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Background: Panarthropods, a major group of invertebrate animals comprised of arthropods, onychophorans, and tardigrades, are the only limb-bearing members of Ecdysozoa. The complexity and versatility of panarthropod paired limbs has prompted great interest in their development to better understand the formation of these structures and the genes involved in this process. However, studies of limb patterning and development are overwhelmingly focused on arthropods, followed by select work on onychophorans but almost entirely lacking for tardigrades. This model organism bias is inherently limited and precludes a comparative analysis of how panarthropod legs originated, have evolved, and the likely limb patterning genes present in the earliest panarthropod ancestors. In this study, we investigated tardigrade homologs of seven arthropod distal limb patterning genes (apterous, aristaless, BarH1, clawless, Lim1, rotund, and spineless) to better characterize tardigrade limb development in a comparative context. Results: We detected homologs of all seven genes in the eutardigrade Hypsibius exemplaris and heterotardigrade Echiniscoides cf. sigismundi suggesting their conservation in both tardigrade lineages. Hybridization chain reaction experiments in H. exemplaris reveal a regionalized expression pattern for the genes aristaless, BarH1, clawless, rotund and spineless. Conclusion: The observed regionalized expression of the distal limb patterning genes in H. exemplaris might reflect the external morphological features of tardigrade legs, such as the distal claws, sensory organs in the proximal region, and specific muscle attachment sites. The comparison between the expression of these limb patterning genes in H. exemplaris relative to other panarthropods suggests their conserved role in the last common panarthropod ancestor, such as establishing the distal limb end and the distribution of sensory structures. Our results support the hypothesis that tardigrade legs are homologous to the distal region of other panarthropod limbs, as suggested by previous work on the expression of leg gap genes in H. exemplaris. [ABSTRACT FROM AUTHOR]

Abstract Background Panarthropods, a major group of invertebrate animals comprised of arthropods, onychophorans, and tardigrades, are the only limb-bearing members of Ecdysozoa. The complexity and versatility of panarthropod paired limbs has prompted great interest in their development to better understand the formation of these structures and the genes involved in this process. However, studies of limb patterning and development are overwhelmingly focused on arthropods, followed by select work on onychophorans but almost entirely lacking for tardigrades. This model organism bias is inherently limited and precludes a comparative analysis of how panarthropod legs originated, have evolved, and the likely limb patterning genes present in the earliest panarthropod ancestors. In this study, we investigated tardigrade homologs of seven arthropod distal limb patterning genes (apterous, aristaless, BarH1, clawless, Lim1, rotund, and spineless) to better characterize tardigrade limb development in a comparative context. Results We detected homologs of all seven genes in the eutardigrade Hypsibius exemplaris and heterotardigrade Echiniscoides cf. sigismundi suggesting their conservation in both tardigrade lineages. Hybridization chain reaction experiments in H. exemplaris reveal a regionalized expression pattern for the genes aristaless, BarH1, clawless, rotund and spineless. Conclusion The observed regionalized expression of the distal limb patterning genes in H. exemplaris might reflect the external morphological features of tardigrade legs, such as the distal claws, sensory organs in the proximal region, and specific muscle attachment sites. The comparison between the expression of these limb patterning genes in H. exemplaris relative to other panarthropods suggests their conserved role in the last common panarthropod ancestor, such as establishing the distal limb end and the distribution of sensory structures. Our results support the hypothesis that tardigrade legs are homologous to the distal region of other panarthropod limbs, as suggested by previous work on the expression of leg gap genes in H. exemplaris.

Tardigrada is an ancient lineage of miniaturized animals. As an outgroup of the well‐studied Arthropoda and Onychophora, studies of tardigrades hold the potential to reveal important insights into body plan evolution in Panarthropoda. Previous studies have revealed interesting facets of tardigrade development and genomics that suggest that a highly compact body plan is a derived condition of this lineage, rather than it representing an ancestral state of Panarthropoda. This conclusion was based on studies of several species from Eutardigrada. We review these studies and expand on them by analyzing the publicly available genome and transcriptome assemblies of Echiniscus testudo, a representative of Heterotardigrada. These new analyses allow us to phylogenetically reconstruct important features of genome evolution in Tardigrada. We use available data from tardigrades to interrogate several recent models of body plan evolution in Panarthropoda. Although anterior segments of panarthropods are highly diverse in terms of anatomy and development, both within individuals and between species, we conclude that a simple one‐to‐one alignment of anterior segments across Panarthropoda is the best available model of segmental homology. In addition to providing important insight into body plan diversification within Panarthropoda, we speculate that studies of tardigrades may reveal generalizable pathways to miniaturization. [ABSTRACT FROM AUTHOR]

Phylum Tardigrada (water bears), well known for their cryptobiosis, includes small invertebrates with four paired limbs and is divided into two classes: Eutardigrada and Heterotardigrada. The evolutionary origin of Tardigrada is known to lie within the lobopodians, which are extinct soft-bodied worms with lobopodous limbs mostly dis- covered at sites of exceptionally well-preserved fossils. Contrary to their closest rel- atives, onychophorans and euarthropods, the origin of morphological characters of tardigrades remains unclear, and detailed comparison with the lobopodians has not been well explored. Here, we present detailed morphological comparison between tar- digrades and Cambrian lobopodians, with a phylogenetic analysis encompassing most of the lobopodians and three panarthropod phyla. The results indicate that the ancestral tardigrades likely had a Cambrian lobopodian–like morphology and shared most recent ancestry with the luolishaniids. Internal relationships within Tardigrada indicate that the ancestral tardigrade had a vermiform body shape without segmental plates, but possessed cuticular structures surrounding the mouth opening, and lobopodous legs ter- minating with claws, but without digits. This finding is in contrast to the long-standing stygarctid- like ancestor hypothesis. The highly compact and miniaturized body plan of tardigrades evolved after the tardigrade lineage diverged from an ancient shared ancestor with the luolishaniids. [ABSTRACT FROM AUTHOR]

Simple Summary: "Anomalocaris" kunmingensis is the most common radiodont in the Guanshan biota, which has been recently reassigned to the family Amplectobeluidae. However, its generic assignment is still uncertain. Our new specimens reveal more characteristic of "Anomalocaris" kunmingensis, warranting the erection of a new genus, Guanshancaris gen. nov. Brachiopod shells bearing embayment injury, and broken trilobites closely associated with the frontal appendages, may indicate durophagous habits of Guanshancaris. The spatial and temporal distribution of Amplectobeluidae, documented in various soft-bodied biota, indicates that this group was restricted to low latitude regions from Cambrian Stage 3 to Drumian, and implies an ecological preference for a shallow-water environment. Radiodonta, an extinct stem-euarthropod group, has been considered as the largest predator of Cambrian marine ecosystems. As one of the radiodont-bearing Konservat-Lagerstätten, the Guanshan biota (South China, Cambrian Stage 4) has yielded a diverse assemblage of soft-bodied and biomineralized taxa that are exclusive to this exceptional deposit. "Anomalocaris" kunmingensis, the most abundant radiodont in the Guanshan biota, was originally assigned to Anomalocaris within the Anomalocarididae. Despite this taxon being formally assigned to the family Amplectobeluidae more recently, its generic assignment remains uncertain. Here, we present new materials of "Anomalocaris" kunmingensis from the Guanshan biota, and reveal that the frontal appendages possess two enlarged endites; all endites bear one posterior auxiliary spine and up to four anterior auxiliary spines; three robust dorsal spines and one terminal spine protrude from the distal part. These new observations, allied with anatomical features illustrated by previous studies, allow us to assign this taxon to a new genus, Guanshancaris gen. nov. Brachiopod shell bearing embayed injury and incomplete trilobites, associated with frontal appendages in our specimens, to some extent confirm Guanshancaris as a possible durophagous predator. The distribution of amplectobeluids demonstrates that this group is restricted to Cambrian Stage 3 to Drumian, and occurs across South China and Laurentia within the tropics/subtropics belt. Moreover, the amount and abundance of amplectobeluids evidently decreases after the Early–Middle Cambrian boundary, which indicates its possible preference for shallow water, referring to its paleoenvironmental distribution and may be influenced by geochemical, tectonic, and climatic variation. [ABSTRACT FROM AUTHOR]

Genome assemblies are growing at an exponential rate and have proved indispensable for studying evolution but the effort has been biased toward vertebrates and arthropods with a particular focus on insects. Onychophora or velvet worms are an ancient group of cryptic, soil dwelling worms noted for their unique mode of prey capture, biogeographic patterns, and diversity of reproductive strategies. They constitute a poorly understood phylum of exclusively terrestrial animals that is sister group to arthropods. Due to this phylogenetic position, they are crucial in understanding the origin of the largest phylum of animals. Despite their significance, there is a paucity of genomic resources for the phylum with only one highly fragmented and incomplete genome publicly available. Initial attempts at sequencing an onychophoran genome proved difficult due to its large genome size and high repeat content. However, leveraging recent advances in long-read sequencing technology, we present here the first annotated draft genome for the phylum. With a total size of 5.6Gb, the gigantism of the Epiperipatus broadwayi genome arises from having high repeat content, intron size inflation, and extensive gene family expansion. Additionally, we report a previously unknown diversity of onychophoran hemocyanins that suggests the diversification of copper-mediated oxygen carriers occurred independently in Onychophora after its split from Arthropoda, parallel to the independent diversification of hemocyanins in each of the main arthropod lineages. [ABSTRACT FROM AUTHOR]

Background: Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane‐bound receptors such as Uncoordianted‐5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. Results: Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated‐5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. Conclusion: Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages. [ABSTRACT FROM AUTHOR]

Abstract Background Wnt genes code for ligands that activate signaling pathways during development in Metazoa. Through the canonical Wnt (cWnt) signaling pathway, these genes regulate important processes in bilaterian development, such as establishing the anteroposterior axis and posterior growth. In Arthropoda, Wnt ligands also regulate segment polarity, and outgrowth and patterning of developing appendages. Arthropods are part of a lineage called Panarthropoda that includes Onychophora and Tardigrada. Previous studies revealed potential roles of Wnt genes in regulating posterior growth, segment polarity, and growth and patterning of legs in Onychophora. Unlike most other panarthropods, tardigrades lack posterior growth, but retain segmentation and appendages. Here, we investigated Wnt genes in tardigrades to gain insight into potential roles that these genes play during development of the highly compact and miniaturized tardigrade body plan. Results We analyzed published genomes for two representatives of Tardigrada, Hypsibius exemplaris and Ramazzottius varieornatus. We identified single orthologs of Wnt4, Wnt5, Wnt9, Wnt11, and WntA, as well as two Wnt16 paralogs in both tardigrade genomes. We only found a Wnt2 ortholog in H. exemplaris. We could not identify orthologs of Wnt1, Wnt6, Wnt7, Wnt8, or Wnt10. We identified most other components of cWnt signaling in both tardigrade genomes. However, we were unable to identify an ortholog of arrow/Lrp5/6, a gene that codes for a Frizzled co-receptor of Wnt ligands. Additionally, we found that some other animals that have lost several Wnt genes and are secondarily miniaturized, like tardigrades, are also missing an ortholog of arrow/Lrp5/6. We analyzed the embryonic expression patterns of Wnt genes in H. exemplaris during developmental stages that span the establishment of the AP axis through segmentation and leg development. We detected expression of all Wnt genes in H. exemplaris besides one of the Wnt16 paralogs. During embryo elongation, expression of several Wnt genes was restricted to the posterior pole or a region between the anterior and posterior poles. Wnt genes were expressed in distinct patterns during segmentation and development of legs in H. exemplaris, rather than in broadly overlapping patterns. Conclusions Our results indicate that Wnt signaling has been highly modified in Tardigrada. While most components of cWnt signaling are conserved in tardigrades, we conclude that tardigrades have lost Wnt1, Wnt6, Wnt7, Wnt8, and Wnt10, along with arrow/Lrp5/6. Our expression data may indicate a conserved role of Wnt genes in specifying posterior identities during establishment of the AP axis. However, the loss of several Wnt genes and the distinct expression patterns of Wnt genes during segmentation and leg development may indicate that combinatorial interactions among Wnt genes are less important during tardigrade development compared to many other animals. Based on our results, and comparisons to previous studies, we speculate that the loss of several Wnt genes in Tardigrada may be related to a reduced number of cells and simplified development that accompanied miniaturization and anatomical simplification in this lineage.

Abstract Background In the vinegar fly Drosophila melanogaster, the homeodomain containing transcription factor Teashirt (Tsh) appears to specify trunk identity in concert with the function of the Hox genes. While in Drosophila there is a second gene closely related to tsh, called tiptop (tio), in other arthropods species only one copy exists (called tio/tsh). The expression of tsh and tio/tsh, respectively, is surprisingly similar among arthropods suggesting that its function as trunk selector gene may be conserved. Other research, for example on the beetle Tribolium castaneum, questions even conservation of Tsh function among insects. The zinc-finger transcription factor Spalt (Sal) is involved in the regulation of Drosophila tsh, but this regulatory interaction does not appear to be conserved in Tribolium either. Whether the function and interaction of tsh and sal as potential trunk-specifiers, however, is conserved is still unclear because comparative studies on sal expression (except for Tribolium) are lacking, and functional data are (if at all existing) restricted to Insecta. Results Here, we provide additional data on arthropod tsh expression, show the first data on onychophoran tio/tsh expression, and provide a comprehensive investigation on sal expression patterns in arthropods and an onychophoran. Conclusions Our data support the idea that tio/tsh genes are involved in the development of “trunk” segments by regulating limb development. Our data suggest further that the function of Sal is indeed unlikely to be conserved in trunk vs head development like in Drosophila, but early expression of sal is in line with a potential homeotic function, at least in Arthropoda.

Radiodonta, an extinct stem-euarthropod group, has been considered as the largest predator of Cambrian marine ecosystems. As one of the radiodont-bearing Konservat-Lagerstätten, the Guanshan biota (South China, Cambrian Stage 4) has yielded a diverse assemblage of soft-bodied and biomineralized taxa that are exclusive to this exceptional deposit. “Anomalocaris” kunmingensis, the most abundant radiodont in the Guanshan biota, was originally assigned to Anomalocaris within the Anomalocarididae. Despite this taxon being formally assigned to the family Amplectobeluidae more recently, its generic assignment remains uncertain. Here, we present new materials of “Anomalocaris” kunmingensis from the Guanshan biota, and reveal that the frontal appendages possess two enlarged endites; all endites bear one posterior auxiliary spine and up to four anterior auxiliary spines; three robust dorsal spines and one terminal spine protrude from the distal part. These new observations, allied with anatomical features illustrated by previous studies, allow us to assign this taxon to a new genus, Guanshancaris gen. nov. Brachiopod shell bearing embayed injury and incomplete trilobites, associated with frontal appendages in our specimens, to some extent confirm Guanshancaris as a possible durophagous predator. The distribution of amplectobeluids demonstrates that this group is restricted to Cambrian Stage 3 to Drumian, and occurs across South China and Laurentia within the tropics/subtropics belt. Moreover, the amount and abundance of amplectobeluids evidently decreases after the Early–Middle Cambrian boundary, which indicates its possible preference for shallow water, referring to its paleoenvironmental distribution and may be influenced by geochemical, tectonic, and climatic variation.

Arthropods are ubiquitous in all modern habitats and yet their origin remains poorly documented. It is widely thought that their segmented and arthrodized body evolved from the annulated vermiform body of a lobopodian ancestor c. 540 Ma. This major transformation included the evolution of sclerotized and articulated appendages from annulated non‐jointed limbs or lobopods. However, this scenario is complicated by the presence in many stem‐group arthropods of body flaps of various origins, characteristics and functions. We describe the new lobopodian Utahnax vannieri gen. et sp. nov. from Drumian strata of the House Range in Utah. Known from an incomplete specimen, this taxon features a vermiform, annulated body flanked by pairs of swimming flaps, except in the caudal region, and a digestive system with putative glands. Extensions of the body cavity into the body flaps of Utahnax suggest that they are modified lobopods similar to the ventral body flaps of radiodonts; as such, they are not homologous to the dorsal body flaps of the stem‐group arthropods Opabinia, Pambdelurion, and possibly Kerygmachela. We discuss the details of the limb anatomy of these three taxa and consider an alternative interpretation for Kerygmachela. Despite morphological similarities in radiodonts, Utahnax, and possibly Kerygmachela, our parsimony and Bayesian inference phylogenetic analyses suggest that ventral body flaps have evolved convergently in those taxa, probably accompanying a shift towards a predominantly swimming predatory lifestyle. Ecological competition with radiodonts is proposed as an explanation for the scarcity of non‐radiodont swimming lobopodians in Cambrian Lagerstätten. [ABSTRACT FROM AUTHOR]

The Furongian period represents an important gap in the fossil record of most groups of non-biomineralizing organisms, owing to a scarcity of Konservat-Lagerstätten of that age. The most significant of these deposits, the Jiangshanian strata of the Sandu Formation near Guole Township (Guangxi, South China), have yielded a moderately abundant, but taxonomically diverse soft-bodied fossil assemblage, which provides rare insights into the evolution of marine life at that time. In this contribution, we report the first discovery of a radiodont fossil from the Guole Konservat-Lagerstätte. The specimen is an incomplete frontal appendage of a possibly new representative of the family Hurdiidae. It is tentatively interpreted as composed of seven podomeres, six of which bearing laminiform endites. The best preserved of these endites is especially long, and it bears short auxiliary spines that greatly vary in size. This is the second occurrence of hurdiids and more generally radiodonts in the Furongian, the first being the external mould of an oral cone from Jiangshanian strata of the Wiśniówka Sandstone Formation in Poland. Restudy of this Polish specimen confirms that it belongs to a hurdiid radiodont and best compares to Peytoia. The family Hurdiidae includes the oldest (basal Cambrian Epoch 2) and youngest (Early Ordovician, possibly Early Devonian) representatives of the Radiodonta and as such, has the longest stratigraphical range of the group. Yet, hurdiids only became prominent components of marine ecosystems during the middle Cambrian (Miaolingian), and their fossil record in younger strata remains limited.

Background: In the vinegar fly Drosophila melanogaster, the homeodomain containing transcription factor Teashirt (Tsh) appears to specify trunk identity in concert with the function of the Hox genes. While in Drosophila there is a second gene closely related to tsh, called tiptop (tio), in other arthropods species only one copy exists (called tio/tsh). The expression of tsh and tio/tsh, respectively, is surprisingly similar among arthropods suggesting that its function as trunk selector gene may be conserved. Other research, for example on the beetle Tribolium castaneum, questions even conservation of Tsh function among insects. The zinc-finger transcription factor Spalt (Sal) is involved in the regulation of Drosophila tsh, but this regulatory interaction does not appear to be conserved in Tribolium either. Whether the function and interaction of tsh and sal as potential trunk-specifiers, however, is conserved is still unclear because comparative studies on sal expression (except for Tribolium) are lacking, and functional data are (if at all existing) restricted to Insecta. Results: Here, we provide additional data on arthropod tsh expression, show the first data on onychophoran tio/tsh expression, and provide a comprehensive investigation on sal expression patterns in arthropods and an onychophoran. Conclusions: Our data support the idea that tio/tsh genes are involved in the development of "trunk" segments by regulating limb development. Our data suggest further that the function of Sal is indeed unlikely to be conserved in trunk vs head development like in Drosophila, but early expression of sal is in line with a potential homeotic function, at least in Arthropoda. [ABSTRACT FROM AUTHOR]

Abstract Background One set of the developmentally important Doublesex and Male-abnormal-3 Related Transcription factors (Dmrt) is subject of intense research, because of their role in sex-determination and sexual differentiation. This likely non-monophyletic group of Dmrt genes is represented by the Drosophila melanogaster gene Doublesex (Dsx), the Caenorhabditis elegans Male-abnormal-3 (Mab-3) gene, and vertebrate Dmrt1 genes. However, other members of the Dmrt family are much less well studied, and in arthropods, including the model organism Drosophila melanogaster, data on these genes are virtually absent with respect to their embryonic expression and function. Results Here we investigate the complete set of Dmrt genes in members of all main groups of Arthropoda and a member of Onychophora, extending our data to Panarthropoda as a whole. We confirm the presence of at least four families of Dmrt genes (including Dsx-like genes) in Panarthropoda and study their expression profiles during embryogenesis. Our work shows that the expression patterns of Dmrt11E, Dmrt93B, and Dmrt99B orthologs are highly conserved among panarthropods. Embryonic expression of Dsx-like genes, however, is more derived, likely as a result of neo-functionalization after duplication. Conclusions Our data suggest deep homology of most of the panarthropod Dmrt genes with respect to their function that likely dates back to their last common ancestor. The function of Dsx and Dsx-like genes which are critical for sexual differentiation in animals, however, appears to be much less conserved.

Abstract Background Morphogen signalling represents a key mechanism of developmental processes during animal development. Previously, several evolutionary conserved morphogen signalling pathways have been identified, and their players such as the morphogen receptors, morphogen modulating factors (MMFs) and the morphogens themselves have been studied. MMFs are factors that regulate morphogen distribution and activity. The interactions of MMFs with different morphogen signalling pathways such as Wnt signalling, Hedgehog (Hh) signalling and Decapentaplegic (Dpp) signalling are complex because some of the MMFs have been shown to interact with more than one signalling pathway, and depending on genetic context, to have different, biphasic or even opposing function. This complicates the interpretation of expression data and functional data of MMFs and may be one reason why data on MMFs in other arthropods than Drosophila are scarce or totally lacking. Results As a first step to a better understanding of the potential roles of MMFs in arthropod development, we investigate here the embryonic expression patterns of division abnormally delayed (dally), dally-like protein (dlp), shifted (shf) and secreted frizzled-related protein 125 (sFRP125) and sFRP34 in the beetle Tribolium castaneum, the spider Parasteatoda tepidariorum, the millipede Glomeris marginata and the onychophoran Euperipatoides kanangrensis. This pioneer study represents the first comprehensive comparative data set of these genes in panarthropods. Conclusions Expression profiles reveal a high degree of diversity, suggesting that MMFs may represent highly evolvable nodes in otherwise conserved gene regulatory networks. Conserved aspects of MMF expression, however, appear to concern function in segmentation and limb development, two of the key topics of evolutionary developmental research.

The Drumian Wheeler Konservat‐Lagerstätte of the House Range of Utah (Wheeler‐HR) has yielded one of the most diverse exceptionally preserved Cambrian biotas of North America. The discovery of soft‐bodied fossils invariably provides precious insights into this biota, given that most of its non‐biomineralizing components are known from very few specimens. This contribution describes some 30 new exceptionally preserved fossils of Wheeler panarthropods. Two new species are recognized, the radiodont Hurdia sp. nov. A and the megacheiran Kanoshoia rectifrons gen. et sp. nov. Along with a species of Leanchoilia,K. rectifrons represents the first confident megacheiran record in these strata. The presence of the radiodont genus Amplectobelua and the isoxyid species Isoxys longissimus is reported outside of the Burgess Shale in Laurentia. New specimens of Caryosyntrips serratus,Naraoia compacta,Messorocaris magna and Mollisonia symmetrica provide insights into the phylogenetic affinities, local spatial distribution and morphological variation of these species hitherto known by single specimens in the Wheeler‐HR. The same is true of new materials of the more common Pahvantia hastata and Perspicaris? dilatus. Formal descriptions of the order Mollisoniida ord. nov. and family Mollisoniidae fam. nov. are also provided. Last, the preservation of body structures other than the dorsal exoskeletons is illustrated for the first time in two common components of the fauna: the agnostid Itagnostus interstrictus and the bivalved euarthropod Pseudoarctolepis sharpi. The new material substantially improves our understanding of the diversity of the Wheeler‐HR biota, and provides new evidence of its distinctiveness relative to the Wheeler biota of the Drum Mountains. [ABSTRACT FROM AUTHOR]

Tardigrades have a miniaturized body plan. Miniaturization in tardigrades is associated with the loss of several organ systems and an intermediate region of their anteroposterior (AP) axis. However, how miniaturization has affected tardigrade legs is unclear. In arthropods and in onychophorans, the leg gap genes are expressed in regionalized proximodistal (PD) patterns in the legs. Functional studies indicate that these genes regulate growth in their respective expression domains and establish PD identities, partly through mutually antagonistic regulatory interactions. Here, we investigated the expression patterns of tardigrade orthologs of the leg gap genes. Rather than being restricted to a proximal leg region, as in arthropods and onychophorans, we detected coexpression of orthologues of homothorax and extradenticle broadly across the legs of the first three trunk segments in the tardigrade Hypsibius exemplaris. We could not identify a dachshund orthologue in tardigrade genomes, a gene that is expressed in an intermediate region of developing legs in arthropods and onychophorans, suggesting that this gene was lost in the tardigrade lineage. We detected Distal-less expression broadly across all developing leg buds in H. exemplaris embryos, unlike in arthropods and onychophorans, in which it exhibits a distally restricted expression domain. The broad expression patterns of the remaining leg gap genes in H. exemplaris legs may reflect the loss of dachshund and the accompanying loss of an intermediate region of the legs in the tardigrade lineage. We propose that the loss of intermediate regions of both the AP and PD body axes contributed to miniaturization of Tardigrada. [ABSTRACT FROM AUTHOR]

Pigment-dispersing factor neuropeptides (PDFs) occur in a wide range of protostomes including ecdysozoans (= molting animals) and lophotrochozoans (mollusks, annelids, flatworms, and allies). Studies in insects revealed that PDFs play a role as coupling factors of circadian pacemaker cells, thereby controlling rest-activity rhythms. While the last common ancestor of protostomes most likely possessed only one pdf gene, two pdf homologs, pdf-I and pdf-II , might have been present in the last common ancestors of Ecdysozoa and Panarthropoda (Onychophora + Tardigrada + Arthropoda). One of these homologs, however, was subsequently lost in the tardigrade and arthropod lineages followed by independent duplications of pdf-I in tardigrades and decapod crustaceans. Due to the ancestral set of two pdf genes, the study of PDFs and their receptor (PDFR) in Onychophora might reveal the ancient organization and function of the PDF/PDFR system in panarthropods. Therefore, we deorphanized the PDF receptor and generated specific antibodies to localize the two PDF peptides and their receptor in the onychophoran Euperipatoides rowelli. We further conducted bioluminescence resonance energy transfer (BRET) experiments on cultured human cells (HEK293T) using an Epac-based sensor (Epac-L) to examine cAMP responses in transfected cells and to reveal potential differences in the interaction of PDF-I and PDF-II with PDFR from E. rowelli. These data show that PDF-II has a tenfold higher potency than PDF-I as an activating ligand. Double immunolabeling revealed that both peptides are co-expressed in E. rowelli but their respective levels of expression differ between specific cells: some neurons express the same amount of both peptides, while others exhibit higher levels of either PDF-I or PDF-II. The detection of the onychophoran PDF receptor in cells that additionally express the two PDF peptides suggests autoreception, whereas spatial separation of PDFR- and PDF-expressing cells supports hormonal release of PDF into the hemolymph. This suggests a dual role of PDF peptides—as hormones and as neurotransmitters/neuromodulators—in Onychophora. [ABSTRACT FROM AUTHOR]

The Sp-family genes encode important transcription factors in animal development. Here we investigate the embryonic expression patterns of the complete set of Sp-genes in the velvet worm Euperipatoides kanangrensis (Onychophora), with a special focus on the Sp6–9 ortholog. In arthropods, Sp6–9, the ortholog of the Drosophila melanogaster D-Sp1 gene plays a conserved role in appendage development. Our data show that the expression of Sp6–9 during the development of the velvet worm is conserved, suggesting that the key function of the Sp6–9 gene dates back to at least the last common ancestor of arthropods and onychophorans and thus likely the last common ancestor of Panarthropoda. [ABSTRACT FROM AUTHOR]

Abstract The Ecdysozoa is a major animal clade whose main uniting feature is a distinctive growth strategy that requires the periodical moulting of the external cuticle. The staggering diversity within Ecdysozoa has prompted substantial efforts to reconstruct their origin and early evolution. Based on palaentological and developmental data, we proposed a scenario for the early evolution of the ecdysozoan clade Panarthropoda (Onychophora, Tardigrada, Euarthropoda), and postulated that a terminal mouth is ancestral for this lineage. In light of the accompanying comment by Claus Nielsen, we take this opportunity to clarify the significance of our argumentation for Panarthropoda in the phylogenetic context of Ecdysozoa, and Bilateria more broadly. We conclude that the ancestral ecdysozoan most likely had an adult terminal mouth, and that the last common ancestors of all the phyla that constitute Ecdysozoa almost certainly also had an adult terminal mouth. The occurrence of a ventral-facing mouth in various adult ecdysozoans – particularly panarthropods – is the result of convergence. Despite the paucity of embryological data on fossil taxa, we contemplate the likelihood that a developmentally early ventral mouth opening could be ancestral for Ecdysozoa, and if so, then this would represent a symplesiomorphy of Bilateria as a whole. Highlights • We reply to Nielsen's proposal that the ancestral Panarthropod had a ventral mouth. • Fossils demonstrate the presence of a terminal anterior facing mouth in adult stem-group scalidophorans and panarthropods. • Palaeontological data suggest that the last common ancestor of Ecdysozoa had an adult terminal mouth. • Extant panarthropod development suggests that palaeoscolecids and lobopodians may have had an embryonic ventral mouth. [ABSTRACT FROM AUTHOR]

The Herefordshire (Silurian) Lagerstätte (approx. 430 Myr BP) has yielded, among many exceptionally preserved invertebrates, a wide range of new genera belonging to crown-group Panarthropoda. Here, we increase this panarthropod diversity with the lobopodian Thanahita distos, a new total-group panarthropod genus and species. This new lobopodian preserves at least nine paired, long, slender appendages, the anterior two in the head region and the posterior seven representing trunk lobopods. The body ends in a short post-appendicular extension. Some of the trunk lobopods bear two claws, others a single claw. The body is covered by paired, tuft-like papillae. Thanahita distos joins only seven other known three-dimensionally preserved lobopodian or onychophoran (velvet worm) fossil specimens and is the first lobopodian to be formally described from the Silurian. Phylogenetic analysis recovered it, together with all described Hallucigenia species, in a sister-clade to crown-group panarthropods. Its placement in a redefined Hallucigeniidae, an iconic Cambrian clade, indicates the survival of this clade to Silurian times.

Tardigrades are a key group for understanding the evolution of the Ecdysozoa, a large clade of molting animals that also includes arthropods and nematodes. However, little is known about most aspects of their basic biology. Neuropeptide and peptide hormone signaling has been extensively studied in arthropods and nematodes (particularly regarding their roles in molting in arthropods), but very little is known about neuropeptide signaling in other ecdysozoans. In this work, different strategies were used to search for neuropeptide and peptide hormone precursors in the genomes of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus . In general, there is a remarkable similarity in the complement of neuropeptides and their sequences between tardigrades and arthropods. The precursors found in tardigrades included homologs of achatin, allatostatins A, B and C, allatotropin, calcitonin, CCHamide, CCRFa, corazonin, crustacean cardioactive peptide, diuretic hormone 31, diuretic hormone 44, ecdysis triggering hormone, eclosion hormone, gonadotropin-releasing hormone (GnRH), GSEFLamide, insulin-like peptides, ion transport peptide, kinin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, RYamide, short neuropeptide F, sulfakinin, tachykinin, trissin and vasopressin. In most cases, homologs of known cognate receptors for each neuropeptide family could only be identified when the precursors were also present in the genome, further supporting their identification. Some neuropeptide precursor genes have undergone several duplications in tardigrades, including allatostatin A and C, corazonin, GnRH, eclosion hormone, sulfakinin and trissin. Furthermore, four novel families of candidate neuropeptide precursors were identified (two of which could also be found in several arthropods). To the best of my knowledge, this work represents the first genome-wide search for neuropeptide precursors in any ecdysozoan species outside arthropods and nematodes, and is a necessary first step towards understanding neuropeptide function in tardigrades. [ABSTRACT FROM AUTHOR]

Background: Due to their phylogenetic position as one of the closest arthropod relatives, studies of the organisation of the nervous system in onychophorans play a key role for understanding the evolution of body segmentation in arthropods. Previous studies revealed that, in contrast to the arthropods, segmentally repeated ganglia are not present within the onychophoran ventral nerve cords, suggesting that segmentation is either reduced or might be incomplete in the onychophoran ventral nervous system. Results: To assess segmental versus non-segmental features in the ventral nervous system of onychophorans, we screened the nerve cords for various markers, including synapsin, serotonin, gamma-aminobutyric acid, RFamide, dopamine, tyramine and octopamine. In addition, we performed retrograde fills of serially repeated commissures and leg nerves to localise the position of neuronal somata supplying those. Our data revealed a mixture of segmental and non-segmental elements within the onychophoran nervous system. Conclusions: We suggest that the segmental ganglia of arthropods evolved by a gradual condensation of subsets of neurons either in the arthropod or the arthropod-tardigrade lineage. These findings are in line with the hypothesis of gradual evolution of segmentation in panarthropods and thus contradict a loss of ancestral segmentation within the onychophoran lineage. [ABSTRACT FROM AUTHOR]

Phylum Tardigrada (water bears), well known for their cryptobiosis, includes small invertebrates with four paired limbs and is divided into two classes: Eutardigrada and Heterotardigrada. The evolutionary origin of Tardigrada is known to lie within the lobopodians, which are extinct soft-bodied worms with lobopodous limbs mostly discovered at sites of exceptionally well-preserved fossils. Contrary to their closest relatives, onychophorans and euarthropods, the origin of morphological characters of tardigrades remains unclear, and detailed comparison with the lobopodians has not been well explored. Here, we present detailed morphological comparison between tardigrades and Cambrian lobopodians, with a phylogenetic analysis encompassing most of the lobopodians and three panarthropod phyla. The results indicate that the ancestral tardigrades likely had a Cambrian lobopodian-like morphology and shared most recent ancestry with the luolishaniids. Internal relationships within Tardigrada indicate that the ancestral tardigrade had a vermiform body shape without segmental plates, but possessed cuticular structures surrounding the mouth opening, and lobopodous legs terminating with claws, but without digits. This finding is in contrast to the long-standing stygarctid-like ancestor hypothesis. The highly compact and miniaturized body plan of tardigrades evolved after the tardigrade lineage diverged from an ancient shared ancestor with the luolishaniids.

ABSTRACT The ever-increasing number of studies that address the origin and evolution of Euarthropoda - whose extant representatives include chelicerates, myriapods, crustaceans and hexapods - are gradually reaching a consensus with regard to the overall phylogenetic relationships of some of the earliest representatives of this phylum. The stem-lineage of Euarthropoda includes numerous forms that reflect the major morphological transition from a lobopodian-type to a completely arthrodized body organization. Several methods of classification that aim to reflect such a complex evolutionary history have been proposed as a consequence of this taxonomic diversity. Unfortunately, this has also led to a saturation of nomenclatural schemes, often in conflict with each other, some of which are incompatible with cladistic-based methodologies. Here, I review the convoluted terminology associated with the classification of stem-group Euarthropoda, and propose a synapomorphy-based distinction that allows 'lower stem-Euarthropoda' (e.g. lobopodians, radiodontans) to be separated from 'upper stem-Euarthropoda' (e.g. fuxianhuiids, Cambrian bivalved forms) in terms of the structural organization of the head region and other aspects of overall body architecture. The step-wise acquisition of morphological features associated with the origins of the crown-group indicate that the node defining upper stem-Euarthropoda is phylogenetically stable, and supported by numerous synapomorphic characters; these include the presence of a deutocerebral first appendage pair, multisegmented head region with one or more pairs of post-ocular differentiated limbs, complete body arthrodization, posterior-facing mouth associated with the hypostome/labrum complex, and post-oral biramous arthropodized appendages. The name 'Deuteropoda' nov. is proposed for the scion (monophyletic group including the crown-group and an extension of the stem-group) that comprises upper stem-Euarthropoda and Euarthropoda. A brief account of common terminological inaccuracies in recent palaeontological studies evinces the utility of Deuteropoda nov. as a reference point for discussing aspects of early euarthropod phylogeny. [ABSTRACT FROM AUTHOR]

Background: The origin of the body plan of modern velvet worms (Onychophora) lies in the extinct lobopodians of the Palaeozoic. Helenodora inopinata, from the Mazon Creek Lagerstätte of Illinois (Francis Creek Shale, Carbondale Formation, Middle Pennsylvanian), has been proposed as an intermediate between the "weird wonders" of the Cambrian seas and modern terrestrial predatory onychophorans. The type material of H. inopinata, however, leaves much of the crucial anatomy unknown. Results: Here we present a redescription of this taxon based on more complete material, including new details of the head and posterior portion of the trunk, informed by the results of experimental decay of extant onychophorans. H. inopinata is indeed best resolved as a stem-onychophoran, but lacks several key features of modern velvet worms including, crucially, those that would suggest a terrestrial mode of life. Conclusions: The presence of H. inopinata in the Carboniferous demonstrates the survival of a Cambrian marine morphotype, and a likely post-Carboniferous origin of crown-Onychophora. Our analysis also demonstrates that taphonomically informed tests of character interpretations have the potential to improve phylogenetic resolution. [ABSTRACT FROM AUTHOR]

[Display omitted] • Hurdiid radiodont appendages first described from the Chengjiang biota. • Insights for understanding character evolution of hurdiids. • Hurdiids are rare in Chengjiang due to unfavorable depositional conditions. • Success of hurdiids relate to their adaption to broad ecological range. Over one hundred arthropod fossil species have been described from the famous Chengjiang Lagerstätte (South China, Cambrian Stage 3, ca. 518 Ma) including a diverse assemblage of radiodonts–a group containing Anomalocaris and its relatives. These iconic stem-group euarthropods include some of the largest animals of the time, and some are known from hundreds of specimens. A longstanding conundrum has been the rarity or absence of hurdiids from Cambrian Series 2 Lagerstätten like Chengjiang. This is because radiodonts are generally common in such deposits and the oldest radiodont ever discovered is a hurdiid. Furthermore, this family displays the widest geographic and temporal ranges of all radiodont families, and the highest diversity. Here we document the first hurdiid frontal appendages from Chengjiang, which display unique features within the family and may provide insights for understanding the character evolution of hurdiid appendages. The palaeoenvironmental distribution of hurdiids suggests that the rarity of hurdiids in Chengjiang may be due to a preference for deeper water environments, and the later success of this family from the Wuliuan onwards may relate to their ability to tolerate cooler water temperatures than other radiodont families. The palaeogeographical, palaeoenvironmental, and stratigraphical patterns observed in hurdiids maybe caused in part by the limited distributions of Konservat-Lagerstätten in the Cambrian as well. [ABSTRACT FROM AUTHOR]

Panarthropoda, the clade comprising the phyla Onychophora, Tardigrada and Euarthropoda, encompasses the largest majority of animal biodiversity. The relationships among the phyla are contested and resolution is key to understanding the evolutionary assembly of panarthropod bodyplans. Molecular phylogenetic analyses generally support monophyly of Onychophora and Euarthropoda to the exclusion of Tardigrada (Lobopodia hypothesis), which is also supported by some analyses of morphological data. However, analyses of morphological data have also been interpreted to support monophyly of Tardigrada and Euarthropoda to the exclusion of Onychophora (Tactopoda hypothesis). Support has also been found for a clade of Onychophora and Tardigrada that excludes Euarthropoda (Protarthropoda hypothesis). Here we show, using a diversity of phylogenetic inference methods, that morphological datasets cannot discriminate statistically between the Lobopodia, Tactopoda and Protarthropoda hypotheses. Since the relationships among the living clades of panarthropod phyla cannot be discriminated based on morphological data, we call into question the accuracy of morphology-based phylogenies of Panarthropoda that include fossil species and the evolutionary hypotheses based upon them.

Background: The tardigrades (water bears) are a cosmopolitan group of microscopic ecdysozoans found in a variety of aquatic and temporarily wet environments. They are members of the Panarthropoda (Tardigrada + Onychophora + Arthropoda), although their exact position within this group remains contested. Studies of embryonic development in tardigrades have been scarce and have yielded contradictory data. Therefore, we investigated the development of the nervous system in embryos of the tardigrade Hypsibius dujardini using immunohistochemical techniques in conjunction with confocal laser scanning microscopy in an effort to gain insight into the evolution of the nervous system in panarthropods. Results: An antiserum against acetylated α-tubulin was used to visualize the axonal processes and general neuroanatomy in whole-mount embryos of the eutardigrade H. dujardini. Our data reveal that the tardigrade nervous system develops in an anterior-to-posterior gradient, beginning with the neural structures of the head. The brain develops as a dorsal, bilaterally symmetric structure and contains a single developing central neuropil. The stomodeal nervous system develops separately and includes at least four separate, ring-like commissures. A circumbuccal nerve ring arises late in development and innervates the circumoral sensory field. The segmental trunk ganglia likewise arise from anterior to posterior and establish links with each other via individual pioneering axons. Each hemiganglion is associated with a number of peripheral nerves, including a pair of leg nerves and a branched, dorsolateral nerve. Conclusions: The revealed pattern of brain development supports a single-segmented brain in tardigrades and challenges previous assignments of homology between tardigrade brain lobes and arthropod brain segments. Likewise, the tardigrade circumbuccal nerve ring cannot be homologized with the arthropod 'circumoral' nerve ring, suggesting that this structure is unique to tardigrades. Finally, we propose that the segmental ganglia of tardigrades and arthropods are homologous and, based on these data, favor a hypothesis that supports tardigrades as the sister group of arthropods. [ABSTRACT FROM AUTHOR]

Introduction Tardigrades are an ancient lineage of microinvertebrates with a unique metameric pattern consisting of a head and four lobopodal leg-bearing segments. While their close relationship to Onychophora and Arthropoda is well established, many questions remain about the structure and origin of the tardigrade metameric pattern. For example, the relationship of the tardigrade head to that of Arthropoda and Onychophora remains a contentious issue. One source of contention stems from disagreement about the structure of the tardigrade brain. The availability of developmental tools for the tardigrade Hypsibius dujardini give this species the potential to clarify questions regarding the relationship of tardigrade segmental patterns to those in Arthropoda and Onychophora. Here we investigate the nervous system, muscle system, and cuticle anatomy of H. dujardini using high-resolution microscopy methods. Results We characterized nervous system anatomy of H. dujardini using a combination of anti-β - tubulin staining and DAPI staining and muscle system anatomy using phalloidin staining. We identified several brain lobes: paired outer lobes, paired inner lobes, and a single horseshoe shaped ventral lobe. We also characterized similarities and differences in the nervous system and muscle system anatomy of the four body segments. Based on these, we detect distinct morphological identities for each segment in this species. Conclusions Based on comparisons of our results to previous reports, we find support for an ancestral tardigrade brain exhibiting architecture similar to that of H. dujardini. Comparisons to other tardigrade species suggest that each segment in the ancestral tardigrade possessed a unique morphological identity, rather than exhibiting strictly homonomous segmentation, and thus that differentiation of anterior segment identities arose prior to the diversification of panarthropodan lineages. This hypothesis can be further tested by examination of the expression boundaries of anterior Hox genes, which differentiate anterior segments through a mechanism conserved between onychophorans and arthropods. Our investigation of H. dujardini segmental morphologies will facilitate developmental genetic studies in this species that promise to illuminate the relationship of the tardigrade metameric pattern to that of other panarthropods. [ABSTRACT FROM AUTHOR]

Screening of a deeply sequenced transcriptome using Illumina sequencing as well as the genome of the tardigrade Hypsibius dujardini revealed a set of five opsin genes. To clarify the phylogenetic position of these genes and to elucidate the evolutionary history of opsins in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we reconstructed the phylogeny of broadly sampled metazoan opsin genes using maximum likelihood and Bayesian inference methods in conjunction with carefully selected substitution models. According to our findings, the opsin repertoire of H. dujardini comprises representatives of all three major bilaterian opsin clades, including one r-opsin, three c-opsins, and a Group 4 opsin (neuropsin/opsin-5). The identification of the tardigrade ortholog of neuropsin/opsin-5 is the first record of this opsin type in a protostome, but our screening of available metazoan genomes revealed that it is also present in other protostomes. Our opsin phylogeny further suggests that two r-opsins, including an “arthropsin,” were present in the last common ancestor of Panarthropoda. Although both r-opsin lineages were retained in Onychophora and Arthropoda, the arthropsin was lost in Tardigrada. The single (most likely visual) r-opsin found in H. dujardini supports the hypothesis of monochromatic vision in the panarthropod ancestor, whereas two duplications of the ancestral panarthropod c-opsin have led to three c-opsins in tardigrades. Although the early-branching nodes are unstable within the metazoans, our findings suggest that the last common ancestor of Bilateria possessed six opsins: Two r-opsins, one c-opsin, and three Group 4 opsins, one of which (Go opsin) was lost in the ecdysozoan lineage. [ABSTRACT FROM AUTHOR]