Morphological phylogenetics of the sea spiders (Arthropoda: Pycnogonida)

Pycnogonids or sea spiders are a group of marine arthropods whose relations to the chelicerates have been an issue of controversy. Higher-level phylogenetic relationships among the lineages of sea spiders are investigated using 36 morphological characters from 37 species from all extant families and a Devonian pycnogonid fossil. This is one of the first attempts to analyze the higher-level relationships of the Pycnogonida using cladistic techniques. Character homoplasy (implied weights) is taken into account to construct a polytomous, most-parsimonious tree in which two major clades within Pycnogonida are obtained. Clade A includes Ammotheidae paraphyletic with Colossendeidae, Austrodecidae and Rhynchothoracidae, and clade B is formed by Nymphonidae, Callipallenidae (apparently paraphyletic), Pycnogonidae and Phoxichilidiidae. The analysis of equally weighted data is presented and helps to identify those characters less consistent. The reduction of the chelifores, palps and ovigers — shown independently within each of the clades as parallel evolution events — challenges the assumption of a gradual mode of reduction within the group, according to analysis of unordered vs ordered characters. Most of the phylogenetic affinities proposed here are compatible with traditional classifications. However, traditional taxonomic characters need to be complemented by sets of anatomical, molecular and developmental data, among others, to produce more robust phylogenetic hypotheses on the higher- and lower-level relationships of the sea spiders.     

Hox genes in sea spiders (Pycnogonida) and the homology of arthropod head segments

The pycnogonids (or sea spiders) are an enigmatic group of arthropods, classified in recent phylogenies as a sister-group of either euchelicerates (horseshoe crabs and arachnids), or all other extant arthropods. Because of their bizarre morpho-anatomy, homologies with other arthropod taxa have been difficult to assess. We review the main morphology-based hypotheses of correspondence between anterior segments of pycnogonids, arachnids and mandibulates. In an attempt to provide new relevant data to these controversial issues, we performed a PCR survey of Hox genes in two pycnogonid species, Endeis spinosa and Nymphon gracile, from which we could recover nine and six Hox genes, respectively. Phylogenetic analyses allowed to identify their orthology relationships. The Deformed gene from E. spinosa and the abdominal-A gene from N. gracile exhibit unusual sequence divergence in their homeodomains, which, in the latter case, may be correlated with the extreme reduction of the posterior region in pycnogonids. Expression patterns of two Hox genes (labial and Deformed) in the E. spinosa protonymphon larva are discussed. The anterior boundaries of their expression domains favour homology between sea spider chelifores, euchelicerates chelicerae and mandibulate (first) antennae, in contradistinction with previously proposed alternative schemes such as the protocerebral identity of sea spider chelifores or the absence of a deutocerebrum in chelicerates. In addition, while anatomical and embryological evidences suggest the possibility that the ovigers of sea spiders could be a duplicated pair of pedipalps, the Hox data support them as modified anterior walking legs, consistent with the classical views.Supplementary material is available for this article at and is accessible for authorized users.Guest editors Jean Deutsch and Gerhard Scholtz     

The chelifores of sea spiders (Arthropoda,Pycnogonida) are the appendages of the deutocerebral segment

SUMMARY Within the last decade, gene expression patterns and neuro‐anatomical data have led to a new consensus concerning the long‐debated association between anterior limbs and neuromeres in the arthropod head. According to this new view, the first appendage in all extant euarthropods is innervated by the second neuromere, the deutocerebrum, whereas the anterior‐most head region bearing the protocerebrum lacks an appendage. This stands in contrast to the clearly protocerebrally targeted “antennae” of Onychophora and to some evidence for protocerebral limbs in fossil euarthropod representatives. Yet, the latter “frontal appendages” or “primary antennae” have most likely been reduced or lost in the lineage, leading to extant taxa. Surprisingly, a recent neuro‐anatomical study on a pycnogonid challenged this evolutionary scenario, reporting a protocerebral innervation of the first appendages, the chelifores. However, this interpretation was soon after questioned by Hox gene expression data. To re‐evaluate the unresolved controversy, we analyzed neuro‐anatomy and neurogenesis in four pycnogonid species using immunohistochemical techniques. We clearly show the postprotocerebral innervation of the chelifores, which is resolved as the plesiomorphic condition in pycnogonids when evaluated against a recently published comprehensive phylogeny. By providing direct morphological support for the deutocerebral status of the cheliforal ganglia, we reconcile morphological and gene expression data and argue for a corresponding position between the anterior‐most appendages in all extant euarthropods. Consequently, other structures have to be scrutinized to illuminate the fate of a presumptive protocerebral appendage in recent euarthropods. The labrum and the “frontal filaments” of some crustaceans are possible candidates for this approach.     

Synopsis of a new collection of sea spiders (Arthropoda: Pycnogonida) from the Ross Sea,Antarctica

The biodiversity research expedition TAN0204 with RS Tangaroa to the Ross Sea in 2004 yielded a new collection of 2,687 specimens of pycnogonids. As much as 25 different species encompassing 14 genera and eight families were identified and their records are discussed herein. The collection is archived in the Marine Invertebrate Collection of the New Zealand National Institute of Water and Atmospheric Research (NIWA). The majority (69%) of specimens are from the Nymphon australe group (Nymphonidae), although species richness and abundance varied among the stations sampled. The collection includes several specimens from polymerous taxa; Pentanymphon antarcticum (Nymphonidae), Decolopoda australis (Colossendeidae) and Pentapycnon bouvieri (Pycnogonidae). All species were classified based on morphological characters, and DNA sequences (from the 18S, 12S, 16S and COI regions) for 21 of the representative morphotypes are given. The DNA sequences confirmed the species-level distinctiveness of these morphotypes. No species new to science were identified, although further detailed morphometric and/or molecular analyses may reveal cryptic or sibling species, especially in species such as the highly abundant Nymphon australe group. An erratum to this article can be found at     

Phylogeny of the sea spiders (Arthropoda, Pycnogonida) based on direct optimization of six loci and morphology

Higher‐level phylogenetics of Pycnogonida has been discussed for many decades but scarcely studied from a cladistic perspective. Traditional taxonomic classifications are yet to be tested and affinities among families and genera are not well understood. Pycnogonida includes more than 1300 species described, but no systematic revisions at any level are available. Previous attempts to propose a phylogeny of the sea spiders were limited in characters and taxon sampling, therefore not allowing a robust test of relationships among lineages. Herein, we present the first comprehensive phylogenetic analysis of the Pycnogonida based on a total evidence approach and Direct Optimization. Sixty‐three pycnogonid species representing all families including fossil taxa were included. For most of the extant taxa more than 6 kb of nuclear and mitochondrial DNA and 78 morphological characters were scored. The most parsimonious hypotheses obtained in equally weighted total evidence analyses show the two most diverse families Ammotheidae and Callipallenidae to be non‐monophyletic. Austrodecidae + Colossendeidae + Pycnogonidae are in the basal most clade, these are morphologically diverse groups of species mostly found in cold waters. The raising of the family Pallenopsidae is supported, while Eurycyde and Ascorhynchus are definitely separated from Ammotheidae. The four fossil taxa are grouped within living Pycnogonida, instead of being an early derived clade. This phylogeny represents a solid framework to work towards the understanding of pycnogonid systematics, providing a data set and a testable hypothesis that indicate those clades that need severe testing, especially some of the deep nodes of the pycnogonid tree and the relationships of ammotheid and callipallenid forms. The inclusion of more rare taxa and additional sources of evidence are necessary for a phylogenetic classification of the Pycnogonida. © The Willi Hennig Society 2006.     

Molecular approach to the phylogenetics of sea spiders (Arthropoda: Pycnogonida) using partial sequences of nuclear ribosomal DNA

The phylogenetic relationships among major evolutionary lineages of the sea spiders (subphylum Pycnogonida) were investigated using partial sequences of nuclear DNA, 18S, and 28S ribosomal genes. Topological differences were obtained with separate analyses of 18S and 28S, and estimates of phylogeny were found to be significantly different between a combined molecular data set (18S and 28S) and a subset of a morphological data matrix analyzed elsewhere. Colossendeidae played a major role in the conflicts; it was closely related to Callipallenidae or Nymphonidae with 18S or 28S, respectively, but related to Ammotheidae according to morphological characters. Austrodecidae was defined as a basal taxon for Pycnogonida by these molecular data. The 18S sequences were surprisingly conserved among pycnogonid taxa, suggesting either an unusual case of slow evolution of the gene, or an unexpected recent divergence of pycnogonid lineages. Notwithstanding difficulties such as non-optimal taxon sampling, this is the first attempt to reconstruct the pycnogonid phylogeny based on DNA. Continued studies of sequences and other characters should increase the reliability of the analyses and our understanding of the phylogenetics of sea spiders.     

Studying sources of incongruence in arthropod molecular phylogenies: Sea spiders (Pycnogonida) as a case study

In this report, we analyze the phylogeny of Pycnogonida using the three nuclear and three mitochondrial markers currently sequenced for studying inter- and intrafamilial relationships within Arthropoda: 18S and 28S rRNA genes, Histone H3, cytochrome c oxidase subunit 1 (CO1), 12S and 16S rRNA genes. We identify several problems in previous studies, due to the use of inappropriate sequences (taxonomic misidentification, DNA contamination, sequencing errors, missing data) or taxa (outgroup choice). Our analyses show that most markers are not powerful to study the phylogeny of sea spiders. The results suggest however a recent diversification of the group (Mesozoic rather than Paleozoic) and the early divergence of Austrodecidae, followed by Colossendeidae, Pycnogonidae and Rhynchothoracidae. Except Ammotheidae and Callipallenidae, all other families were recovered as monophyletic. Analyses of synonymous sites in CO1 sequences reveal an extreme heterogeneity of nucleotide composition within sea spiders, as six unrelated species show a reverse strand-specific bias. We therefore suggest that several independent reversals of asymmetric mutational constraints occurred during the evolution of Pycnogonida, as a consequence of genomic inversions involving either the control region or a fragment containing the CO1 gene. These hypotheses are supported by the comparison of two complete mitochondrial genomes of sea spiders (Achelia bituberculata and Nymphon gracile) with that of Limulus.     

The coincident time-space patterns of septate junction development in normal and exogastrulated sea urchin embryos

Earlier studies have shown that two types of septate junction are formed during early sea urchin morphogenesis. One type is the straight, unbranched, double septum septate (SUDS) which is found in the ectodermal layer throughout early development. The second type is formed only in cells which invaginate to become endoderm and to form the digestive tract. This junction is characterized by pleated, anastomosing, single septum septates (PASS). In order to ascertain in which parts of the digestive tract these junctions are formed, we studied exogastrulae because the endoderm is everted and forms constricted areas of the gut which are easily recognizable. Our results show that, in control embryos, SUDS septates are found in the mouth, esophagus and coelom and that PASS septates are found in the stomach, intestine and anus. These junctional types are also found in the same areas in exogastrulae; SUDS septates are found in the stomadeum, esophagus and coelom, and PASS septates are found in the stomach and intestine. The transition from SUDS to PASS junctions takes place within the same time period in exogastrulae as in normal embryos, i.e., from the time of mid-gastrulation through the pluteus stage. These results indicate that septate junction formation in the sea urchin embryo digestive tract may be genetically programmed in terms of both time and spatial location. This program is not altered either by the major dislocation of cells from their normal position within the embryo or from normal contacts with neighboring cells.     

The complete mitochondrial genome of the Antarctic sea spider Ammothea carolinensis (Chelicerata; Pycnogonida)

Mitochondria are responsible for the oxidative phosphorylation process. Accordingly, putatively adaptive changes in their genomic features have been variously associated with major eco-physiological shifts in animal evolution, including increased metabolic rates and heat adaptation. Antarctic pycnogonids offer an interesting system to test whether the selective pressure for heat production and increased aerobic metabolism may be driving genomic changes like: (a) unusual compositional biases at the nucleotide and amino acid level, possibly related to cold adaptation; (b) an accelerated rate of mutations/genomic rearrangements, possibly related to the mutagenic effects of oxygen intermediates. The complete mitochondrial genome (mtDNA) of the Antarctic sea spider Ammothea carolinensis Leach, 1814 (Arthropoda: Pycnogonida), the type species for the genus Ammothea, has been determined and is here compared to known genomes from Antarctic and temperate species. We describe a marked heterogeneity in base composition skewness parameters as well as a strong signature of purifying selection toward an increase in thymines at second codon positions, possibly associated with an increased stability of hydrophobic inter-membrane domains. We further observe a fairly high rate of genomic changes, including a possible hot spot of recombination at the level of tRNA-Q. Nevertheless, these features do not seem to be restricted to the two Antarctic pycnogonids analyzed, as to suggest a causal relationship between cold adaptation and genomic changes, and are better interpreted as basal features shared by the entire group. The relevance of the newly determined sequence for the phylogeny of pycnogonids, including its base composition and genomic rearrangements, is further discussed.     

A clarification of the two types of invertebrate pleated septate junction

It is confirmed that there are two distinct variations of invertebrate septate junction. The first of these, the ‘lower invertebrate pleated septate junction’, is described fully using conventional thin section, lanthanum tracer and freeze-fracture techniques. The second type, the well-known pleated septate junction characteristic of the molluscs and athropods, is renamed the ‘mollusc-arthropod pleated septate junction’, and is described briefly to allow easier comparison between the two variations. As both types have now been studied in a range of invertebrate phyla the results can be used as a basis for discussing their respective phylogenetic positions. The lower invertebrate pleated septate junction occurs in several groups in the minor phyla immediately above the Coelenterata and in the lower phyla of both the deuterostome and proterostome lineages. The mollusc-arthropod pleated septate junction is restricted to the Mollusca and Arthropoda as its name implies.     

Formation of the septate junction in regenerating planarian gastrodermis

Septate junctions develop initially just basad from apical junctional complexes at the apical ends of regenerating gastrodermal cells. The first morphological indication of differentiation of the junction is the appearance of gentle undulations of the plasma membranes of apposing cells. Subsequently dense dots develop at fairly regular intervals at the cytoplasmic surface of one cell, while SER cisternae become localized opposite them near the surface of the apposing cell. The dense dots are associated with bulges which narrow the intercellular space. Later the dense dots are replaced by filaments aligned along the inner leaflet of the parent cell. Strands of amorphous deposits form connections between SER cisternae and the sister membrane on the opposite side of the junction. Ruthenium red staining provides information on precursors which occupy the intercellular space between the apposed plasma membranes. As development of the junction progresses, ruthenium red stains only the newly formed septa but not the interseptal matrix. Regular arrangement of individual septa seems to be completed under the control of V-projections from both of their surfaces. Precursors for the structural material of the septa may be a secretory product derived from the SER. Dense dots and their derived filaments probably serve as reinforcing material for strengthening the cell membrane of the junction.     

Freeze fracture studies on the annelid septate junction

Summary Freeze-fractured preparations of septate junctions between epidermal cells of annelids (Lumbricus terrestris and Tubifex spec.) have been investigated. In Lumbricus the protoplasmic face (PF) of the plasma membrane is characterized by variously arranged rows of particles. Apically the rows take an undulating course and often are separated by wide distances. In the basal part of the junction the rows run closely together and more or less in parallel. The diameter of the particles measures 80–120 Å, the distance between two particles (centre to centre) is 150–250 Å. Additionally striking rows of large particles (long diameter 150–200 Å). Are to be observed mainly near the basal part of the junction. In Tubifex both faces of the plasma membrane could be studied in detail. The protoplasmic face (PF) contains rows of distinct individual particles (mean diameter 100–150 Å, centre to centre distance approx. 250 Å) whereas the particles of the extracellular face (EF, mean diameter 200–250 Å) usually form continuous strands in which the individual particles seem to fuse. The density of arrangement of the strands varies considerably. Additionally ladder-shaped membrane structures have been observed in plasma membranes of this species.     

Predatory behavior of giant Antarctic sea spiders (Colossendeis) in nearshore environments

Pycnogonids in the genus Colossendeis are found in the deep sea and Southern Ocean. Although the genus contains the largest and most conspicuous species of sea spiders, little is known about their ecology or behavior. We documented two species feeding on a variety of benthic and pelagic invertebrates during three diving field seasons at McMurdo Station, Antarctica. Individuals of one species, Colossendeis megalonyx, fed on a variety of pelagic organisms, particularly the pteropod Clione antarctica. We used video to document rapid capture of individuals of C. antarctica by captive specimens of C. megalonyx in the laboratory, and we suggest that, at least in the nearshore environment, pelagic invertebrates are an important food source for this and potentially other pycnogonid species.     

The septate junction limits mobility of lipophilic markers in plasma membranes ofHydra vulgaris (attenuata)

Summary Fluorescent lipophilic probes were used to study the role of septate junctions in maintaining distinct apical and basolateral domains of plasma membranes in epithelial cells of hydra. In short-term experiments, a 16-carbon chain aminofluorescein probe (AFC₁₆) was localized to the apical plasma membranes of ectodermal and endodermal epithelial cells when presented in the culture medium or injected into the gastric lumen, but did not demarcate basolateral membranes. In longer term experiments, basolateral membranes were stained and the staining was independent of temperature conditions. A dual 18-carbon chain indocarbocyanine probe (DiIC₁₈) gradually diffused across the septate junction to label basolateral membranes at room temperature, but not at 4°C. DiIC₁₈ also filled and stained certain mounted nematocytes. The results indicate that in hydra, lipophilic probes may be limited in mobility within the membrane plane by the septate junctions in a manner similar to vertebrate tight junctions, and that apical membranes of mature nematocytes are differentially permeable.     

A new species of Endeis (Pycnogonida) from West Africa

A new species of pycnogonid, Endeis picta sp. nov. , is described from the coast of Ghana, occuring in a Balanus tintinnabulum fouling community on a ship's hull. The species is close to E. straughani Clark.     

Role of the septate junction in the regulation of paracellular transepithelial flow

A comparison of the distribution of septate junctions in invertebrate epithelia and tight junctions in vertebrate systems suggests that these structures may be functionally analogous. This proposition is supported by the internal design of each junction which constitutes a serial arrangement of structures crossing the intercellular space between cells to effectively provide resistance to the paracellular flow of water and small molecules. We have tested the validity of such an analogy by examining whether the osmotic sensitivity of the septate junctions of planarian epidermis follow the rather striking pattern observed for the junctions of very tight vertebrate epithelia (e.g. toad urinary bladder). It has been found that the septate junctions in this system respond in similar fashion to their vertebrate counterparts, blistering with accumulated fluid when the medium outside the epidermis is made hypertonic with small, water-soluble molecules. We conclude that the two types of junction probably are functionally analogous and that, in each case, this rectified structural response to transepithelial osmotic gradients may be indicative of the role of such structures in the transport function of epithelia.     

Evidence for intercellular cohesion in the septate junction of the protozoon Gregarina

Injection of Lucifer Yellow dye was used to establish whether septate junctions formed a permeability barrier between primite and satellite gamonts in the syzygy of the protozoon Gregarina. The fluorescent dye did not pass from one cell to the other, thus suggesting that the septate junction served only for mechanical adhesion. Later on in development, when the gametocyst had formed, the septate junction vanished and interruptions were observed between opposing cell membranes. At this stage the fluorescent dye was able to pass freely into the conjugated cells.