Epimorphic development in tropical shallow-water Nymphonidae (Arthropoda: Pycnogonida) revealed by fluorescence imaging.

BACKGROUND: Extant lineages of sea spiders (Pycnogonida) exhibit different types of development. Most commonly, pycnogonids hatch as a minute, feeding protonymphon larva with subsequent anamorphic development. However, especially in cold water habitats at higher latitudes and in the deep sea, some taxa have large, lecithotrophic larvae, or even undergo extended embryonic development with significantly advanced postlarval hatching stages. Similar biogeographic trends are observed in other marine invertebrates, often referred to as "Thorson's rule". RESULTS: To expand our knowledge on the developmental diversity in the most speciose pycnogonid genus Nymphon, we studied the developmental stages of the two tropical representatives N. floridanum and N. micronesicum., We compared classical scanning electron microscopy with fluorescence-based approaches to determine which imaging strategy is better suited for the ethanol-fixed material available. Both species show epimorphic development and hatch as an advanced, lecithotrophic postlarval instar possessing the anlagen of all body segments. Leg pairs 1-3 show a considerable degree of differentiation at hatching, but their proximal regions remain coiled and hidden under the cuticle of the hatching instar. The adult palp and oviger are not anteceded by three-articled larval limbs, but differentiate directly from non-articulated limb buds during postembryonic development. CONCLUSIONS: Fluorescence imaging yielded more reliable morphological data than classical scanning electron microscopy, being the method of choice for maximal information gain from rare and fragile sea spider samples fixed in high-percentage ethanol. The discovery of epimorphic development with lecithotrophic postlarval instars in two small Nymphon species from tropical shallow-water habitats challenges the notion that this developmental pathway represents an exclusive cold-water adaptation in Nymphonidae. Instead, close phylogenetic affinities to the likewise more direct-developing Callipallenidae hint at a common evolutionary origin of this trait in the clade Nymphonoidea (Callipallenidae + Nymphonidae). The lack of functional palpal and ovigeral larval limbs in callipallenids and postlarval hatchers among nymphonids may be a derived character of Nymphonoidea. To further test this hypothesis, a stable and well-resolved phylogenetic backbone for Nymphonoidea is key.

Comprehensive Species Sampling and Sophisticated Algorithmic Approaches Refute the Monophyly of Arachnida.

Deciphering the evolutionary relationships of Chelicerata (arachnids, horseshoe crabs, and allied taxa) has proven notoriously difficult, due to their ancient rapid radiation and the incidence of elevated evolutionary rates in several lineages. Although conflicting hypotheses prevail in morphological and molecular data sets alike, the monophyly of Arachnida is nearly universally accepted, despite historical lack of support in molecular data sets. Some phylotranscriptomic analyses have recovered arachnid monophyly, but these did not sample all living orders, whereas analyses including all orders have failed to recover Arachnida. To understand this conflict, we assembled a data set of 506 high-quality genomes and transcriptomes, sampling all living orders of Chelicerata with high occupancy and rigorous approaches to orthology inference. Our analyses consistently recovered the nested placement of horseshoe crabs within a paraphyletic Arachnida. This result was insensitive to variation in evolutionary rates of genes, complexity of the substitution models, and alternative algorithmic approaches to species tree inference. Investigation of sources of systematic bias showed that genes and sites that recover arachnid monophyly are enriched in noise and exhibit low information content. To test the impact of morphological data, we generated a 514-taxon morphological data matrix of extant and fossil Chelicerata, analyzed in tandem with the molecular matrix. Combined analyses recovered the clade Merostomata (the marine orders Xiphosura, Eurypterida, and Chasmataspidida), but merostomates appeared nested within Arachnida. Our results suggest that morphological convergence resulting from adaptations to life in terrestrial habitats has driven the historical perception of arachnid monophyly, paralleling the history of numerous other invertebrate terrestrial groups.

Phylogenomic Resolution of Sea Spider Diversification through Integration of Multiple Data Classes.

Despite significant advances in invertebrate phylogenomics over the past decade, the higher-level phylogeny of Pycnogonida (sea spiders) remains elusive. Due to the inaccessibility of some small-bodied lineages, few phylogenetic studies have sampled all sea spider families. Previous efforts based on a handful of genes have yielded unstable tree topologies. Here, we inferred the relationships of 89 sea spider species using targeted capture of the mitochondrial genome, 56 conserved exons, 101 ultraconserved elements, and 3 nuclear ribosomal genes. We inferred molecular divergence times by integrating morphological data for fossil species to calibrate 15 nodes in the arthropod tree of life. This integration of data classes resolved the basal topology of sea spiders with high support. The enigmatic family Austrodecidae was resolved as the sister group to the remaining Pycnogonida and the small-bodied family Rhynchothoracidae as the sister group of the robust-bodied family Pycnogonidae. Molecular divergence time estimation recovered a basal divergence of crown group sea spiders in the Ordovician. Comparison of diversification dynamics with other marine invertebrate taxa that originated in the Paleozoic suggests that sea spiders and some crustacean groups exhibit resilience to mass extinction episodes, relative to mollusk and echinoderm lineages.

First description of epimorphic development in Antarctic Pallenopsidae (Arthropoda, Pycnogonida) with insights into the evolution of the four-articled sea spider cheliphore.

BACKGROUND: Sea spiders (Pycnogonida) are an abundant faunal element of the Southern Ocean (SO). Several recent phylogeographical studies focused on the remarkably diverse SO pycnogonid fauna, resulting in the identification of new species in previously ill-defined species complexes, insights into their genetic population substructures, and hypotheses on glacial refugia and recolonization events after the last ice age. However, knowledge on the life history of many SO pycnogonids is fragmentary, and early ontogenetic stages often remain poorly documented. This impedes assessing the impact of different developmental pathways on pycnogonid dispersal and distributions and also hinders pycnogonid-wide comparison of developmental features from a phylogenetic-evolutionary angle. RESULTS: Using scanning electron microscopy (SEM) and fluorescent nuclear staining, we studied embryonic stages and postembryonic instars of three SO representatives of the taxon Pallenopsidae (Pallenopsis villosa, P. hodgsoni, P. vanhoeffeni), the development of which being largely unknown. The eggs are large and yolk-rich, and the hatching stage is an advanced lecithotrophic instar that stays attached to the father for additional molts. The first free-living instar is deduced to possess at least three functional walking leg pairs. Despite gross morphological similarities between the congeners, each instar can be reliably assigned to a species based on body size, shape of ocular tubercle and proboscis, structure of the attachment gland processes, and seta patterns on cheliphore and walking legs. CONCLUSIONS: We encourage combination of SEM with fluorescent markers in developmental studies on ethanol-preserved and/or long term-stored pycnogonid material, as this reveals internal differentiation processes in addition to external morphology. Using this approach, we describe the first known cases of pallenopsid development with epimorphic tendencies, which stand in contrast to the small hatching larvae in other Pallenopsidae. Evaluation against current phylogenetic hypotheses indicates multiple gains of epimorphic development within Pycnogonida. Further, we suggest that the type of development may impact pycnogonid distribution ranges, since free-living larvae potentially have a better dispersal capability than lecithotrophic attaching instars. Finally, we discuss the bearing of pycnogonid cheliphore development on the evolution of the raptorial first limb pair in Chelicerata and support a multi-articled adult limb as the plesiomorphic state of the chelicerate crown group, arising ontogenetically via postembryonic segmentation of a three-articled embryonic limb.

Genetic signature of Last Glacial Maximum regional refugia in a circum-Antarctic sea spider.

The evolutionary history of Antarctic organisms is becoming increasingly important to understand and manage population trajectories under rapid environmental change. The Antarctic sea spider Nymphon australe, with an apparently large population size compared with other sea spider species, is an ideal target to look for molecular signatures of past climatic events. We analysed mitochondrial DNA of specimens collected from the Antarctic continent and two Antarctic islands (AI) to infer past population processes and understand current genetic structure. Demographic history analyses suggest populations survived in refugia during the Last Glacial Maximum. The high genetic diversity found in the Antarctic Peninsula and East Antarctic (EA) seems related to multiple demographic contraction-expansion events associated with deep-sea refugia, while the low genetic diversity in the Weddell Sea points to a more recent expansion from a shelf refugium. We suggest the genetic structure of N. australe from AI reflects recent colonization from the continent. At a local level, EA populations reveal generally low genetic differentiation, geographically and bathymetrically, suggesting limited restrictions to dispersal. Results highlight regional differences in demographic histories and how these relate to the variation in intensity of glaciation-deglaciation events around Antarctica, critical for the study of local evolutionary processes. These are valuable data for understanding the remarkable success of Antarctic pycnogonids, and how environmental changes have shaped the evolution and diversification of Southern Ocean benthic biodiversity.

Upper limits to body size imposed by respiratory-structural trade-offs in Antarctic pycnogonids.

Across metazoa, surfaces for respiratory gas exchange are diverse, and the size of those surfaces scales with body size. In vertebrates with lungs and gills, surface area and thickness of the respiratory barrier set upper limits to rates of metabolism. Conversely, some organisms and life stages rely on cutaneous respiration, where the respiratory surface (skin, cuticle, eggshell) serves two primary functions: gas exchange and structural support. The surface must be thin and porous enough to transport gases but strong enough to withstand external forces. Here, we measured the scaling of surface area and cuticle thickness in Antarctic pycnogonids, a group that relies on cutaneous respiration. Surface area and cuticle thickness scaled isometrically, which may reflect the dual roles of cuticle in gas exchange and structural support. Unlike in vertebrates, the combined scaling of these variables did not match the scaling of metabolism. To resolve this mismatch, larger pycnogonids maintain steeper oxygen gradients and higher effective diffusion coefficients of oxygen in the cuticle. Interactions among scaling components lead to hard upper limits in body size, which pycnogonids could evade only with some other evolutionary innovation in how they exchange gases.

Respiratory gut peristalsis by sea spiders.

The fundamental constraint shaping animal systems for internal gas transport is the slow pace of diffusion [1]. In response, most macroscopic animals have evolved systems for driving internal flows using muscular pumps or cilia. In arthropods, aside from terrestrial lineages that exchange gases via tracheal systems, most taxa have a dorsal heart that drives O2-carrying hemolymph through peripheral vessels and an open hemocoel [2], with O2 often bound to respiratory proteins. Here we show that pycnogonids (sea spiders), a basal group of marine arthropods [3], use a previously undescribed mechanism of internal O2 transport: flows of gut fluids and hemolymph driven by peristaltic contractions of a space-filling system of gut diverticula. This observation fundamentally expands the known range of gas-transport systems in extant arthropods.

Astronomical and atmospheric impacts on deep-sea hydrothermal vent invertebrates.

Ocean tides and winter surface storms are among the main factors driving the dynamics and spatial structure of marine coastal species, but the understanding of their impact on deep-sea and hydrothermal vent communities is still limited. Multidisciplinary deep-sea observatories offer an essential tool to study behavioural rhythms and interactions between hydrothermal community dynamics and environmental fluctuations. Here, we investigated whether species associated with a Ridgeia piscesae tubeworm vent assemblage respond to local ocean dynamics. By tracking variations in vent macrofaunal abundance at different temporal scales, we provide the first evidence that tides and winter surface storms influence the distribution patterns of mobile and non-symbiotic hydrothermal species (i.e. pycnogonids Sericosura sp. and Polynoidae polychaetes) at more than 2 km depth. Local ocean dynamics affected the mixing between hydrothermal fluid inputs and surrounding seawater, modifying the environmental conditions in vent habitats. We suggest that hydrothermal species respond to these habitat modifications by adjusting their behaviour to ensure optimal living conditions. This behaviour may reflect a specific adaptation of vent species to their highly variable habitat.

From egg to "no-body": an overview and revision of developmental pathways in the ancient arthropod lineage Pycnogonida.

BACKGROUND: Arthropod diversity is unparalleled in the animal kingdom. The study of ontogeny is pivotal to understand which developmental processes underlie the incredible morphological disparity of arthropods and thus to eventually unravel evolutionary transformations leading to their success. Work on laboratory model organisms has yielded in-depth data on numerous developmental mechanisms in arthropods. Yet, although the range of studied taxa has increased noticeably since the advent of comparative evolutionary developmental biology (evo-devo), several smaller groups remain understudied. This includes the bizarre Pycnogonida (sea spiders) or "no-bodies", a taxon occupying a crucial phylogenetic position for the interpretation of arthropod development and evolution. RESULTS: Pycnogonid development is variable at familial and generic levels and sometimes even congeneric species exhibit different developmental modes. Here, we summarize the available data since the late 19th century. We clarify and resolve terminological issues persisting in the pycnogonid literature and distinguish five developmental pathways, based on (1) type of the hatching stage, (2) developmental-morphological features during postembryonic development and (3) selected life history characteristics. Based on phylogenetic analyses and the fossil record, we discuss plausible plesiomorphic features of pycnogonid development that allow comparison to other arthropods. These features include (1) a holoblastic, irregular cleavage with equal-sized blastomeres, (2) initiation of gastrulation by a single bottle-shaped cell, (3) the lack of a morphologically distinct germ band during embryogenesis, (4) a parasitic free-living protonymphon larva as hatching stage and (5) a hemianamorphic development during the postlarval and juvenile phases. Further, we propose evolutionary developmental trajectories within crown-group Pycnogonida. CONCLUSIONS: A resurgence of studies on pycnogonid postembryonic development has provided various new insights in the last decades. However, the scarcity of modern-day embryonic data - including the virtual lack of gene expression and functional studies - needs to be addressed in future investigations to strengthen comparisons to other arthropods and arthropod outgroups in the framework of evo-devo. Our review may serve as a basis for an informed choice of target species for such studies, which will not only shed light on chelicerate development and evolution but furthermore hold the potential to contribute important insights into the anamorphic development of the arthropod ancestor.

Regional differentiation and extensive hybridization between mitochondrial clades of the Southern Ocean giant sea spider Colossendeis megalonyx.

Assessing the enormous diversity of Southern Ocean benthic species and their evolutionary histories is a central task in the era of global climate change. Based on mitochondrial markers, it was recently suggested that the circumpolar giant sea spider Colossendeis megalonyx comprises a complex of at least six cryptic species with mostly small and non-overlapping distribution ranges. Here, we expand the sampling to include over 500 mitochondrial COI sequences of specimens from around the Antarctic. Using multiple species delimitation approaches, the number of distinct mitochondrial OTUs increased from six to 15-20 with our larger dataset. In contrast to earlier studies, many of these clades show almost circumpolar distributions. Additionally, analysis of the nuclear internal transcribed spacer region for a subset of these specimens showed incongruence between nuclear and mitochondrial results. These mito-nuclear discordances suggest that several of the divergent mitochondrial lineages can hybridize and should not be interpreted as cryptic species. Our results suggest survival of C. megalonyx during Pleistocene glaciations in multiple refugia, some of them probably located on the Antarctic shelf, and emphasize the importance of multi-gene datasets to detect the presence of cryptic species, rather than their inference based on mitochondrial data alone.

New Sericosura (Pycnogonida:Ammotheidae) from deep-sea hydrothermal vents in the Southern Ocean.

Three new species of Sericosura (Pycnogonida: Ammotheidae) are described from recently discovered hydrothermal vents in the East Scotia Ridge, Southern Ocean: Sericosura bamberi sp. nov., S. dimorpha sp. nov. and S. curva sp. nov. The eleven species known to date in the genus Sericosura are all inhabitants of chemosynthetic environments in different oceans around the world. Morphology and preliminary DNA data from the COI locus suggest the East Scotia Ridge pycnogonids have relatively close evolutionary affinities with species known from the East Pacific Rise and the Mid-Atlantic Ridge. This finding highlights the importance of Sericosura as a characteristic taxon of hydrothermal vents and the great potential of this genus for global scale ecological and evolutionary studies of hydrothermal vents fauna. The use of pycnogonid DNA data combined with recent models explaining biogeographic provinces along the mid-ocean ridge system should prove extremely useful to understanding the patterns of diversification of endemic fauna from chemosynthetic environments and from the deep-sea in general.

New species of Australian Pseudopallene (Pycnogonida: Callipallenidae) based on live colouration, morphology and DNA.

Callipallenid sea spiders are commonly found in southern Australian waters. The genera Pseudopallene and Stylopallene are frequent epibionts of arborescent bryozoan colonies of Orthoscuticella spp. and Amathia spp. Based on external morphology, colouration patterns, and sequence divergence of nuclear and mitochondrial markers, we differentiate species of Pseudopallene and Stylopallene from southeast Tasmania, New South Wales and Victoria. In this paper we describe five species new to science: P. tasmania, P. gracilis, P. constricta, P. flava and P. harrisi, and propose the 'variabilis'- complex, which is yet to be resolved based on additional material. Also, two species previously known from Southern Australia are reported as new records for Tasmania. The use of molecular data in resolving species identities of Australian Pycnogonida appears crucial, particularly in the understanding of the amazing, yet undiscovered diversity of brightly colourful callipallenid species. The genus Pseudopallene previously known from 20 species distributed in both southern and northern hemispheres now includes 14 Australian endemic species and at least one species complex yet to be resolved.

Morphogenesis of Pseudopallene sp. (Pycnogonida, Callipallenidae) II: postembryonic development.

Pycnogonida (sea spiders) are bizarre marine arthropods that are nowadays most frequently considered as being the sister group to all other chelicerates. The majority of pycnogonid species develops via a protonymphon larva with only three pairs of limbs affiliated with the future head region. Deviating from this, the hatching stage of some representatives shows already an advanced degree of trunk differentiation. Using scanning electron microscopy, fluorescent nucleic staining, and bright-field stereomicroscopy, postembryonic development of Pseudopallene sp. (Callipallenidae), a pycnogonid with an advanced hatching stage, is described. Based on external morphology, six postembryonic stages plus a sub-adult stage are distinguished. The hatching larva is lecithotrophic and bears the chelifores as only functional appendage pair and unarticulated limb buds of walking leg pairs 1 and 2. Palpal and ovigeral larval limbs are absent. Differentiation of walking leg pairs 3 and 4 is sequential. Apart from the first pair, each walking leg goes through a characteristic sequence of three externally distinct stages with two intermittent molts (limb bud-seven podomeres-nine podomeres). First external signs of oviger development are detectable in postembryonic stage 3 bearing three articulated walking leg pairs. Following three more molts, the oviger has attained adult podomere composition. The advanced hatching stages of different callipallenids are compared and the inclusive term "walking leg-bearing larva" is suggested, as opposed to the behavior-based name "attaching larva". Data on temporal and structural patterns of walking leg differentiation in other pycnogonids are reviewed and discussed. To facilitate comparisons of walking leg differentiation patterns across many species, we propose a concise notation in matrix fashion. Due to deviating structural patterns of oviger differentiation in another callipallenid species as well as within other pycnogonid taxa, evolutionary conservation of characteristic stages of oviger development is not apparent even in closely related species.

Morphogenesis of Pseudopallene sp. (Pycnogonida, Callipallenidae) I: embryonic development.

Embryonic development of Pycnogonida (sea spiders) is poorly understood in comparison to other euarthropod lineages with well-established model organisms. However, given that pycnogonids potentially represent the sister group to chelicerates or even to all other euarthropods, their development might yield important data for the reconstruction of arthropod evolution. Using scanning electron microscopy, fluorescent nucleic staining and immunohistochemistry, the general course of embryonic morphogenesis in Pseudopallene sp. (Callipallenidae), a pycnogonid with prolonged embryonic development, is described. A staging system comprising ten stages is presented, which can be used in future studies addressing specific developmental processes. The initially slit-like stomodeum anlage forms at the anterior end of an eight-shaped germ band and predates proboscis outgrowth. The latter process is characterized by the protrusion of three cell populations that are subsequently involved in pharynx formation. In later stages, the proboscis assumes distally a horseshoe-like shape. At no time, a structure corresponding to the euarthropod labrum is detectable. Based on the complete lack of palpal and ovigeral embryonic limbs and the early differentiation of walking leg segments 1 and 2, the existence of an embryonized protonymphon stage during callipallenid development is rejected. The evolution of pycnogonid hatching stages, especially within Callipallenidae and Nymphonidae, is re-evaluated in the light of recent phylogenetic analyses. Specifically, the re-emergence of the ancestral protonymphon larva (including re-development of palpal and ovigeral larval limbs) and a possible re-appearance of adult palps in the nymphonid lineage are discussed. This challenges the perception of pycnogonid head appendage evolution as being driven by reduction events alone.

The mitochondrial genome of Colossendeis megalonyx supports a basal position of Colossendeidae within the Pycnogonida.

We present the almost complete (16,007 bp) mitochondrial genome of a Colossendeis megalonyx specimen from the Southern Ocean and discuss gene order and tRNA structure in a comparative phylogenetic context. Our data suggest a basal position of the colossendeid lineage corroborating earlier phylogenetic studies but disagreeing with results of a recently published study that supported a highly derived sister-group relationship of Colossendeidae and Nymphonidae. Our results, together with BLAST searches and phylogenetic comparisons, indicate that the specimen presented as Colossendeis sp. in a series of recent studies had been misidentified. It has now been identified as a nymphonid species.

Oxygen hypothesis of polar gigantism not supported by performance of Antarctic pycnogonids in hypoxia.

Compared to temperate and tropical relatives, some high-latitude marine species are large-bodied, a phenomenon known as polar gigantism. A leading hypothesis on the physiological basis of gigantism posits that, in polar water, high oxygen availability coupled to low metabolic rates relieves constraints on oxygen transport and allows the evolution of large body size. Here, we test the oxygen hypothesis using Antarctic pycnogonids, which have been evolving in very cold conditions (-1.8-0 degrees C) for several million years and contain spectacular examples of gigantism. Pycnogonids from 12 species, spanning three orders of magnitude in body mass, were collected from McMurdo Sound, Antarctica. Individual sea spiders were forced into activity and their performance was measured at different experimental levels of dissolved oxygen (DO). The oxygen hypothesis predicts that, all else being equal, large pycnogonids should perform disproportionately poorly in hypoxia, an outcome that would appear as a statistically significant interaction between body size and oxygen level. In fact, although we found large effects of DO on performance, and substantial interspecific variability in oxygen sensitivity, there was no evidence for sizexDO interactions. These data do not support the oxygen hypothesis of Antarctic pycnogonid gigantism and suggest that explanations must be sought in other ecological or evolutionary processes.

Microscopic anatomy of pycnogonida: II. Digestive system. III. Excretory system.

The digestive system of several species of sea spiders (Pycnogonida, Arthropoda) was studied by electron microscopy. It is composed of the foregut inside a long proboscis, a midgut and a hindgut. Lips near the three jaws at the tip of the proboscis receive several hundred ductules originating from salivary glands. These previously undetected glands open on the lips, a fluted, projecting ridge at the external hinge line of the jaws, i.e., to the outside of the mouth. This disposition suggests affinities to the chelicerate line. The trigonal esophagus within the proboscis contains a complex, setose filter device, operated by dedicated muscles, that serves to reduce ingested food to subcellular dimensions. The midgut has diverticula into the bases of all legs. Its cells differentiate from the basal layer and contain a bewildering array of secretion droplets, lysosomes and phagosomes. In the absence of a hepatopancreas, the midgut serves both digestive and absorptive functions. The cuticle-lined hindgut lies in the highly reduced, peg-like abdomen. Traditionally, pycnogonids have been claimed to have no excretory organ at all. Such a structure, however, has been located in at least one ammotheid, Nymphopsis spinosissima, in which a simple, but standard, excretory gland has been found in the scape of the chelifore. It consists of an end sac, a straight proximal tubule, a short distal tubule, and a raised nephropore. The end sac is a thin-walled and polygonal chamber, about 150 microm in cross section, suspended in the hemocoel of the appendage, its edges radially tethered to the cuticle at more than half a dozen locations. This wall consists of a filtration basement membrane, 1-4 microm thick, facing the hemocoel, and internally of a continuous carpet of podocytes and their pedicels. The podocytes, measuring maximally 10 by 15 microm, have complex contents, of which a labyrinthine system of connected intracellular channels stands out. These coated cisternae open into a central vacuole that often rivals the nucleus in size. The design of the organ closely approximates that of the primitive crustacean Hutchinsoniella macracantha.

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.

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.