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 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.

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.

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.

Concatenated analysis sheds light on early metazoan evolution and fuels a modern "urmetazoon" hypothesis.

For more than a century, the origin of metazoan animals has been debated. One aspect of this debate has been centered on what the hypothetical "urmetazoon" bauplan might have been. The morphologically most simply organized metazoan animal, the placozoan Trichoplax adhaerens, resembles an intriguing model for one of several "urmetazoon" hypotheses: the placula hypothesis. Clear support for a basal position of Placozoa would aid in resolving several key issues of metazoan-specific inventions (including, for example, head-foot axis, symmetry, and coelom) and would determine a root for unraveling their evolution. Unfortunately, the phylogenetic relationships at the base of Metazoa have been controversial because of conflicting phylogenetic scenarios generated while addressing the question. Here, we analyze the sum of morphological evidence, the secondary structure of mitochondrial ribosomal genes, and molecular sequence data from mitochondrial and nuclear genes that amass over 9,400 phylogenetically informative characters from 24 to 73 taxa. Together with mitochondrial DNA genome structure and sequence analyses and Hox-like gene expression patterns, these data (1) provide evidence that Placozoa are basal relative to all other diploblast phyla and (2) spark a modernized "urmetazoon" hypothesis.

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.

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.

A phylogenomic approach to resolve the arthropod tree of life.

Arthropods were the first animals to conquer land and air. They encompass more than three quarters of all described living species. This extraordinary evolutionary success is based on an astoundingly wide array of highly adaptive body organizations. A lack of robustly resolved phylogenetic relationships, however, currently impedes the reliable reconstruction of the underlying evolutionary processes. Here, we show that phylogenomic data can substantially advance our understanding of arthropod evolution and resolve several conflicts among existing hypotheses. We assembled a data set of 233 taxa and 775 genes from which an optimally informative data set of 117 taxa and 129 genes was finally selected using new heuristics and compared with the unreduced data set. We included novel expressed sequence tag (EST) data for 11 species and all published phylogenomic data augmented by recently published EST data on taxonomically important arthropod taxa. This thorough sampling reduces the chance of obtaining spurious results due to stochastic effects of undersampling taxa and genes. Orthology prediction of genes, alignment masking tools, and selection of most informative genes due to a balanced taxa-gene ratio using new heuristics were established. Our optimized data set robustly resolves major arthropod relationships. We received strong support for a sister group relationship of onychophorans and euarthropods and strong support for a close association of tardigrades and cycloneuralia. Within pancrustaceans, our analyses yielded paraphyletic crustaceans and monophyletic hexapods and robustly resolved monophyletic endopterygote insects. However, our analyses also showed for few deep splits that were recently thought to be resolved, for example, the position of myriapods, a remarkable sensitivity to methods of analyses.

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 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.

A phylogenomic approach to resolve the basal pterygote divergence.

One of the most fascinating Bauplan transitions in the animal kingdom was the invention of insect wings, a change that also contributed to the success and enormous diversity of this animal group. However, the origin of insect flight and the relationships of basal winged insect orders are still controversial. Three hypotheses have been proposed to explain the phylogeny of winged insects: 1) the traditional Palaeoptera hypothesis (Ephemeroptera + Odonata, Neoptera), 2) the Metapterygota hypothesis (Ephemeroptera, Odonata + Neoptera), and 3) the Chiastomyaria hypothesis (Odonata, Ephemeroptera + Neoptera). Neither phylogenetic analyses of single genes nor even multiple marker systems (e.g., molecular markers + morphological characters) have yet been able to conclusively resolve basal pterygote divergences. A possible explanation for the lack of resolution is that the divergences took place in the mid-Devonian within a short period of time and attempts to solve this problem have been confounded by the major challenge of finding molecular markers to accurately track these short ancient internodes. Although phylogenomic data are available for Neoptera and some wingless (apterygote) orders, they are lacking for the crucial Odonata and Ephemeroptera orders. We adopt a multigene approach including data from two new expressed sequence tag projects-from the orders Ephemeroptera (Baetis sp.) and Odonata (Ischnura elegans)-to evaluate the potential of phylogenomic analyses in clarifying this unresolved issue. We analyzed two data sets that differed in represented taxa, genes, and overall sequence lengths: maxspe (15 taxa, 125 genes, and 31,643 amino acid positions) and maxgen (8 taxa, 150 genes, and 42,541 amino acid positions). Maximum likelihood and Bayesian inference analyses both place the Odonata at the base of the winged insects. Furthermore, statistical hypotheses testing rejected both the Palaeoptera and the Metapterygota hypotheses. The comprehensive molecular data set developed here provides conclusive support for odonates as the most basal winged insect order (Chiastomyaria hypothesis). Data quality assessment indicates that proteins involved in cellular processes and signaling harbor the most informative phylogenetic signal.

Multiple dicer genes in the early-diverging metazoa.

Dicer proteins are highly conserved, are present in organisms ranging from plants to metazoans, and are essential components of the RNA interference pathway. Although the complement of Dicer proteins has been investigated in many "higher" metazoans, there has been no corresponding characterization of Dicer proteins in any early-branching metazoan. We cloned partial cDNAs of genes belonging to the Dicer family from the anthozoan cnidarian Nematostella vectensis and two distantly related haplotypes (species lineages) of the Placozoa (Trichoplax adhaerens 16S haplotype 1 [H1] and Placozoa sp. [H2]). We also identified Dicer genes in the hydrozoan Hydra magnipapillata and the demosponge Amphimedon queenslandica with the use of publicly available sequence databases. Two Dicer genes are present in each cnidarian species, whereas five Dicer genes each are found in the Porifera and Placozoa. Phylogenetic analyses comparing these and other metazoan Dicers suggest an ancient duplication event of a "Proto-Dicer" gene. We show that the Placozoa is the only known metazoan phylum which contains both representatives of this duplication event and that the multiple Dicer genes of the "basal" metazoan phyla represent lineage-specific duplications. There is a striking diversity of Dicer genes in basal metazoans, in stark contrast to the single Dicer gene found in most higher metazoans. This new data has allowed us to formulate new hypotheses regarding the evolution of metazoan Dicer proteins and their possible functions in the early diverging metazoan phyla. We theorize that the multiple placozoan Dicer genes fulfill a specific biological requirement, such as an immune defense strategy against viruses.

An integrative approach to species discovery in odonates: from character-based DNA barcoding to ecology.

Modern taxonomy requires an analytical approach incorporating all lines of evidence into decision-making. Such an approach can enhance both species identification and species discovery. The character-based DNA barcode method provides a molecular data set that can be incorporated into classical taxonomic data such that the discovery of new species can be made in an analytical framework that includes multiple sources of data. We here illustrate such a corroborative framework in a dragonfly model system that permits the discovery of two new, but visually cryptic species. In the African dragonfly genus Trithemis three distinct genetic clusters can be detected which could not be identified by using classical taxonomic characters. In order to test the hypothesis of two new species, DNA-barcodes from different sequence markers (ND1 and COI) were combined with morphological, ecological and biogeographic data sets. Phylogenetic analyses and incorporation of all data sets into a scheme called taxonomic circle highly supports the hypothesis of two new species. Our case study suggests an analytical approach to modern taxonomy that integrates data sets from different disciplines, thereby increasing the ease and reliability of both species discovery and species assignment.

Red drifters and dark residents: the phylogeny and ecology of a Plio-Pleistocene dragonfly radiation reflects Africa's changing environment (Odonata, Libellulidae, Trithemis).

In the last few million years, tropical Africa has experienced pronounced climatic shifts with progressive aridification. Such changes must have had a great impact on freshwater biota, such as Odonata. With about forty species, Trithemis dominates dragonfly communities across Africa, from rain-pools to streams, deserts to rainforests, and lowlands to highlands. Red-bodied species tend to favor exposed, standing and often temporary waters, have strong dispersal capacities, and some of the largest geographic ranges in the genus. Those in cooler habitats, like forest streams, are generally dark-bodied and more sedentary. We combined molecular analyses of ND1, 16S, and ITS (ITSI, 5.8S, and ITSII) with morphological, ecological, and geographical data for 81% of known Trithemis species, including three Asian and two Madagascan endemics. Using molecular clock analyses, the genus's origin was estimated 6-9Mya, with multiple lineages arising suddenly around 4Mya. Open stagnant habitats were inferred to be ancestral and the rise of Trithemis may have coincided with savannah-expansion in the late Miocene. The adaptation of red species to more ephemeral conditions leads to large ranges and limited radiation within those lineages. By contrast, three clades of dark species radiated in the Plio-Pleistocene, each within distinct ecological confines: (1) lowland streams, (2) highland streams, and (3) swampy habitats on alternating sides of the Congo-Zambezi watershed divide; together giving rise to the majority of species diversity in the genus. During Trithemis evolution, multiple shifts from open to more forested habitats and from standing to running waters occurred. Allopatry by habitat fragmentation may be the dominant force in speciation, but possibly genetic divergence across habitat gradients was also involved. The study demonstrates the importance of combining ecological and phylogenetic data to understand the origin of biological diversity under great environmental change.

On the value of Elongation factor-1alpha for reconstructing pterygote insect phylogeny.

Pterygota are traditionally divided in two lineages, the "Palaeoptera" and Neoptera. Despite several efforts neither morphology nor molecular systematics have resolved the phylogeny of the pterygote insects. Too few markers have yet been identified for adequately tracking mesozoic-aged divergences. We tested the Elongation factor-1alpha for its phylogenetic value in pterygote insect systematics. This highly conserved nuclear protein-coding gene has previously been reported to be useful in other groups for phylogenetic analyses at the intraordinal level as well as at the interordinal level. The analyses suggest that EF-1alpha DNA sequences as well as intron positions provide informative markers for pterygote phylogenetics.

Can comprehensive background knowledge be incorporated into substitution models to improve phylogenetic analyses? A case study on major arthropod relationships.

BACKGROUND: Whenever different data sets arrive at conflicting phylogenetic hypotheses, only testable causal explanations of sources of errors in at least one of the data sets allow us to critically choose among the conflicting hypotheses of relationships. The large (28S) and small (18S) subunit rRNAs are among the most popular markers for studies of deep phylogenies. However, some nodes supported by this data are suspected of being artifacts caused by peculiarities of the evolution of these molecules. Arthropod phylogeny is an especially controversial subject dotted with conflicting hypotheses which are dependent on data set and method of reconstruction. We assume that phylogenetic analyses based on these genes can be improved further i) by enlarging the taxon sample and ii) employing more realistic models of sequence evolution incorporating non-stationary substitution processes and iii) considering covariation and pairing of sites in rRNA-genes. RESULTS: We analyzed a large set of arthropod sequences, applied new tools for quality control of data prior to tree reconstruction, and increased the biological realism of substitution models. Although the split-decomposition network indicated a high noise content in the data set, our measures were able to both improve the analyses and give causal explanations for some incongruities mentioned from analyses of rRNA sequences. However, misleading effects did not completely disappear. CONCLUSION: Analyses of data sets that result in ambiguous phylogenetic hypotheses demand for methods, which do not only filter stochastic noise, but likewise allow to differentiate phylogenetic signal from systematic biases. Such methods can only rely on our findings regarding the evolution of the analyzed data. Analyses on independent data sets then are crucial to test the plausibility of the results. Our approach can easily be extended to genomic data, as well, whereby layers of quality assessment are set up applicable to phylogenetic reconstructions in general.

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.

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.

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.