainly, automated methods have been used, an automated secondary structure guided alignment approach (RNAsalsa) and the software Aliscore for alignment masking. Further, mixed RNA/DNA models have been applied to avoid artifacts due to interdependence and covariation of paired sites of rRNA genes. Concurrently, reconstruction methods have been used that account for variation of evolutionary rates among lineages (non-stationarity). Although split-decomposition networks indicated conflicting signal in the data set, analyses modeling non-stationary statistically outperform stationary approaches. Topologies show strong support for a pancrustacean clade. The placement of some myriapod orders remains suspicious, a sister group of hexapods cannot robustly be resolved. Analyses taking non-stationarity into account unequivocally propose monophyletic Hexapoda. Relationships among entognathous primary wingless hexapods are resolved and Ectognatha are maximally supported. Within endopterygotes, hymenopterans are strongly proposed as a sister group of remaining holometabolous insects. Again, advanced methods in data quality assessment and modeling pay off its effort

Signatures o DNA Methylation across Insects Suggest Reduced DNA Methylation Levels in Holometabola

It has been experimentally shown that DNA methylation is involved in the regulation of gene expression and the silencing of transposable element activity in eukaryotes. The variable levels of DNA methylation among different insect species indicate an evolutionarily flexible role of DNA methylation in insects, which due to a lack of comparative data is not yet well-substantiated. Here, we use computational methods to trace signatures of DNA methylation across insects by analyzing transcriptomic and genomic sequence data from all currently recognized insect orders. We conclude that: 1) a functional methylation system relying exclusively on DNA methyltransferase 1 is widespread across insects. 2) DNA methylation has potentially been lost or extremely reduced in species belonging to springtails (Collembola), flies and relatives (Diptera), and twisted-winged parasites (Strepsiptera). 3) Holometabolous insects display signs of reduced DNA methylation levels in protein-coding sequences compared with hemimetabolous insects. 4) Evolutionarily conserved insect genes associated with housekeeping functions tend to display signs of heavier DNA methylation in comparison to the genomic/transcriptomic background. With this comparative study, we provide the much needed basis for experimental and detailed comparative analyses required to gain a deeper understanding on the evolution and function of DNA methylation in insects

Parametric and non-parametric masking of randomness in sequence alignments can be improved and leads to better resolved trees

<p>Abstract</p> <p>Background</p> <p>Methods of alignment masking, which refers to the technique of excluding alignment blocks prior to tree reconstructions, have been successful in improving the signal-to-noise ratio in sequence alignments. However, the lack of formally well defined methods to identify randomness in sequence alignments has prevented a routine application of alignment masking. In this study, we compared the effects on tree reconstructions of the most commonly used profiling method (GBLOCKS) which uses a predefined set of rules in combination with alignment masking, with a new profiling approach (ALISCORE) based on Monte Carlo resampling within a sliding window, using different data sets and alignment methods. While the GBLOCKS approach excludes variable sections above a certain threshold which choice is left arbitrary, the ALISCORE algorithm is free of <it>a priori </it>rating of parameter space and therefore more objective.</p> <p>Results</p> <p>ALISCORE was successfully extended to amino acids using a proportional model and empirical substitution matrices to score randomness in multiple sequence alignments. A complex bootstrap resampling leads to an even distribution of scores of randomly similar sequences to assess randomness of the observed sequence similarity. Testing performance on real data, both masking methods, GBLOCKS and ALISCORE, helped to improve tree resolution. The sliding window approach was less sensitive to different alignments of identical data sets and performed equally well on all data sets. Concurrently, ALISCORE is capable of dealing with different substitution patterns and heterogeneous base composition. ALISCORE and the most relaxed GBLOCKS gap parameter setting performed best on all data sets. Correspondingly, Neighbor-Net analyses showed the most decrease in conflict.</p> <p>Conclusions</p> <p>Alignment masking improves signal-to-noise ratio in multiple sequence alignments prior to phylogenetic reconstruction. Given the robust performance of alignment profiling, alignment masking should routinely be used to improve tree reconstructions. Parametric methods of alignment profiling can be easily extended to more complex likelihood based models of sequence evolution which opens the possibility of further improvements.</p

Identifying genetic markers for a range of phylogenetic utility–From species to family level

Resolving the phylogenetic relationships of closely related species using a small set of loci is challenging as sufficient information may not be captured from a limited sample of the genome. Relying on few loci can also be problematic when conflict between gene-trees arises from incomplete lineage sorting and/or ongoing hybridization, problems especially likely in recently diverged lineages. Here, we developed a method using limited genomic resources that allows identification of many low copy candidate loci from across the nuclear and chloroplast genomes, design probes for target capture and sequence the captured loci. To validate our method we present data from Eucalyptus and Melaleuca, two large and phylogenetically problematic genera within the Myrtaceae family. With one annotated genome, one transcriptome and two whole-genome shotgun sequences of one Eucalyptus and four Melaleuca species, respectively, we identified 212 loci representing 263 kbp for targeted sequence capture and sequencing. Of these, 209 were successfully tested from 47 samples across five related genera of Myrtaceae. The average percentage of reads mapped back to the reference was 57.6% with coverage of more than 20 reads per position across 83.5% of the data. The methods developed here should be applicable across a large range of taxa across all kingdoms. The core methods are very flexible, providing a platform for various genomic resource availabilities and are useful from shallow to deep phylogenies

Identifying genetic markers for a range of phylogenetic utility–From species to family level

Resolving the phylogenetic relationships of closely related species using a small set of loci is challenging as sufficient information may not be captured from a limited sample of the genome. Relying on few loci can also be problematic when conflict between gene-trees arises from incomplete lineage sorting and/or ongoing hybridization, problems especially likely in recently diverged lineages. Here, we developed a method using limited genomic resources that allows identification of many low copy candidate loci from across the nuclear and chloroplast genomes, design probes for target capture and sequence the captured loci. To validate our method we present data from Eucalyptus and Melaleuca, two large and phylogenetically problematic genera within the Myrtaceae family. With one annotated genome, one transcriptome and two whole-genome shotgun sequences of one Eucalyptus and four Melaleuca species, respectively, we identified 212 loci representing 263 kbp for targeted sequence capture and sequencing. Of these, 209 were successfully tested from 47 samples across five related genera of Myrtaceae. The average percentage of reads mapped back to the reference was 57.6% with coverage of more than 20 reads per position across 83.5% of the data. The methods developed here should be applicable across a large range of taxa across all kingdoms. The core methods are very flexible, providing a platform for various genomic resource availabilities and are useful from shallow to deep phylogenies

A minimum reporting standard for multiple sequence alignments

Multiple sequence alignments (MSAs) play a pivotal role in studies of molecular sequence data, but nobody has developed a minimum reporting standard (MRS) to quantify the completeness of MSAs. We present an MRS that relies on four simple completeness metrics. The metrics are implemented in AliStat, a program developed to support the MRS. A survey of published MSAs illustrates the benefits and unprecedented transparency offered by the MRS.CSIRO

Oxidative stress and senescence in social insects:A significant but inconsistent link?

The life-prolonging effects of antioxidants have long entered popular culture, but the scientific community still debates whether free radicals and the resulting oxidative stress negatively affect longevity. Social insects are intriguing models for analysing the relationship between oxidative stress and senescence because life histories differ vastly between long-lived reproductives and the genetically similar but short-lived workers. Here, we present the results of an experiment on the accumulation of oxidative damage to proteins, and a comparative analysis of the expression of 20 selected genes commonly involved in managing oxidative damage, across four species of social insects: a termite, two bees and an ant. Although the source of analysed tissue varied across the four species, our results suggest that oxidative stress is a significant factor in senescence and that its manifestation and antioxidant defenses differ among species, making it difficult to find general patterns. More detailed and controlled investigations on why responses to oxidative stress may differ across social species may lead to a better understanding of the relations between oxidative stress, antioxidants, social life history and senescence. This article is part of the theme issue 'Ageing and sociality: why, when and how does sociality change ageing patterns

Development and evaluation of a custom bait design based on 469 single-copy protein-coding genes for exon capture of isopods (Philosciidae: Haloniscus)

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies