New records on the rich loriciferan fauna of Trezen ar Skoden (Roscoff, France): Description of two new species of Nanaloricus and the new genus Scutiloricus.

Loricifera is a phylum of microscopic animals that inhabit marine environments worldwide. Named after their conspicuous and protective lorica, the phylum was first described from Roscoff (France) in 1983 and, hitherto, it contains only 40 species. Based on data collected from Roscoff during the past four decades, we here describe two new species of Nanaloricus, namely Nanaloricus valdemari sp. nov. and Nanaloricus mathildeae sp. nov., as well as a new genus and species, Scutiloricus hugoi gen. et sp. nov. Adults of N. valdemari sp. nov. are distinguished by a pair of unique cuticular ridges, here referred to as longitudinal stripes, spanning laterally along the anterior two thirds of the dorsal lorical plate. N. mathildeae sp. nov. is characterized by strong sexual dimorphism. Specifically, the branches composing the multiform male clavoscalids are much broader as compared to other Nanaloricus species. The two new Nanaloricus species are both characterized by unique sensory organs associated with the double trichoscalids. The size and exact position of these organs differ between the two species. Adults of Scutiloricus hugoi gen. et sp. nov. are characterized by, among other features, a square lorica composed of six cuticular plates with a total of 14 anterior spikes, of which 12 have transverse cuticular ridges and thus appear fenestrated; laterodorsal flosculi arranged linearly; a posterior lorical region characterized by an anal field with a small anal cone flanked by a pair of spurs. Notably, mature females are characterized by a pair of seminal receptacles, a character not previously reported in Loricifera. We discuss the new findings and compare N. valdemari sp. nov. and N. mathildeae sp.nov. with other species assigned to genus Nanaloricus. The distinguishing features of Scutiloricus hugoi gen. et sp. nov. are discussed from a comparative perspective with the other genera of family Nanaloricidae.

High Mountain Echiniscid (Heterotardigrada) Fauna of Taiwan.

Taiwan lies at the transitional zone between the East Palaearctic and Oriental regions, which translates into both Palaearctic and Indomalayan taxa being present on the island. Furthermore, large habitat heterogeneity and high mountains contributed to the rise of conditions favouring allopatric speciation and the emergence of endemic species. The tardigrade fauna of Taiwan is poorly studied, and the aim of this contribution is to provide new data on the members of the family Echiniscidae, the largest limno-terrestrial group of the class Heterotardigrada, found at high elevations in central Taiwan. We report 11 species grouped in 5 genera: Claxtonia (1 species), Echiniscus (3 species), Hypechiniscus (1 species), Nebularmis (2 species), and Pseudechiniscus (4 species). All are new to Taiwan, including 5 species that are new to science, 4 or which are described herein by means of integrative taxonomy: Hypechiniscus crassus sp. nov. (the exarmatus morphogroup), Pseudechiniscus (Meridioniscus) dreyeri sp. nov., Pseudechiniscus (Pseudechiniscus) formosus sp. nov., and Pseudechiniscus (Pseudechiniscus) totoro sp. nov. The new findings also help to clarify the description of Echiniscus clevelandi Beasley, 1999, and supplement the phylogenies of the Echiniscus virginicus complex and of the genera Hypechiniscus, Nebularmis and Pseudechiniscus.

Comparative myoanatomy of Tardigrada: new insights from the heterotardigrades Actinarctus doryphorus (Tanarctidae) and Echiniscoides sigismundi (Echiniscoididae).

BACKGROUND: Tardigrada is a group of microscopic invertebrates distributed worldwide in permanent and temporal aquatic habitats. Famous for their extreme stress tolerance, tardigrades are also of interest due to their close relationship with Arthropoda and Cycloneuralia. Despite recent efforts in analyzing the musculature of a number of tardigrade species, data on the class Heterotardigrada remain scarce. Aiming to expand the current morphological framework, and to promote the use of muscular body plans in elucidating tardigrade phylogeny, the myoanatomy of two heterotardigrades, Actinarctus doryphorus and Echiniscoides sigismundi, was analyzed by cytochemistry, scanning electron and confocal laser scanning microscopy and 3D imaging. We discuss our findings with reference to other tardigrades and internal phylogenetic relationships of the phylum. RESULTS: We focus our analyses on the somatic musculature, which in tardigrades includes muscle groups spanning dorsal, ventral, and lateral body regions, with the legs being musculated by fibers belonging to all three groups. A pronounced reduction of the trunk musculature is seen in the dorsoventrally compressed A. doryphorus, a species that generally has fewer cuticle attachment sites as compared to E. sigismundi and members of the class Eutardigrada. Interestingly, F-actin positive signals were found in the head appendages of A. doryphorus. Our analyses further indicate that cross-striation is a feature common to the somatic muscles of heterotardigrades and that E. sigismundi-as previously proposed for other echiniscoidean heterotardigrades-has relatively thick somatic muscle fibers. CONCLUSIONS: We provide new insights into the myoanatomical differences that characterize distinct evolutionary lineages within Tardigrada, highlighting characters that potentially can be informative in future phylogenetic analyses. We focus our current analyses on the ventral trunk musculature. Our observations suggest that seven paired ventromedian attachment sites anchoring a large number of muscles can be regarded as part of the ground pattern of Tardigrada and that fusion and reduction of cuticular attachment sites is a derived condition. Specifically, the pattern of these sites differs in particular details between tardigrade taxa. In the future, a deeper understanding of the tardigrade myoanatomical ground pattern will require more investigations in order to include all major tardigrade lineages.

Molecular palaeontology illuminates the evolution of ecdysozoan vision.

Colour vision is known to have arisen only twice-once in Vertebrata and once within the Ecdysozoa, in Arthropoda. However, the evolutionary history of ecdysozoan vision is unclear. At the molecular level, visual pigments, composed of a chromophore and a protein belonging to the opsin family, have different spectral sensitivities and these mediate colour vision. At the morphological level, ecdysozoan vision is conveyed by eyes of variable levels of complexity; from the simple ocelli observed in the velvet worms (phylum Onychophora) to the marvellously complex eyes of insects, spiders, and crustaceans. Here, we explore the evolution of ecdysozoan vision at both the molecular and morphological level; combining analysis of a large-scale opsin dataset that includes previously unknown ecdysozoan opsins with morphological analyses of key Cambrian fossils with preserved eye structures. We found that while several non-arthropod ecdysozoan lineages have multiple opsins, arthropod multi-opsin vision evolved through a series of gene duplications that were fixed in a period of 35-71 million years (Ma) along the stem arthropod lineage. Our integrative study of the fossil and molecular record of vision indicates that fossils with more complex eyes were likely to have possessed a larger complement of opsin genes.

Revision of Testechiniscus Kristensen, 1987 (Heterotardigrada: Echiniscidae) refutes the polar-temperate distribution of the genus.

The family Echiniscidae comprises limno-terrestrial heterotardigrades with a strongly sclerotised dorsum, typically covered with plates. Among other members of the Echiniscus evolutionary line, the genus Testechiniscus Kristensen, 1987 stands out with well-developed ventral armature and polygonal sculpturing of the dorsal plates. It has alleged bipolar distribution (with satellite alpine records in the Holarctic). Thanks to fresh material from terra typica (Svalbard), we integratively redescribe (i.e. using light and electron microscopy imaging, morphometry, and molecular methods) the nominal species for the genus, Testechiniscus spitsbergensis (Scourfield, 1897). A comparison of the neotype series with a number of Holarctic records revealed morphological variability suggesting that the species may encompass several taxa, which, most likely, will be possible to delineate primarily with molecular tools. Moreover, based on material from Simien Mountains (Northern Ethiopia), we describe a new sibling subspecies, Testechiniscus spitsbergensis tropicalis ssp. nov. Extensive morphometric datasets are provided for the genus members for the first time. A new generic definition is proposed, embracing the two subspecies of T. spitsbergensis and T. laterculus (Schuster et al., 1980), but excluding two circum-Antarctic species, T. macronyx (Richters, 1907) and T. meridionalis (Murray, 1906). The later species are likely to be erected in the future as separate genera, and their autapomorphies are described here. In the light of our findings, the genus          Testechiniscus should be recognised as a native element of the Northern Hemisphere, with mainly circum-Arctic distribution and additional, insular alpine records from the Nearctic, Palearctic and Eastern Afrotropic.

Integrative taxonomy identifies Macrobiotus papei, a new tardigrade species of the Macrobiotus hufelandi complex (Eutardigrada: Macrobiotidae) from the Udzungwa Mountains National Park (Tanzania).

In this paper we describe Macrobiotus papei, a new species of the Macrobiotus hufelandi complex from the Udzungwa Mountains National Park in Tanzania. Our research included the traditional morphology-based taxonomic analysis, supported by morphometrics, light and scanning electron microscopy imaging, as well as analysis of nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) marker. The molecular sequences provided a more accurate description and independent verification of the taxonomic status of M. papei sp. nov. Such integrative approach requires a considerable number of individuals and eggs, which we obtained by establishing a laboratory culture of the new species. Our analyses revealed that M. papei sp. nov., by having the flexible filaments on the terminal discs of the egg processes, is most similar to Macrobiotus paulinae Stec et al., 2015 from Africa and Macrobiotus polypiformis Roszkowska et al., 2017 from South America. However, the new species can be easily distinguished from both these species by patches of cuticular granulation on the external and internal surface of legs I-III (only external patches present in in M. paulinae and M. polypiformis), two separate lateral transversal ridges with a rounded median tooth between instead of a single thin ventral transverse ridge in the third band of teeth in the oral cavity, smooth terminal discs and filaments on the egg processes (terminal discs and filaments covered by aggregations of small microgranules in M. paulinae and M. polypiformis), and by some morphometric characters of both adults and eggs.

The challenge of proving the existence of metazoan life in permanently anoxic deep-sea sediments.

The demonstration of the existence of metazoan life in absence of free oxygen is one of the most fascinating and difficult challenges in biology. Danovaro et al. (2010) discovered three new species of the Phylum Loricifera, living in the anoxic sediments of the L'Atalante, a deep-hypersaline anoxic basin of the Mediterranean Sea. Multiple and independent analyses based on staining, incorporation of radiolabeled substrates, CellTracker Green incorporation experiments and ultra-structure analyses, allowed Danovaro et al. (2010) to conclude that these animals were able to spend their entire life cycle under anoxic conditions. Bernhard et al. (2015) investigated the same basin. Due to technical difficulties in sampling operations, they could not collect samples from the permanently anoxic sediment, and sampled only the redoxcline portion of the L'Atalante basin. They found ten individuals of Loricifera and provided alternative interpretations of the results of Danovaro et al. (2010). Here we analyze these interpretations, and present additional evidence indicating that the Loricifera encountered in the anoxic basin L'Atalante were actually alive at the time of sampling. We also discuss the reliability of different methodologies and approaches in providing evidence of metazoans living in anoxic conditions, paving the way for future investigations.This paper is a response to Bernhard JM, Morrison CR, Pape E, Beaudoin DJ, Todaro MA, Pachiadaki MG, Kormas KAr, Edgcomb VG. 2015. Metazoans of redoxcline sediments in Mediterranean deep-sea hypersaline anoxic basins. BMC Biology 2015 13:105.See research article at http://bmcbiol.biomedcentral.com/articles/10.1186/s12915-015-0213-6.

Tardigrades of Sweden; an updated check-list.

Tardigrades occur worldwide and in a variety of ecosystems and habitats representing an important component of the micrometazoan biodiversity. Several studies documenting the occurrence of tardigrades in Sweden have been published since the first reports in early 1900, but no comprehensive summary of these studies have been published. We compiled the available information on recorded tardigrades from Sweden, using material from published studies and museum and university collections. In total, our review document 101 species of tardigrades that have been recorded from Sweden (an updated checklist of tardigrades from Sweden will be available online), of which 14 species are new records for the country. The highest number of species was recorded in the northernmost province of Lappland and the more southern provinces of Uppland and Skåne, while much lower species numbers are reported from the middle part of Sweden. This pattern probably represents biased sampling activities of biologists rather than real differences in biodiversity of tardigrades. In view of the few studies that have been made on tardigrade biodiversity in Sweden, the relatively high number of tardigrade species recorded, representing almost a tenth of the species recorded worldwide, indicates that many more species remain to be found. In this respect, more studies of the marine ecosystems along the Swedish west coast and the long Baltic Sea coastline would be of particular interest.

Dendrogramma, new genus, with two new non-bilaterian species from the marine bathyal of southeastern Australia (Animalia, Metazoa incertae sedis)--with similarities to some medusoids from the Precambrian Ediacara.

A new genus, Dendrogramma, with two new species of multicellular, non-bilaterian, mesogleal animals with some bilateral aspects, D. enigmatica and D. discoides, are described from the south-east Australian bathyal (400 and 1000 metres depth). A new family, Dendrogrammatidae, is established for Dendrogramma. These mushroom-shaped organisms cannot be referred to either of the two phyla Ctenophora or Cnidaria at present, because they lack any specialised characters of these taxa. Resolving the phylogenetic position of Dendrogramma depends much on how the basal metazoan lineages (Ctenophora, Porifera, Placozoa, Cnidaria, and Bilateria) are related to each other, a question still under debate. At least Dendrogramma must have branched off before Bilateria and is possibly related to Ctenophora and/or Cnidaria. Dendrogramma, therefore, is referred to Metazoa incertae sedis. The specimens were fixed in neutral formaldehyde and stored in 80% ethanol and are not suitable for molecular analysis. We recommend, therefore, that attempts be made to secure new material for further study. Finally similarities between Dendrogramma and a group of Ediacaran (Vendian) medusoids are discussed.

Life cycle and morphology of a cambrian stem-lineage loriciferan.

Cycloneuralians form a rich and diverse element within Cambrian assemblages of exceptionally preserved fossils. Most resemble priapulid worms whereas other Cycloneuralia (Nematoda, Nematomorpha, Kinorhyncha, Loricifera), well known at the present day, have little or no fossil record. First reports of Sirilorica Peel, 2010 from the lower Cambrian Sirius Passet fauna of North Greenland described a tubular lorica covering the abdomen and part of a well developed introvert with a circlet of 6 grasping denticles near the lorica. The introvert is now known to terminate in a narrow mouth tube, while a conical anal field is also developed. Broad muscular bands between the plates in the lorica indicate that it was capable of movement by rhythmic expansion and contraction of the lorica. Sirilorica is regarded as a macrobenthic member of the stem-lineage of the miniaturised, interstitial, present day Loricifera. Like loriciferans, Sirilorica is now known to have grown by moulting. Evidence of the life cycle of Sirilorica is described, including a large post-larval stage and probably an initial larva similar to that of the middle Cambrian fossil Orstenoloricusshergoldii.

Ultrastructure and morphology of the cycliophoran female.

Knowledge on the morphology of the cycliophoran female has mostly been based on observations of immature females in brood chambers of feeding stages. With the use of light- and transmission electron microscopy, the morphology and ultrastructure of the free and fully mature female of Cycliophora is described now for the first time. The external morphology is characterized by a ciliation consisting of an anteroventral ciliated field, a posterior ciliated tuft, and four sensory structures extending anteriorly from the anteroventral ciliated field. In addition, a small ciliated structure in the midventral region is interpreted as a round-shaped gonopore. Internally, a bilateral cerebral ganglion is situated in the anterior region and a large oocyte is located medially in the body. Several glands are present anteriorly, while posteriorly a pair of glands is associated with the ciliated tuft. Dorsal and ventral longitudinal muscles, as well as, dorsoventral muscles are identified by electron microscopy. Muscle fibers attach to the endocuticle via the epidermis, by means of attachment fibers. An unknown endosymbiont is present throughout the body of the female. We discuss the functional implications of the morphological and ultrastructural aspects of the cycliophoran female. Finally, we compare this life cycle stage with that fromother phyla, suggested as phylogenetically close.

Evidence for simultaneous sound production in the bowhead whale (Balaena mysticetus).

Simultaneous production of two harmonically independent sounds, the two-voice phenomenon, is a well-known feature in bird song. Some toothed whales can click and whistle simultaneously, and a few studies have also reported simultaneous sound production by baleen whales. The mechanism for sound production in toothed whales has been largely uncovered within the last three decades, whereas mechanism for sound production in baleen whales remains poorly understood. This study provides three lines of evidence from recordings made in 2008 and 2009 in Disko Bay, Western Greenland, strongly indicating that bowhead whales are capable of simultaneous dual frequency sound production. This capability may function to enable more complex singing in an acoustically mediated reproductive advertisement display, as has been suggested for songbirds, and/or have significance in individual recognition.

The first metazoa living in permanently anoxic conditions.

BACKGROUND: Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity. RESULTS: During the last ten years three oceanographic expeditions were conducted to search for the presence of living fauna in the sediments of the deep anoxic hypersaline L'Atalante basin (Mediterranean Sea). We report here that the sediments of the L'Atalante basin are inhabited by three species of the animal phylum Loricifera (Spinoloricus nov. sp., Rugiloricus nov. sp. and Pliciloricus nov. sp.) new to science. Using radioactive tracers, biochemical analyses, quantitative X-ray microanalysis and infrared spectroscopy, scanning and transmission electron microscopy observations on ultra-sections, we provide evidence that these organisms are metabolically active and show specific adaptations to the extreme conditions of the deep basin, such as the lack of mitochondria, and a large number of hydrogenosome-like organelles, associated with endosymbiotic prokaryotes. CONCLUSIONS: This is the first evidence of a metazoan life cycle that is spent entirely in permanently anoxic sediments. Our findings allow us also to conclude that these metazoans live under anoxic conditions through an obligate anaerobic metabolism that is similar to that demonstrated so far only for unicellular eukaryotes. The discovery of these life forms opens new perspectives for the study of metazoan life in habitats lacking molecular oxygen.

Cycliophoran dwarf males break the rule: high complexity with low cell numbers.

Complexity of metazoan bodyplans is commonly assumed to be correlated to the absolute number of cells and the number of cell types present in a species (1). Sexually mature individuals of the smallest free-living animals have a minimum of several hundred somatic cells, and only secondarily simplified parasitic or commensal species range below this threshold. Males of the two hitherto described representatives of the phylum Cycliophora (2), with a body length of about 40 microm, are among the smallest existing free-living metazoans, yet they exhibit an amazingly complex bodyplan. Herein, we show that only a few dozen cells account for this complexity. We conclude therefore that metazoan complexity is not obligatorily correlated with body size or with the overall cell number of an individual. Accordingly, the earliest multicellular animals on Earth, which most probably were small individuals, may have had more complex bodyplans than commonly assumed.

Cyclomorphosis in Tardigrada: adaptation to environmental constraints.

Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice formation proceeds rapidly at a crystallization temperature of around -20 degrees C, revealing extensive supercooling in both stages, while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage specimens (350-500 microm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was approximately 950 mOsm kg(-1). Exposure to hypo-osmotic stress in 2 and 10 ppt caused (1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.

Myoanatomy of the marine tardigrade Halobiotus crispae (Eutardigrada: Hypsibiidae).

The muscular architecture of Halobiotus crispae (Eutardigrada: Hypsibiidae) was examined by means of fluorescent-coupled phalloidin in combination with confocal laser scanning microscopy and computer-aided three-dimensional reconstruction, in addition to light microscopy (Nomarski), scanning electron microscopy, and transmission electron microscopy (TEM). The somatic musculature of H. crispae is composed of structurally independent muscle fibers, which can be divided into a dorsal, ventral, dorsoventral, and a lateral musculature. Moreover, a distinct leg musculature is found. The number and arrangement of muscles differ in each leg. Noticeably, the fourth leg contains much fewer muscles when compared with the other legs. Buccopharyngeal musculature (myoepithelial muscles), intestinal musculature, and cloacal musculature comprise the animal's visceral musculature. TEM of stylet and leg musculature revealed ultrastructural similarities between these two muscle groups. Furthermore, microtubules are found in the epidermal cells of both leg and stylet muscle attachments. This would indicate that the stylet and stylet glands are homologues to the claw and claw glands, respectively. When comparing with previously published data on both heterotardigrade and eutardigrade species, it becomes obvious that eutardigrades possess very similar numbers and arrangement of muscles, yet differ in a number of significant details of their myoanatomy. This study establishes a morphological framework for the use of muscular architecture in elucidating tardigrade phylogeny.

Three-dimensional reconstruction of the musculature of various life cycle stages of the cycliophoran Symbion americanus.

Cycliophora is a very recently described phylum of acoelomate metazoans with a complex life cycle and a phylogenetic position that has been under debate ever since its discovery in 1995. Symbion americanus, which lives attached to the mouthparts of the American lobster, Homarus americanus, represents the second species described for the phylum. Aiming to increase the morphological knowledge about this cryptic clade, the present study describes the muscle arrangement of the feeding stage, the attached Prometheus larva with the dwarf male inside, the free living male, the Pandora larva, and the chordoid larva of S. americanus using actin staining and confocal laser scanning microscopy. 3D reconstructions of the muscular systems are presented. In the feeding stage, circular muscles compose the buccal funnel aperture. In addition, a pair of muscles runs longitudinally in the buccal funnel. A complex sphincter was found just proximally to the anus, and six longitudinal muscles run from the trunk constriction ("neck") in basal direction. The musculature of the larval stages and the dwarf male is very complex and includes longitudinal muscles that run dorsally and ventrally. In addition, we found dorso-ventral muscles. The male has a complex posterior muscle apparatus in the vicinity of the penis. In this stage, X- and V-shaped structures were identified on the dorsal and the ventral side, respectively. Pandora and chordoid larvae possess additional circular muscles. We discuss our findings with respect to muscle elements of other metazoan groups and the chordoid larva of Symbion pandora.

Urnaloricus gadi nov. gen. et nov. sp. (Loricifera, Urnaloricidae nov. fam.), an aberrant Loricifera with a viviparous pedogenetic life cycle.

A new species of Loricifera, Urnaloricus gadi nov. gen. et nov. sp., is described from the Faroe Bank, located Southwest of the Faroe Islands, North Atlantic. The new species does not fit into any known families of Loricifera and therefore it is grouped into a new family Urnaloricidae nov. fam. The new species is characterized by having a very complicated life cycle that involves a large cyst-like mega-larva, two reduced larval instars and the Higgins-larvae eating their maternal stage from within. An adult stage is missing. This form of reproduction is called viviparous pedogenesis and normally is found only in nematodes and insects. In the life cycle of Urnaloricidae nov. fam., there are two types of free-living larval stages: a Higgins-larva and a mega-larva. The latter is found in two different forms, a pre- and a cyst-forming mega-larva. Additionally, there are two reduced life history stages, the reduced larval stage (probably a postlarva) and the ghost-larval stage inside the cyst-forming mega-larva. The external morphology of the two forms of mega-larvae is much reduced, e.g., the introvert has only a few rows of scalids when compared with the Higgins-larva. The pre mega-larva is free-living and can sometimes be covered with coccoliths. Internally, a large ovary with a few oocytes, a digestive system, and an internal armature with retracted scalids are present. The pre mega-larva presumably molts into a cyst-forming mega-larva and thereby the ovary is now seen inside the cyst-forming mega-larva. The cyst-forming mega-larva has the same structures as in the pre mega-larva though here the scalids are protruded and there is a gonopore. Inside the cyst-forming mega-larva the ovary produces more oocytes and begins to fill out the entire lumen. At this stage the cyst-forming mega-larva molts first to the presumed postlarval stage, and then this stage molts to a ghost-larva. Hence, the ovary now matures inside the ghost-larva, which is surrounded by both the cuticle of the reduced postlarval stage and the cuticle of the cyst-forming mega-larva. The oocytes mature into eggs, and then into embryos and finally into Higgins-larvae while reabsorbing all the tissue of their maternal stage, the ghost-larva. During this maturation the cuticle of the cyst-forming mega-larva starts to harden and become cyst-like. The fully developed Higgins-larvae emerge through the gonopore of the cyst-forming mega-larva by penetrating the thin cuticles of the ghost-larva and the postlarva. The embryos have holoblastic radial cleavage and later a fluid-filled blastocoel is formed. The eggshells are extremely elastic; hence, they can become very elongated as the embryos mature into Higgins-larvae.

Broad phylogenomic sampling improves resolution of the animal tree of life.

Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.