Four myriapod relatives - but who are sisters? No end to debates on relationships among the four major myriapod subgroups.

BACKGROUND: Phylogenetic relationships among the myriapod subgroups Chilopoda, Diplopoda, Symphyla and Pauropoda are still not robustly resolved. The first phylogenomic study covering all subgroups resolved phylogenetic relationships congruently to morphological evidence but is in conflict with most previously published phylogenetic trees based on diverse molecular data. Outgroup choice and long-branch attraction effects were stated as possible explanations for these incongruencies. In this study, we addressed these issues by extending the myriapod and outgroup taxon sampling using transcriptome data. RESULTS: We generated new transcriptome data of 42 panarthropod species, including all four myriapod subgroups and additional outgroup taxa. Our taxon sampling was complemented by published transcriptome and genome data resulting in a supermatrix covering 59 species. We compiled two data sets, the first with a full coverage of genes per species (292 single-copy protein-coding genes), the second with a less stringent coverage (988 genes). We inferred phylogenetic relationships among myriapods using different data types, tree inference, and quartet computation approaches. Our results unambiguously support monophyletic Mandibulata and Myriapoda. Our analyses clearly showed that there is strong signal for a single unrooted topology, but a sensitivity of the position of the internal root on the choice of outgroups. However, we observe strong evidence for a clade Pauropoda+Symphyla, as well as for a clade Chilopoda+Diplopoda. CONCLUSIONS: Our best quartet topology is incongruent with current morphological phylogenies which were supported in another phylogenomic study. AU tests and quartet mapping reject the quartet topology congruent to trees inferred with morphological characters. Moreover, quartet mapping shows that confounding signal present in the data set is sufficient to explain the weak signal for the quartet topology derived from morphological characters. Although outgroup choice affects results, our study could narrow possible trees to derivatives of a single quartet topology. For highly disputed relationships, we propose to apply a series of tests (AU and quartet mapping), since results of such tests allow to narrow down possible relationships and to rule out confounding signal.

The cephalopod arm crown: appendage formation and differentiation in the Hawaiian bobtail squid Euprymna scolopes.

BACKGROUND: Cephalopods are a highly derived class of molluscs that adapted their body plan to a more active and predatory lifestyle. One intriguing adaptation is the modification of the ventral foot to form a bilaterally symmetric arm crown, which constitutes a true morphological novelty in evolution. In addition, this structure shows many diversifications within the class of cephalopods and therefore offers an interesting opportunity to study the molecular underpinnings of the emergence of phenotypic novelties and their diversification. Here we use the sepiolid Euprymna scolopes as a model to study the formation and differentiation of the decabrachian arm crown, which consists of four pairs of sessile arms and one pair of retractile tentacles. We provide a detailed description of arm crown formation in order to understand the basic morphology and the developmental dynamics of this structure. RESULTS: We show that the morphological formation of the cephalopod appendages occurs during distinct phases, including outgrowth, elongation, and tissue differentiation. Early outgrowth is characterized by uniform cell proliferation, while the elongation of the appendages initiates tissue differentiation. The latter progresses in a gradient from proximal to distal, whereas cell proliferation becomes restricted to the distal-most end of the arm. Differences in the formation of arms and tentacles exist, with the tentacles showing an expedite growth rate and higher complexity at younger stages. CONCLUSION: The early outgrowth and differentiation of the E. scolopes arm crown shows similarities to the related, yet derived cephalopod Octopus vulgaris. Parallels in the growth and differentiation of appendages seem to exist throughout the animal kingdom, raising the question of whether these similarities reflect a recruitment of similar molecular patterning pathways.

Skin toxins in coral-associated Gobiodon species (Teleostei: Gobiidae) affect predator preference and prey survival.

Predation risk is high for the many small coral reef fishes, requiring successful sheltering or other predator defence mechanisms. Coral-dwelling gobies of the genus Gobiodon live in close association with scleractinian corals of the genus Acropora. Earlier studies indicated that the low movement frequency of adult fishes and the development of skin toxins (crinotoxicity) are predation avoidance mechanisms. Although past experiments showed that predators refuse food prepared with goby skin mucus, direct predator-prey interactions have not been studied. The present study compares the toxicity levels of two crinotoxic coral gobies - Gobiodon histrio, representative of a conspicuously coloured species, and Gobiodon sp.3 with cryptic coloration - using a standard bioassay method. The results show that toxin levels of both species differ significantly shortly after mucus release but become similar over time. Predator preferences were tested experimentally in an aquarium in which the two gobies and a juvenile damselfish Chromis viridis were exposed to the small grouper Epinephelus fasciatus. Video-analysis revealed that although coral gobies are potential prey, E. fasciatus clearly preferred the non-toxic control fish (C. viridis) over Gobiodon. When targeting a goby, the predator did not prefer one species over the other. Contrary to our expectations that toxic gobies are generally avoided, gobies were often captured, but they were expelled quickly, repeatedly and alive. This unusual post-capture avoidance confirms that these gobies have a very good chance of surviving attacks in the field due to their skin toxins. Nonetheless, some gobies were consumed: the coral shelter may therefore also provide additional protection, with toxins protecting them mainly during movement between corals. In summary, chemical deterrence by crinotoxic fishes seems to be far more efficient in predation avoidance than in physical deterrence involving body squamation and/or strong fin spines.

Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.

INTRODUCTION: Nematostella vectensis, a member of the cnidarian class Anthozoa, has been established as a promising model system in developmental biology, but while information about the genetic regulation of embryonic development is rapidly increasing, little is known about the cellular organization of the various cell types in the adult. Here, we studied the anatomy and development of the muscular system of N. vectensis to obtain further insights into the evolution of muscle cells. RESULTS: The muscular system of N. vectensis is comprised of five distinct muscle groups, which are differentiated into a tentacle and a body column system. Both systems house longitudinal as well as circular portions. With the exception of the ectodermal tentacle longitudinal muscle, all muscle groups are of endodermal origin. The shape and epithelial organization of muscle cells vary considerably between different muscle groups. Ring muscle cells are formed as epitheliomuscular cells in which the myofilaments are housed in the basal part of the cell, while the apical part is connected to neighboring cells by apical cell-cell junctions. In the longitudinal muscles of the column, the muscular part at the basal side is connected to the apical part by a long and narrow cytoplasmic bridge. The organization of these cells, however, remains epitheliomuscular. A third type of muscle cell is represented in the longitudinal muscle of the tentacle. Using transgenic animals we show that the apical cell-cell junctions are lost during differentiation, resulting in a detachment of the muscle cells to a basiepithelial position. These muscle cells are still located within the epithelium and outside of the basal matrix, therefore constituting basiepithelial myocytes. We demonstrate that all muscle cells, including the longitudinal basiepithelial muscle cells of the tentacle, initially differentiate from regular epithelial cells before they alter their epithelial organisation. CONCLUSIONS: A wide range of different muscle cell morphologies can already be found in a single animal. This suggests how a transition from an epithelially organized muscle system to a mesenchymal could have occurred. Our study on N. vectensis provides new insights into the organisation of a muscle system in a non-bilaterian organism.

Where taxonomy based on subtle morphological differences is perfectly mirrored by huge genetic distances: DNA barcoding in Protura (Hexapoda).

BACKGROUND: Protura is a group of tiny, primarily wingless hexapods living in soil habitats. Presently about 800 valid species are known. Diagnostic characters are very inconspicuous and difficult to recognize. Therefore taxonomic work constitutes an extraordinary challenge which requires special skills and experience. Aim of the present pilot project was to examine if DNA barcoding can be a useful additional approach for delimiting and determining proturan species. METHODOLOGY AND PRINCIPAL FINDINGS: The study was performed on 103 proturan specimens, collected primarily in Austria, with additional samples from China and Japan. The animals were examined with two markers, the DNA barcoding region of the mitochondrial COI gene and a fragment of the nuclear 28S rDNA (Divergent Domain 2 and 3). Due to the minuteness of Protura a modified non-destructive DNA-extraction method was used which enables subsequent species determination. Both markers separated the examined proturans into highly congruent well supported clusters. Species determination was performed without knowledge of the results of the molecular analyses. The investigated specimens comprise a total of 16 species belonging to 8 genera. Remarkably, morphological determination in all species exactly mirrors molecular clusters. The investigation revealed unusually huge genetic COI distances among the investigated proturans, both maximal intraspecific distances (0-21.3%), as well as maximal congeneric interspecifical distances (up to 44.7%). CONCLUSIONS: The study clearly demonstrates that the tricky morphological taxonomy in Protura has a solid biological background and that accurate species delimitation is possible using both markers, COI and 28S rDNA. The fact that both molecular and morphological analyses can be performed on the same individual will be of great importance for the description of new species and offers a valuable new tool for biological and ecological studies, in which proturans have generally remained undetermined at species level.

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.

Take time to smell the frogs: vocal sac glands of reed frogs (Anura: Hyperoliidae) contain species-specific chemical cocktails.

Males of all reed frog species (Anura: Hyperoliidae) have a prominent, often colourful, gular patch on their vocal sac, which is particularly conspicuous once the vocal sac is inflated. Although the presence, shape, and form of the gular patch are well-known diagnostic characters for these frogs, its function remains unknown. By integrating biochemical and histological methods, we found strong evidence that the gular patch is a gland producing volatile compounds, which might be emitted while calling. Volatile compounds were confirmed by gas chromatography-mass spectrometry in the gular glands in 11 species of the hyperoliid genera Afrixalus, Heterixalus, Hyperolius, and Phlyctimantis. Comparing the gular gland contents of 17 specimens of four sympatric Hyperolius species yielded a large variety of 65 compounds in species-specific combinations. We suggest that reed frogs might use a complex combination of at least acoustic and chemical signals in species recognition and mate choice.

The placental analogue and the pattern of sexual reproduction in the cheilostome bryozoan Bicellariella ciliata (Gymnolaemata).

BACKGROUND: Matrotrophy or extraembryonic nutrition - transfer of nutrients from mother to embryo during gestation - is well known and thoroughly studied among vertebrates, but still poorly understood in invertebrates. The current paper focuses on the anatomy and ultrastructure of the oogenesis and placentotrophy as well as formation of the brood chamber (ovicell) in the cheilostome bryozoan Bicellariella ciliata (Linnaeus, 1758). Our research aimed to combine these aspects of the sexual reproduction into an integral picture, highlighting the role of the primitive placenta-like system in the evolution of bryozoan reproductive patterns. RESULTS: Follicular and nutrimentary provisioning of the oocyte occur during oogenesis. Small macrolecithal oocytes are produced, and embryos are nourished in the ovicell via a simple placental analogue (embryophore). Every brooding episode is accompanied by the hypertrophy of the embryophore, which collapses after larval release. Nutrients are released and uptaken by exocytosis (embryophore) and endocytosis (embryo). Embryos lack specialized area for nutrient uptake, which occurs through the whole epidermal surface. The volume increase between the ripe oocyte and the larva is ca. 10-fold. CONCLUSIONS: The ovicell is a complex organ (not a special polymorph as often thought) consisting of an ooecium (protective capsule) and an ooecial vesicle (plugging the entrance to the brooding cavity) that develop from the distal and the fertile zooid correspondingly. Combination of macrolecithal oogenesis and extraembryonic nutrition allows attributing B. ciliata to species with reproductive pattern IV. However, since its oocytes are small, this species represents a previously undescribed variant of this pattern, which appears to represent a transitional state from the insipient matrotrophy (with large macrolecithal eggs) to substantial one (with small microlecithal ones). Altogether, our results substantially added and corrected the data obtained by the previous authors, providing a new insight in our understanding of the evolution of matrotrophy in invertebrates.

Organogenesis in the budding process of the freshwater bryozoan Cristatella mucedo Cuvier, 1798 (Bryozoa, Phylactolaemata).

The phylogenetic position of bryozoans has been disputed for decades, and molecular phylogenetic analyzes have not unequivocally clarified their position within the Bilateria. As probably the most basal bryozoans, Phylactolaemata is the most promising taxon for large-scale phylogenetic comparisons. These comparisons require extending the morphological and developmental data by investigating different phylactolaemate species to identify basal characters and resolve in-group phylogeny. Accordingly, we analyzed the bud development and the organogenesis of the freshwater bryozoan Cristatella mucedo, with special focus on the formation of the digestive tract and differentiation of the coelomic compartments. Most parts of the digestive tract are formed as an outpocketing at the future anal side growing towards the mouth area. The ganglion is formed by an invagination between the anlagen of the mouth and anus. The lophophoral arms develop as paired lateral protrusions into the lumen of the bud and are temporarily connected by a median, thin bridge. All coelomic compartments are confluent during their development and also in the adult. The epistome coelom develops by fusion of two peritoneal infolds between the gut loop and overgrows the ganglion medially. The coelomic ring canal on the oral side develops by two lateral ingrowths and supplies the oral tentacles. On the forked canal, supplying the innermost row of tentacles above the epistome, a bladder-shaped swelling, probably with excretory function, is present in some adults. It remains difficult to draw comparisons to other phyla because only few studies have dealt with budding of potentially related taxa in more detail. Nonetheless, our results show that comparative organogenesis can contribute to phylactolaemate systematics and, when more data are available, possibly to that of other bryozoan classes and bilaterian phyla.

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.

Vaginal oestrus during the reproductive and non-reproductive period in European ground squirrels.

Most hibernating animals are thought to be monoestrous because reproductive activity is subject to strong time constraints. In previous studies, female European ground squirrels (Spermophilus citellus) turned out to have elevated oestradiol levels during late lactation and after weaning, indicating ovarian activity during summer. Therefore, we monitored vaginal cytology, endocrine changes, and vulval development in semi-free-living female European ground squirrels throughout one active season. Vaginal oestrus, defined by the predominance of cornified cells in smear samples, was found during the mating period shortly after vernal emergence. This phase was followed by metoestrus, characterised by the appearance of leukocytes, and a subsequent anoestrous phase. During weaning or postlactation, a second vaginal oestrus was documented in all experimental females, again followed by a metoestrous and an anoestrous phase lasting until hibernation. In line with the second vaginal oestrus, plasma oestradiol concentrations peaked during postlactation. Progesterone levels were elevated from gestation to postlactation, and titres were marginally higher during vaginal oestrus in summer than in spring. Vulval swelling was more pronounced during the first than the second vaginal oestrus. The second oestrous cycle was non-reproductive, as males were sexually inactive with regressed testes during summer. We assume that the second oestrous cycle and the accompanying endocrine changes have beneficial effects on prehibernatory fattening and reproductive performance in the subsequent season. This might allow females to become oestrous immediately after emergence from hibernation in spring.

Follicular development in European ground squirrels (Spermophilus citellus) in different phases of the annual cycle.

In seasonally breeding mammals, in particular hibernators, reproduction underlies severe energetic and temporal constraints to enable the allocation of sufficient body fat reserves before winter. Thus, the timing of conception in spring can be crucial in terms of reproductive success. This study investigates follicular development in European ground squirrels (Spermophilus citellus) in three phases of the annual cycle: at vernal emergence, after weaning the offspring and shortly before hibernation. The animals were kept in outdoor enclosures within the natural habitat of the species. They were captured in weekly intervals, weighed and reproductive status was determined. Unilateral ovariectomy was scheduled such that the three periods were sampled. Numbers and diameters of tertiary follicles (TF) and corpora lutea (CL) in each ovary were determined, and plasma oestradiol and progesterone levels were analysed. The highest numbers of TF, including Graafian follicles, were found in ovaries at vernal emergence. During post-lactation, the number of TF was lower and active CL appeared in the investigated ovaries. Shortly before hibernation, active CL were present, but luteolysis had started in some individuals. Both oestradiol and progesterone secretion peaked after the termination of lactation and decreased before hibernation. The results demonstrate a second oestrus cycle in European ground squirrels after weaning, including an active luteal phase. This non-reproductive oestrus cycle with its endocrine output is an intriguing phenomenon. It may positively affect both prehibernatory fattening and reproduction in the subsequent season.

Reproductive effort and costs of reproduction in female European ground squirrels.

Reproductive effort, factors affecting reproductive output and costs of reproduction were studied in primiparous yearling compared to multiparous older female European ground squirrels (Spermophilus citellus). Yearling females weaned smaller litters than older ones. Litter size increased with posthibernation body mass at the expense of slightly lighter young for yearling but not for older mothers. In older females, on the other hand, emergence body mass influenced offspring mass, whereas litter size was affected by oestrus date. High reproductive effort entailed reproductive costs in terms of reduced subsequent fecundity but not subsequent survival for both yearling and older females. The production of large litters and long duration of lactation delayed subsequent oestrus, which, in turn, correlated negatively with litter size. During the second half of lactation, oestradiol levels were significantly elevated, indicating the initiation of follicular maturation processes. Oestradiol levels during that time correlated negatively with current, but positively with subsequent litter size. We therefore assume that inhibitory effects of lactation on gonadal development may mediate the negative relationship between reproductive effort and subsequent reproductive timing in adults. This effect is absent in yearlings because they are reproducing for the first time. Reproductive output in yearlings was influenced by interactions between structural growth and puberty.