Lower Ordovician synziphosurine reveals early euchelicerate diversity and evolution.

Euchelicerata is a clade of arthropods comprising horseshoe crabs, scorpions, spiders, mites and ticks, as well as the extinct eurypterids (sea scorpions) and chasmataspidids. The understanding of the ground plans and relationships between these crown-group euchelicerates has benefited from the discovery of numerous fossils. However, little is known regarding the origin and early evolution of the euchelicerate body plan because the relationships between their Cambrian sister taxa and synziphosurines, a group of Silurian to Carboniferous stem euchelicerates with chelicerae and an unfused opisthosoma, remain poorly understood owing to the scarce fossil record of appendages. Here we describe a synziphosurine from the Lower Ordovician (ca. 478 Ma) Fezouata Shale of Morocco. This species possesses five biramous appendages with stenopodous exopods bearing setae in the prosoma and a fully expressed first tergite in the opisthosoma illuminating the ancestral anatomy of the group. Phylogenetic analyses recover this fossil as a member of the stem euchelicerate family Offacolidae, which is characterized by biramous prosomal appendages. Moreover, it also shares anatomical features with the Cambrian euarthropod Habelia optata, filling the anatomical gap between euchelicerates and Cambrian stem taxa, while also contributing to our understanding of the evolution of euchelicerate uniramous prosomal appendages and tagmosis.

A reappraisal of Paleozoic horseshoe crabs from Russia and Ukraine.

Xiphosura are extant marine chelicerates that have displayed apparent morphological conservatism and remarkable survivorship across their ~ 480 Ma fossil record. The easily recognisable features that are known to even the earliest xiphosurans-a crescentic prosoma and often trapezoidal thoracetron (opisthosoma)-have generated debate surrounding their origins and taxonomic significance. This interest resulted in the description of numerous horseshoe crab species during the early to mid-twentieth century, particularly in Russia, that have remained unrevised since their original publications and unconsidered in the light of recent phylogenetic hypotheses. Here, we re-examine the non-belinurid taxa housed within the Chernyshev Central Museum for Geological Exploration in Saint Petersburg. We present the first formal diagnosis of Bellinuroopsis rossicus, erect Shpineviolimulus jakovlevi (Glushenko and Ivanov, 1961) comb. nov., to contain the species formerly described as 'Paleolimulus' jakovlevi and refer Paleolimulus juresanensis to Paleolimulidae incertae sedis. Phylogenetic analysis places S. jakovlevi at the base of Limulina. This position, coupled with a prosomal shield that is notably larger than the thoracetron, and lack of hypertrophied genal spines, suggests that this morphology may represent the ancestral austrolimulid shape. As an extension of this revision, we assessed the general austrolimulid morphological characters and uncovered two possible groups of these bizarre xiphosurids.

The draft genome of horseshoe crab Tachypleus tridentatus reveals its evolutionary scenario and well-developed innate immunity.

BACKGROUND: Horseshoe crabs are ancient marine arthropods with a long evolutionary history extending back approximately 450 million years, which may benefit from their innate immune systems. However, the genetic mechanisms underlying their abilities of distinguishing and defending against invading microbes are still unclear. RESULTS: Here, we describe the 2.06 Gbp genome assembly of Tachypleus tridentatus with 24,222 predicted protein-coding genes. Comparative genomics shows that T. tridentatus and the Atlantic horseshoe crab Limulus polyphemus have the most orthologues shared among two species, including genes involved in the immune-related JAK-STAT signalling pathway. Divergence time dating results show that the last common ancestor of Asian horseshoe crabs (including T. tridentatus and C. rotundicauda) and L. polyphemus appeared approximately 130 Mya (121-141), and the split of the two Asian horseshoe crabs was dated to approximately 63 Mya (57-69). Hox gene analysis suggests two clusters in both horseshoe crab assemblies. Surprisingly, selective analysis of immune-related gene families revealed the high expansion of conserved pattern recognition receptors. Genes involved in the IMD and JAK-STAT signal transduction pathways also exhibited a certain degree of expansion in both genomes. Intact coagulation cascade-related genes were present in the T. tridentatus genome with a higher number of coagulation factor genes. Moreover, most reported antibacterial peptides have been identified in T. tridentatus with their potentially effective antimicrobial sites. CONCLUSIONS: The draft genome of T. tridentatus would provide important evidence for further clarifying the taxonomy and evolutionary relationship of Chelicerata. The expansion of conserved immune signalling pathway genes, coagulation factors and intact antimicrobial peptides in T. tridentatus constitutes its robust and effective innate immunity for self-defence in marine environments with an enormous number of invading pathogens and may affect the quality of the adaptive properties with regard to complicated marine environments.

Untangling the Gordian knot-further resolving the super-species complex of 300-million-year-old xiphosurids by reconstructing their ontogeny.

The group Xiphosurida (horseshoe "crabs") is today only represented by four species. However, in the fossil record, several dozen species have been described, especially from the Carboniferous (about 300 million years ago). Several species have been interpreted as representatives of Euproops or Belinurus, but there is ongoing discussion which of these species are valid and how they can be differentiated. Recent studies suggested that differences in the timing of individual development could provide information for species distinction, exemplified by studies on Euproops danae (Mazon Creek, USA) and Euproops sp. ("Piesproops"; Piesberg, Germany). For this study, we reinvestigated all Carboniferous xiphosurids from the British Coal Measures stored in the collections of the Natural History Museum London. Size comparisons of the specimens revealed nine size groups; the smaller specimens were originally labelled as Belinurus, the larger ones as Euproops. The nine size groups exhibit five different morphotypes differing in structures surrounding the posterior shield (= thoracetron): spines of different lengths and, in larger specimens, a more or less developed flange. Two of these morphotypes show significantly longer spines than the remaining specimens and could be conspecific as E. anthrax. The remaining specimens are interpreted as growth series of another species, presumably of E. rotundatus. An ontogenetic flange formation is also known from E. danae and the "Piesproops", but the timing differs between all three species. In E. rotundatus, the flange develops rather late, but then comparably abruptly, which makes this development more metamorphic in relation to development in the other species.

Xiphosurid from the Tournaisian (Carboniferous) of Scotland confirms deep origin of Limuloidea.

Horseshoe crabs are archetypal marine chelicerates with a fossil record extending from the Lower Ordovician to today. The major horseshoe crab groups are thought to have arisen in the middle to late Palaeozoic. Here we present the oldest known limuloid from the lower Carboniferous (Tournaisian stage, c. 350 million years ago) of Scotland: Albalimulus bottoni gen. et sp. nov. A comprehensive phylogenetic analysis supports the placement of A. bottoni as a representative of the extant family Limulidae and 100 million years older than any other limulid taxon. The use of geometric morphometric analyses corroborate the erection of the new taxon and illustrates the exploitation of morphospace by xiphosurids. This new taxon highlights the complex evolutionary history of xiphosurids and the importance of documenting these unique Palaeozoic individuals.

On the appendicular anatomy of the xiphosurid Tachypleus syriacus and the evolution of fossil horseshoe crab appendages.

Xiphosurida-crown group horseshoe crabs-are a group of morphologically conservative marine chelicerates (at least since the Jurassic). They represent an idealised example of evolutionary stasis. Unfortunately, body fossils of horseshoe crabs seldom preserve appendages and their associated features; thus, an important aspect of their morphology is absent in explorations of their conservative Bauplan. As such, fossil horseshoe crab appendages are rarely considered within a comparative framework: previous comparisons have focussed almost exclusively on extant taxa to the exclusion of extinct taxa. Here, we examine eight specimens of the xiphosurid Tachypleus syriacus (Woodward, 1879) from the Cenomanian (ca 100 Ma) Konservat-Lagerstätten of Lebanon, five of which preserve the cephalothoracic and thoracetronic appendages in exceptional detail. Comparing these appendages of T. syriacus with other fossil xiphosurids highlights the conserved nature of appendage construction across Xiphosurida, including examples of Austrolimulidae, Paleolimulidae, and Limulidae. Conversely, Belinuridae have more elongate cephalothoracic appendages relative to body length. Differences in appendage sizes are likely related to the freshwater and possible subaerial life modes of belinurids, contrasting with the primarily marine habits of other families. The morphological similarity of T. syriacus to extant members of the genus indicates that the conserved nature of the generic lineage can be extended to ecological adaptations, notably burrowing, swimming, possible diet, and sexual dimorphism.

Chromosomal level reference genome of Tachypleus tridentatus provides insights into evolution and adaptation of horseshoe crabs.

Horseshoe crabs including Tachypleus tridentatus are a group of marine arthropods and living fossil species which have existed on the earth for ~500 million years. However, the genetic mechanisms underlying their unique adaptive ability are still unclear. Here, we assembled the first chromosome-level T. tridentatus genome, and proved that this genome is of high quality with contig N50 1.69 Mb. By comparison with other arthropods, some gene families of T. tridentatus experienced significant expansion, which are related to several signaling pathways, endonuclease activities, and metabolic processes. Based on the results of comparative analysis of genomics and 27 transcriptomes from nine tissues, we found that the expanded Dscam genes are located at key hub positions of immune network. Furthermore, the Dscam genes showed higher levels of expression in the yellow connective tissue (the birthplace of blood cells with strong differentiation capability) than the other eight tissues. Besides, the Dscam genes are positively correlated with the expression of the core immunity gene, clotting factor B, which is involved in the coagulation cascade reaction. The effective and unusual immune ability endowed by the expansion and expression of Dscam genes in the horseshoe crab may be a factor that makes the species have a strong environmental adaptability within ~500 million years. The high quality chromosome level genome of a horseshoe crab and unique genomic features reported in this study provide important data resources for future studies on the evolution of marine ecological systems.

Opsins and Their Expression Patterns in the Xiphosuran Limulus polyphemus.

The American horseshoe crab Limulus polyphemus (Linnaeus, 1758) is one of four extant species of xiphosuran chelicerates, the sister group to arachnids. Because of their position in the arthropod family tree and because they exhibit many plesiomorphic characteristics, Xiphosura are considered a proxy for the euchelicerate ancestor and therefore important for understanding the evolution and diversification of chelicerates and arthropods. Limulus polyphemus is the most extensively studied xiphosuran, and its visual system has long been a focus of studies critical for our understanding of basic mechanisms of vision and the evolution of visual systems in arthropods. Building upon a wealth of information about the anatomy and physiology of its visual system, advances in genetic approaches have greatly expanded possibilities for understanding its biochemistry. This review focuses on studies of opsin expression in L. polyphemus, which have been significantly advanced by the availability of transcriptomes and a recent high-quality assembly of its genome. These studies show that the repertoire of expressed opsins in L. polyphemus is far larger than anticipated, that the regulation of their expression in rhabdoms is far more complex than anticipated, and that photosensitivity may be distributed widely throughout the L. polyphemus central nervous system. The visual system of L. polyphemus is now arguably the best understood among chelicerates, and as such, it is a critical resource for furthering our understanding of the evolution and diversification of visual systems in arthropods.

An "ancient" complexity? Evolutionary morphology of the circulatory system in Xiphosura.

Horseshoe crabs (Xiphosura) have been an object of zoological research for almost 200 years. Although some morphological work on the circulatory system has been done, the three-dimensional structure of this complex organ system has never been shown satisfactorily and some crucial questions remain unanswered. Here, the circulatory systems of juveniles of the horseshoe crab taxa Limulus polyphemus and Carcinoscorpius rotundicauda were investigated using a combination of an injection method and micro-computed tomography. Data were processed and 3D-visualized using reconstruction software. Furthermore, the heart was examined using scanning electron microscopy. Additionally, the histology of some structures was investigated via light microscopy and transmission electron microscopy. The results show the high degree of complexity of the arterial and lacunar systems of Xiphosura and provide insights into their three-dimensional structure and relationship to other organ systems such as the central nervous system. We show that the major lacunae, previously described as vessel-like - though indeed highly ramified - can clearly be distinguished from arteries in histological sections because they have no distinct walls. Similarities and differences between the xiphosuran species and arachnids are highlighted and possible phylogenetic implications and evolutionary scenarios discussed.

The horseshoe crab of the genus Limulus: living fossil or stabilomorph?

A new horseshoe crab species, Limulus darwini, is described from the uppermost Jurassic (ca. 148 Ma) near-shore sediments of the Kcynia Formation, central Poland. The only extant species Limulus polyphemus (Linnaeus) inhabits brackish-marine, shallow water environments of the east coast of the United States. Here it is shown that there are no important morphological differences between the Kcynia Formation specimens and extant juvenile representatives of the genus Limulus. The palaeoecological setting inhabited by the new species and the trophic relationships of extant horseshoe crabs are discussed in an attempt to determine the potential range of food items ingested by these Mesozoic xiphosurans. In this paper we propose the adoption of a new term stabilomorphism, this being: an effect of a specific formula of adaptative strategy among organisms whose taxonomic status does not exceed genus-level. A high effectiveness of adaptation significantly reduces the need for differentiated phenotypic variants in response to environmental changes and provides for long-term evolutionary success.

A holomorph approach to xiphosuran evolution--a case study on the ontogeny of Euproops.

Specimens of Euproops sp. (Xiphosura, Chelicerata) from the Carboniferous Piesberg quarry near Osnabrück, Germany, represent a relatively complete growth series of 10 stages. Based on this growth sequence, morphological changes throughout the ontogeny can be identified. The major change affects the shape of the epimera of the opisthosoma. In earlier stages, they appear very spine-like, whereas in later stages the bases of these spine-like structures become broader; the broadened bases are then successively drawn out distally. In the most mature stage known, the epimera are of trapezoidal shape and approach each other closely to form a complete flange around the thoracetron (=fused tergites of the opisthosoma). These ontogenetic changes question the taxonomic status of different species of Euproops, as the latter appear to correspond to different stages of the ontogenetic series reconstructed from the Piesberg specimens. This means that supposed separate species could, in fact, represent different growth stages of a single species. It could alternatively indicate that heterochrony (=evolutionary change of developmental timing) plays an important role in the evolution of Xiphosura. We propose a holomorph approach, i.e., reconstructing ontogenetic sequences for fossil and extant species as a sound basis for a taxonomic, phylogenetic, and evolutionary discussion of Xiphosura.

Molecular phylogeny of extant horseshoe crabs (Xiphosura, Limulidae) indicates Paleogene diversification of Asian species.

Horseshoe crabs are marine invertebrates well known for their exceptionally low rates of diversification during their entire evolutionary history. Despite the low species diversity in the group, the phylogenetic relationships among the extant species, especially among the three Asian species are still unresolved. Here we apply a new set of molecular genetic data in combination with a wide geographic sampling of the intra-specific diversity to reinvestigate the evolutionary history among the four living limulid xiphosurans. Our analysis of the intraspecific diversity reveals low levels of connectivity among Carcinoscorpius rotundicauda lineages, which can be explained by the estuarine-bound ecology of this species. Moreover, a clear genetic break across the Thai-Malay Peninsula suggests the presence of cryptic species in C. rotundicauda. The limulid phylogeny finds strong support for a monophyletic genus Tachypleus and a diversification of the three Asian species during the Paleogene period, with speciation events well separated in time by several million years. The tree topology suggests that the three Asian species originated in central South East Asia from a marine stem group that inhabited the shallow coastal waters between the Andaman Sea, Vietnam, and Borneo. In this region C. rotundicauda probably separated from the Tachypleus stem group by invading estuarine habitats, while Tachypleus tridentatus most likely migrated northeast along the Southern coast of China and towards Japan.

Arginine kinase from Nautilus pompilius, a living fossil. Site-directed mutagenesis studies on the role of amino acid residues in the Guanidino specificity region.

Arginine kinases were isolated from the cephalopods Nautilus pompilius, Octopus vulgaris, and Sepioteuthis lessoniana, and the cDNA-derived amino acid sequences have been determined. Although the origin and evolution of cephalopods have long been obscure, this work provides the first molecular evidence for the phylogenetic position of Cephalopoda in molluscan evolution. A crystal structure for Limulus arginine kinase showed that four amino acid residues (Ser(63), Gly(64), Val(65), and Tyr(68)) are hydrogen-bonded with the substrate arginine. We introduced three independent mutations, Ser(63) --> Gly, Ser(63) --> Thr, and Tyr(68) --> Ser, in Nautilus arginine kinase. One of the mutants had a considerably reduced substrate affinity, accompanied by a decreased V(max). In other mutants, the activity was lost almost completely. It is known that substantial conformational changes take place upon substrate binding in arginine kinase. We hypothesize that the hydrogen bond between Asp(62) and Arg(193) stabilizes the closed, substrate-bound state. Site-directed mutagenesis studies strongly support this hypothesis. The mutant (Asp(62) --> Gly or Arg(193) --> Gly), which destabilizes the maintenance of the closed state and/or perhaps disrupts the unique topology of the catalytic pocket, showed only a very weak activity (0.6-1.5% to the wild-type).

Phylogenetic relationship among horseshoe crab species: effect of substitution models on phylogenetic analyses.

The horseshoe crabs, known as living fossils, have maintained their morphology almost unchanged for the past 150 million years. The little morphological differentiation among horseshoe crab lineages has resulted in substantial controversy concerning the phylogenetic relationship among the extant species of horseshoe crabs, especially among the three species in the Indo-Pacific region. Previous studies suggest that the three species constitute a phylogenetically unresolvable trichotomy, the result of a cladogenetic process leading to the formation of all three Indo-Pacific species in a short geological time. Data from two mitochondrial genes (for 16S ribosomal rRNA and cytochrome oxidase subunit I) and one nuclear gene (for coagulogen) in the four species of horseshoe crabs and outgroup species were used in a phylogenetic analysis with various substitution models. All three genes yield the same tree topology, with Tachypleus-gigas and Carcinoscorpius-rotundicauda grouped together as a monophyletic taxon. This topology is significantly better than all the alternatives when evaluated with the RELL (resampling estimated log-likelihood) method.

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes.

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.