Studying Placozoa WBR in the Simplest Metazoan Animal, Trichoplax adhaerens.

Placozoans are a promising model system to study fundamental regeneration processes in a morphologically and genetically very simple animal. We here provide a brief introduction to the enigmatic Placozoa and summarize the state of the art of animal handling and experimental manipulation possibilities.

The damselfly genus Megaloprepus (Odonata: Pseudostigmatidae): Revalidation and delimitation of species-level taxa including the description of one new species.

As the longest-winged odonate species of the extant world, Megaloprepus caerulatus (Drury, 1782) has received attention by many entomologists. While the behavior and ecology of this species has been subject of intense studies, biogeography and species status throughout its distributional range in old-growth Neotropical forests are less well known. For tropical forests, this information is a sine qua non when estimating the impact of degradation and climate change. Recent population genetic analyses, quantitative morphometric, and traditional taxonomic studies rediscovered a complex composed of cryptic species within the genus Megaloprepus Rambur, 1842up until now still regarded as a monotypic genus. Here we introduce one new species Megaloprepus diaboli sp. nov. from the southern Pacific coast of Costa Rica and from the central Caribbean coast of Honduras and Guatemala. The holotype is from the Corcovado National Park, Costa Rica (N82855.62 W833513.92), and was deposited at the National Museum of Costa Rica. Aside from M. caerulatus, two formerly described and later refused species within the genus were reevaluated and consequently raised to species status: Megaloprepus latipennis Selys, 1860 is found in the northeastern regions of Mesoamerica and Megaloprepus brevistigma Selys, 1860 in South America east of the Andes. Morphological descriptions of selected specimens (holotype of M. diaboli, lectotype of M. latipennis, and the mature males of M. brevistigma and M. caerulatus) are provided. Diagnostic features of the four species are illustrated, discussed, and summarized in a key to adult males.

The enigmatic Placozoa part 1: Exploring evolutionary controversies and poor ecological knowledge.

The placozoan Trichoplax adhaerens is a tiny hairy plate and more simply organized than any other living metazoan. After its original description by F.E. Schulze in 1883, it attracted attention as a potential model for the ancestral state of metazoan organization, the "Urmetazoon". Trichoplax lacks any kind of symmetry, organs, nerve cells, muscle cells, basal lamina, and extracellular matrix. Furthermore, the placozoan genome is the smallest (not secondarily reduced) genome of all metazoan genomes. It harbors a remarkably rich diversity of genes and has been considered the best living surrogate for a metazoan ancestor genome. The phylum Placozoa presently harbors three formally described species, while several dozen "cryptic" species are yet awaiting their description. The phylogenetic position of placozoans has recently become a contested arena for modern phylogenetic analyses and view-driven claims. Trichoplax offers unique prospects for understanding the minimal requirements of metazoan animal organization and their corresponding malfunctions.

The enigmatic Placozoa part 2: Exploring evolutionary controversies and promising questions on earth and in space.

The placozoan Trichoplax adhaerens has been bridging gaps between research disciplines like no other animal. As outlined in part 1, placozoans have been subject of hot evolutionary debates and placozoans have challenged some fundamental evolutionary concepts. Here in part 2 we discuss the exceptional genetics of the phylum Placozoa and point out some challenging model system applications for the best known species, Trichoplax adhaerens.

The first complete mitochondrial genome of the migratory dragonfly Pantala flavescens Fabricius, 1798 (Libellulidae: Odonata).

Pantala flavescens is the world's most abundant and widely distributed dragonfly and with its outstanding migratory capacity an important model system to study insect migration at the evolutionary base of winged insects. We here report on the first complete mitochondrial genome (mitogenome) of P. flavescens sampled from a population in Rufiji River, Tanzania. The mitogenome is 14,853 bp long with an AT-biased base composition (72.7% A + T) and encodes a typical set of 13 protein-coding genes (PCGs), 22 tRNAs, and two rRNAs. The control region (CR) (171 bp) is the shortest reported in any anisopteran odonate, so far. Phylogenetic analyses support the placement of P. flavescens within the Libellulidae.

Transcriptome profiling with focus on potential key genes for wing development and evolution in Megaloprepus caerulatus, the damselfly species with the world's largest wings.

The evolution, development and coloration of insect wings remains a puzzling subject in evolutionary research. In basal flying insects such as Odonata, genomic research regarding bauplan evolution is still rare. Here we focus on the world's largest odonate species-the "forest giant" Megaloprepus caerulatus, to explore its potential for looking deeper into the development and evolution of wings. A recently discovered cryptic species complex in this genus previously considered monotypic is characterized by morphological differences in wing shape and color patterns. As a first step toward understanding wing pattern divergence and pathways involved in adaptation and speciation at the genomic level, we present a transcriptome profiling of M. caerulatus using RNA-Seq and compare these data with two other odonate species. The de novo transcriptome assembly consists of 61,560 high quality transcripts and is approximately 93% complete. For almost 75% of the identified transcripts a possible function could be assigned: 48,104 transcripts had a hit to an InterPro protein family or domain, and 28,653 were mapped to a Gene Ontology term. In particular, we focused on genes related to wing development and coloration. The comparison with two other species revealed larva-specific genes and a conserved 'core' set of over 8,000 genes forming orthologous clusters with Ischnura elegans and Ladona fulva. This transcriptome may provide a first point of reference for future research in odonates addressing questions surrounding the evolution of wing development, wing coloration and their role in speciation.

Comparative transcriptomics reveal developmental turning points during embryogenesis of a hemimetabolous insect, the damselfly Ischnura elegans.

Identifying transcriptional changes during embryogenesis is of crucial importance for unravelling evolutionary, molecular and cellular mechanisms that underpin patterning and morphogenesis. However, comparative studies focusing on early/embryonic stages during insect development are limited to a few taxa. Drosophila melanogaster is the paradigm for insect development, whereas comparative transcriptomic studies of embryonic stages of hemimetabolous insects are completely lacking. We reconstructed the first comparative transcriptome covering the daily embryonic developmental progression of the blue-tailed damselfly Ischnura elegans (Odonata), an ancient hemimetabolous representative. We identified a "core" set of 6,794 transcripts - shared by all embryonic stages - which are mainly involved in anatomical structure development and cellular nitrogen compound metabolic processes. We further used weighted gene co-expression network analysis to identify transcriptional changes during Odonata embryogenesis. Based on these analyses distinct clusters of transcriptional active sequences could be revealed, indicating that embryos at different development stages have their own transcriptomic profile according to the developmental events and leading to sequential reprogramming of metabolic and developmental genes. Interestingly, a major change in transcriptionally active sequences is correlated with katatrepsis (revolution) during mid-embryogenesis, a 180° rotation of the embryo within the egg and specific to hemimetabolous insects.

Long-term genetic monitoring of a riverine dragonfly, Orthetrum coerulescens (Odonata: Libellulidae]: Direct anthropogenic impact versus climate change effects.

Modern conservationists call for long term genetic monitoring datasets to evaluate and understand the impact of human activities on natural ecosystems and species on a global but also local scale. However, long-term monitoring datasets are still rare but in high demand to correctly identify, evaluate and respond to environmental changes. In the presented study, a population of the riverine dragonfly, Orthetrum coerulescens (Odonata: Libellulidae), was monitored over a time period from 1989 to 2013. Study site was an artificial irrigation ditch in one of the last European stone steppes and "nature heritage", the Crau in Southern France. This artificial riverine habitat has an unusual high diversity of odonate species, prominent indicators for evaluating freshwater habitats. A clearing of the canal and destruction of the bank vegetation in 1996 was assumed to have great negative impact on the odonate larval and adult populations. Two mitochondrial markers (CO1 & ND1) and a panel of nuclear microsatellite loci were used to assess the genetic diversity. Over time they revealed a dramatic decline in diversity parameters between the years 2004 and 2007, however not between 1996 and 1997. From 2007 onwards the population shows a stabilizing trend but has not reached the amount of genetic variation found at the beginning of this survey. This decline cannot be referred to the clearing of the canal or any other direct anthropogenic impact. Instead, it is most likely that the populations' decay was due to by extreme weather conditions during the specific years. A severe drought was recorded for the summer months of these years, leading to reduced water levels in the canal causing also other water parameters to change, and therefore impacting temperature sensitive riverine habitat specialists like the O. coerulescens in a significant way. The data provide important insights into population genetic dynamics and metrics not always congruent with traditional monitoring data (e.g. abundance); a fact that should be regarded with caution when management plans for developed landscapes are designed.

The marker choice: Unexpected resolving power of an unexplored CO1 region for layered DNA barcoding approaches.

The potential of DNA barcoding approaches to identify single species and characterize species compositions strongly depends on the marker choice. The prominent "Folmer region", a 648 basepair fragment at the 5' end of the mitochondrial CO1 gene, has been traditionally applied as a universal DNA barcoding region for metazoans. In order to find a suitable marker for biomonitoring odonates (dragonflies and damselflies), we here explore a new region of the CO1 gene (CO1B) for DNA barcoding in 51 populations of 23 dragonfly and damselfly species. We compare the "Folmer region", the mitochondrial ND1 gene (NADH dehydrogenase 1) and the new CO1 region with regard to (i) speed and reproducibility of sequence generation, (ii) levels of homoplasy and (iii) numbers of diagnostic characters for discriminating closely related sister taxa and populations. The performances of the gene regions regarding these criteria were quite different. Both, the amplification of CO1B and ND1 was highly reproducible and CO1B showed the highest potential for discriminating sister taxa at different taxonomic levels. In contrast, the amplification of the "Folmer region" using the universal primers was difficult and the third codon positions of this fragment have experienced nucleotide substitution saturation. Most important, exploring this new barcode region of the CO1 gene identified a higher discriminating power between closely related sister taxa. Together with the design of layered barcode approaches adapted to the specific taxonomic "environment", this new marker will further enhance the discrimination power at the species level.

The most primitive metazoan animals, the placozoans, show high sensitivity to increasing ocean temperatures and acidities.

The increase in atmospheric carbon dioxide (CO2) leads to rising temperatures and acidification in the oceans, which directly or indirectly affects all marine organisms, from bacteria to animals. We here ask whether the simplest-and possibly also the oldest-metazoan animals, the placozoans, are particularly sensitive to ocean warming and acidification. Placozoans are found in all warm and temperate oceans and are soft-bodied, microscopic invertebrates lacking any calcified structures, organs, or symmetry. We here show that placozoans respond highly sensitive to temperature and acidity stress. The data reveal differential responses in different placozoan lineages and encourage efforts to develop placozoans as a potential biomarker system.

MtDNA: The small workhorse of evolutionary studies.

The double-stranded, circular mitochondrial DNA (mtDNA), which is present in all eukaryotic life forms, was initially discovered and characterized in the last century and has been widely used in evolutionary studies. Since then, a large number of studies have taken advantage of the genetic information encoded in this genome. Because of its small size in animals (in general), the technical ease of manipulating mitochondrial genome and the dynamics of its evolutionary change, this genome has been the workhorse of evolutionary studies over the past three decades. However, the ease with which nuclear DNA can be manipulated due to next generation sequencing (NGS) methods, has recently caused an expected dip in the use of mtDNA in evolutionary studies.  This review examines the future of mitochondrial DNA as a useful tool in studies centered around evolution.

The complete mitochondrial genome of the neotropical helicopter damselfly Megaloprepus caerulatus (Odonata: Zygoptera) assembled from next generation sequencing data.

Odonata (dragonflies and damselflies) is a small order at the base of flying insects (Pterygota). Resolving family-level phylogenetic relationships within this order receives great attention. Hereby, genetic data already resulted in various changes, which are however still under discussion. Mitochondrial genomes may further enhance such phylogenies. This study presents the complete mitochondrial genome of the Neotropical damselfly Megaloprepus caerulatus based on next generation sequencing (NGS) data on total genomic DNA. The total length comprises 16,094 bp and includes the standard metazoan set of 37 genes together with a 1376 bp long A + T rich (control) region. Gene content, gene arrangement and base frequency are consistent with other odonate mitochondrial genomes. It further contains four intergenic spacer regions, indicating a possible family specific feature for the Coenagrionidae and its close relatives.

Short read sequencing assembly revealed the complete mitochondrial genome of Ischnura elegans Vander Linden, 1820 (Odonata: Zygoptera).

Damselflies of the genus Ischnura emerge as organisms with high potential in ecological, evolutionary and developmental research at the base of flying insects. Ischnura elegans and Ischnura hastata are for example one of the few odonate species where a complete life cycle over generations can be reared under laboratory conditions. We here report the complete mitochondrial genome of Ischnura elegans as a valuable genomic resource for future eco-evo-devo studies at the base of flying insects. The genome has a total length of 15,962 bp and displays all typical features of Odonata (dragonflies and damselflies) mitochondrial genomes in gene content and order as well as A + T content. Start and stop codons of all protein-coding genes are consistent. Most interestingly, we found four intergenic spacer regions and a long A + T rich (control) region of 1196 bp, which is almost double the size of the close relative Ischnura pumilio. We assume that the adequate insert size and iterative mapping may be more efficient in assembling this duplicated and repetitive region.

The complete mitochondrial genome of the emperor dragonfly Anax imperator LEACH, 1815 (Odonata : Aeshnidae) via NGS sequencing.

Here we report the complete mitochondrial genome of the emperor dragonfly, Anax imperator (Odonata: Aeshnidae) as the first of its genus. Data were generated via next generation sequencing (NGS) and assembled using an iterative approach. The typical metazoan set of 37 genes (13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes) was detected in the same gene order as in other odonate mitogenomes. However, only three intergenic spacer regions are present in A. imperator lacking the distinct s5 spacer, which was regarded as informative feature of the odonate suborder Anisoptera (dragonflies) but absent in Zygoptera (damselflies). With 16,087 bp, it is the longest anisopteran mitogenome to date, mainly due to the long A + T-rich control region of 1291 bp.

Some 'ant'swers: Application of a layered barcode approach to problems in ant taxonomy.

DNA barcoding has emerged as a routine tool in modern taxonomy. Although straightforward, this approach faces new challenges, when applied to difficult situation such as defining cryptic biodiversity. Ants are prime examples for high degrees of cryptic biodiversity due to complex population differentiation, hybridization and speciation processes. Here, we test the DNA barcoding region, cytochrome c oxidase 1 and two supplementary markers, 28S ribosomal DNA and long-wavelength rhodopsin, commonly used in ant taxonomy, for their potential in a layered, character-based barcoding approach across different taxonomic levels. Furthermore, we assess performance of the character-based barcoding approach to determine cryptic species diversity in ants. We found (i) that the barcode potential of a specific genetic marker varied widely among taxonomic levels in ants; (ii) that application of a layered, character-based barcode for identification of specimens can be a solution to taxonomical challenging groups; (iii) that the character-based barcoding approach allows us to differentiate specimens even within locations based on pure characters. In summary, (layered) character-based barcoding offers a reliable alternative for problematic species identification in ants and can be used as a fast and cost-efficient approach to estimate presence, absence or frequency of cryptic species.

A comprehensive analysis of bilaterian mitochondrial genomes and phylogeny.

About 2800 mitochondrial genomes of Metazoa are present in NCBI RefSeq today, two thirds belonging to vertebrates. Metazoan phylogeny was recently challenged by large scale EST approaches (phylogenomics), stabilizing classical nodes while simultaneously supporting new sister group hypotheses. The use of mitochondrial data in deep phylogeny analyses was often criticized because of high substitution rates on nucleotides, large differences in amino acid substitution rate between taxa, and biases in nucleotide frequencies. Nevertheless, mitochondrial genome data might still be promising as it allows for a larger taxon sampling, while presenting a smaller amount of sequence information. We present the most comprehensive analysis of bilaterian relationships based on mitochondrial genome data. The analyzed data set comprises more than 650 mitochondrial genomes that have been chosen to represent a profound sample of the phylogenetic as well as sequence diversity. The results are based on high quality amino acid alignments obtained from a complete reannotation of the mitogenomic sequences from NCBI RefSeq database. However, the results failed to give support for many otherwise undisputed high-ranking taxa, like Mollusca, Hexapoda, Arthropoda, and suffer from extreme long branches of Nematoda, Platyhelminthes, and some other taxa. In order to identify the sources of misleading phylogenetic signals, we discuss several problems associated with mitochondrial genome data sets, e.g. the nucleotide and amino acid landscapes and a strong correlation of gene rearrangements with long branches.

A comparative analysis of complete mitochondrial genomes among Hexapoda.

With respect to bauplan radiation, species and taxa richness, hexapods have an unassailable lead. But still, the phylogenetic relationships among the orders and infraorders remain a matter of discussion. The rapidly increasing mitochondrial genome sequences from diverse insect species provide the opportunity to explore miscellaneous evolutionary questions in the superclass Hexapoda. A combined primary sequence analyses of the complete available data set has not yet been performed. Until now phylogenetic analyses of subsets of selected taxa resulted to strong supported topologies showing in some instances discrepancies between morphological and nuclear data. This circumstance started the discussion about the limits of complete mitochondrial genomes for inferring deep hexapod relationships. By using the hitherto densest taxon sampling of Hexapoda our analyses resulted in discrepancies to the current phylogenetic hypotheses based on morphological and nuclear data, e.g. monophyly of hexapods and some hexapods orders, e.g. Diptera, Hemiptera and Orthoptera. Nonetheless, compared to previously published studies that strongly support systematically erroneous groups using a sparse taxon sampling, our analyses had no support for theses discrepancies. Consequently, we highly recommend interpreting mt-genome based phylogenies with incomplete representation of major orders/taxa particularly for hexapods with cautions although the inferred relationships are highly supported.

Insect phylogenomics: exploring the source of incongruence using new transcriptomic data.

The evolution of the diverse insect lineages is one of the most fascinating issues in evolutionary biology. Despite extensive research in this area, the resolution of insect phylogeny especially of interordinal relationships has turned out to be still a great challenge. One of the challenges for insect systematics is the radiation of the polyneopteran lineages with several contradictory and/or unresolved relationships. Here, we provide the first transcriptomic data for three enigmatic polyneopteran orders (Dermaptera, Plecoptera, and Zoraptera) to clarify one of the most debated issues among higher insect systematics. We applied different approaches to generate 3 data sets comprising 78 species and 1,579 clusters of orthologous genes. Using these three matrices, we explored several key mechanistic problems of phylogenetic reconstruction including missing data, matrix selection, gene and taxa number/choice, and the biological function of the genes. Based on the first phylogenomic approach including these three ambiguous polyneopteran orders, we provide here conclusive support for monophyletic Polyneoptera, contesting the hypothesis of Zoraptera + Paraneoptera and Plecoptera + remaining Neoptera. In addition, we employ various approaches to evaluate data quality and highlight problematic nodes within the Insect Tree that still exist despite our phylogenomic approach. We further show how the support for these nodes or alternative hypotheses might depend on the taxon- and/or gene-sampling.

Isolation of Hox cluster genes from insects reveals an accelerated sequence evolution rate.

Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution.