The ovary structure and oogenesis in the basal crustaceans and hexapods. Possible phylogenetic significance

Recent large-scale phylogenetic analyses of exclusively molecular or combined molecular and morphological characters support a close relationship between Crustacea and Hexapoda. The growing consensus on this phylogenetic link is reflected in uniting both taxa under the name Pancrustacea or Tetraconata. Several recent molecular phylogenies have also indicated that the monophyletic hexapods should be nested within paraphyletic crustaceans. However, it is still contentious exactly which crustacean taxon is the sister group to Hexapoda. Among the favored candidates are Branchiopoda, Malacostraca, Remipedia and Xenocarida (Remipedia + Cephalocarida). In this context, we review morphological and ultrastructural features of the ovary architecture and oogenesis in these crustacean groups in search of traits potentially suitable for phylogenetic considerations. We have identified a suite of morphological characters which may prove useful in further comparative studies.     

A new view of insect-crustacean relationships I. Inferences from neural cladistics and comparative neuroanatomy

Traditional hypotheses regarding the relationships of the major arthropod lineages focus on suites of comparable characters, often those that address features of the exoskeleton. However, because of the enormous morphological variety among arthropods, external characters may lead to ambiguities of interpretation and definition, particularly when species have undergone evolutionary simplification and reversal. Here we present the results of a cladistic analysis using morphological characters associated with brains and central nervous systems, based on the evidence that cerebral organization is generally robust over geological time. Well-resolved, strongly supported phylogenies were obtained from a neuromorphological character set representing a variety of discrete neuroanatomical traits. Phylogenetic hypotheses from this analysis support many accepted relationships, including monophyletic Chelicerata, Myriapoda, and Hexapoda, paraphyletic Crustacea and the union of Hexapoda and Crustacea (Tetraconata). They also support Mandibulata (Myriapoda + Tetraconata). One problematic result, which can be explained by symplesiomorphies that are likely to have evolved in deep time, is the inability to resolve Onychophora as a taxon distinct from Arthropoda. Crucially, neuronal cladistics supports the heterodox conclusion that both Hexapoda and Malacostraca are derived from a common ancestor that possessed a suite of discrete neural centers comprising an elaborate brain. Remipedes and copepods, both resolved as basal to Branchiopoda share a neural ground pattern with Malacostraca. These findings distinguish Hexapoda (Insecta) from Branchiopoda, which is the sister group of the clade Malacostraca + Hexapoda. The present study resolves branchiopod crustaceans as descendents of an ancestor with a complex brain, which means that they have evolved secondary simplification and the loss or reduction of numerous neural systems.     

The Tetraconata concept: hexapod-crustacean relationships and the phylogeny of Crustacea

A growing body of evidence indicates that Crustacea and Hexapoda are sister groups, rather than Hexapoda and Myriapoda. Some recent molecular data even suggest that Mandibulata is not monophyletic, with Myriapoda and Chelicerata instead being sister groups. Here, arguments for homology of the mandible throughout mandibulate arthropods and for a monophyletic Mandibulata will be presented, as well as arguments supporting the taxon Tetraconata (i.e. Crustacea + Hexapoda). The latter include molecular data (nuclear and mitochondrial ribosomal RNAs and protein coding genes), and morphological characters such as ommatidial structure, the presence of neuroblasts and a very similar axonogenesis of pioneer neurons. However, crustaceans are insufficiently sampled for the molecular data, and studies of neurogenesis are lacking for many crustacean taxa. Remipedia, Cephalocarida and Maxillopoda are particularly problematic. This is important for the entire problem, because monophyly of the Crustacea has not yet been proven beyond doubt and several molecular analyses suggest a paraphyletic Crustacea. Here, arguments for the monophyly of the Crustacea are reviewed and two alternatives for the relationships between the five higher taxa Remipedia, Cephalocarida, Maxillopoda, Branchiopoda and Malacostraca are discussed: the Entomostraca concept sensu Walossek with Malacostraca as sister group to Cephalocarida, Maxillopoda and Branchiopoda, and the Thoracopoda concept sensu Hessler with Cephalocarida, Branchiopoda and Malacostraca forming a monophylum.     

Evolution of eye development in arthropods: phylogenetic aspects

The architecture of the adult arthropod visual system for many decades has contributed important character sets that are useful for reconstructing the phylogenetic relationships within this group. In the current paper we explore whether aspects of eye development can also contribute new arguments to the discussion of arthropod phylogeny. We review the current knowledge on eye formation in Trilobita, Xiphosura, Myriapoda, Hexapoda, and Crustacea. All euarthropod taxa share the motif of a proliferation zone at the side of the developing eye field that contributes new eye elements. Two major variations of this common motif can be distinguished: 1. The “row by row type” of Trilobita, Xiphosura, and Diplopoda. In this type, the proliferation zone at the side of the eye field generates new single, large elements with a high and variable cell number, which are added to the side of the eye and extend rows of existing eye elements. Cell proliferation, differentiation and ommatidial assembly seem to be separated in time but spatially confined within the precursors of the optic units which grow continuously once they are formed (intercalary growth). 2. The “morphogenetic front type” of eye formation in Crustacea + Hexapoda (Tetraconata). In this type, there is a clear temporal and spatial separation of the formation and differentiation processes. Proliferation and the initial steps of pattern formation take place in linear and parallel mitotic and morphogenetic fronts (the mitotic waves and the morphogenetic furrow/transition zone) and numerous but small new elements with a strictly fixed set of cells are added to the eye field. In Tetraconata, once formed, the individual ommatidia do not grow any more. Scutigeromorph chilopods take an intermediate position between these two major types. We suggest that the “row by row type” as seen in Trilobita, Xiphosura and Diplopoda represents the plesiomorphic developmental mode of eye formation from the euarthropod ground pattern whereas the “morphogenetic front type” is apomorphic for the Tetraconata. Our data are discussed with regard to two competing hypotheses on arthropod phylogeny, the “Tracheata” versus “Tetraconata” concept. The modes of eye development in Myriapoda is more parsimonious to explain in the Tetraconata hypothesis so that our data raise the possibility that myriapod eyes may not be secondarily reconstructed insect eyes as the prevailing hypothesis suggests.     

On the phylogenetic position of insects in the Pancrustacea clade

The current views on the phylogeny of arthropods are at odds with the traditional system, which recognizes four independent arthropod classes: Chelicerata, Crustacea, Myriapoda, and Insecta. There is compelling evidence that insects comprise a monophyletic lineage with Crustacea within a larger clade named Pancrustacea, or Tetraconata. However, which crustacean group is the closest living relative of insects is still an open question. In recent phylogenetic trees constructed on the basis of large gene sequence data insects are placed together with primitive crustaceans, the Branchiopoda. This topology is often suspected to be a result of the long branch attraction artifact. We analyzed concatenated data on 77 ribosomal proteins, elongation factor 1A (EF1A), initiation factor 5A (eIF5A), and several other nuclear and mitochondrial proteins. Analyses of nuclear genes confirm the monophyly of Hexapoda, the clade uniting entognath and ectognath insects. The hypothesis of the monophyly of Hexapoda and Branchiopoda is supported in the majority of analyses. The Maxillopoda, another clade of Entomostraca, occupies a sister position to the Hexapoda + Branchiopoda group. Higher crustaceans, the Malacostraca, in most analyses appear a more basal lineage within the Pancrustacea. We report molecular synapomorphies in low homoplastic regions, which support the clade Hexapoda + Branchiopoda + Maxillopoda and the monophyletic Malacostraca including Phyllocarida. Thus, the common origin of Hexapoda and Branchiopoda and their position within Entomostraca are suggested to represent bona fide phylogenetic relationships rather than computational artifacts.     

A new view of insect-crustacean relationships II. Inferences from expressed sequence tags and comparisons with neural cladistics

The enormous diversity of Arthropoda has complicated attempts by systematists to deduce the history of this group in terms of phylogenetic relationships and phenotypic change. Traditional hypotheses regarding the relationships of the major arthropod groups (Chelicerata, Myriapoda, Crustacea, and Hexapoda) focus on suites of morphological characters, whereas phylogenomics relies on large amounts of molecular sequence data to infer evolutionary relationships. The present discussion is based on expressed sequence tags (ESTs) that provide large numbers of short molecular sequences and so provide an abundant source of sequence data for phylogenetic inference. This study presents well-supported phylogenies of diverse arthropod and metazoan outgroup taxa obtained from publicly-available databases. An in-house bioinformatics pipeline has been used to compile and align conserved orthologs from each taxon for maximum likelihood inferences. This approach resolves many currently accepted hypotheses regarding internal relationships between the major groups of Arthropoda, including monophyletic Hexapoda, Tetraconata (Crustacea + Hexapoda), Myriapoda, and Chelicerata sensu lato (Pycnogonida + Euchelicerata). "Crustacea" is a paraphyletic group with some taxa more closely related to the monophyletic Hexapoda. These results support studies that have utilized more restricted EST data for phylogenetic inference, yet they differ in important regards from recently published phylogenies employing nuclear protein-coding sequences. The present results do not, however, depart from other phylogenies that resolve Branchiopoda as the crustacean sister group of Hexapoda. Like other molecular phylogenies, EST-derived phylogenies alone are unable to resolve morphological convergences or evolved reversals and thus omit what may be crucial events in the history of life. For example, molecular data are unable to resolve whether a Hexapod-Branchiopod sister relationship infers a branchiopod-like ancestry of the Hexapoda, or whether this assemblage originates from a malacostracan-like ancestor, with the morphologically simpler Branchiopoda being highly derived. Whereas this study supports many internal arthropod relationships obtained by other sources of molecular data, other approaches are required to resolve such evolutionary scenarios. The approach presented here turns out to be essential: integrating results of molecular phylogenetics and neural cladistics to infer that Branchiopoda evolved simplification from a more elaborate ancestor. Whereas the phenomenon of evolved simplification may be widespread, it is largely invisible to molecular techniques unless these are performed in conjunction with morphology-based strategies.     

Neurophylogeny: Architecture of the nervous system and a fresh view on arthropod phyologeny

The phylogenetic relationships within the Arthropoda have beencontroversial for more than a century. Today, comparative studieson the structure and development of the nervous system contributeimportant arguments to this discussion, so that the term "neurophylogeny"was coined for this discipline. The large number of recent studieson the nervous system in various nonmodel arthropods indicatesthat we are far advanced in the process of analyzing the cellulararchitecture of the arthropod nervous system in a depth thatwill ultimately provide characters at a level of resolutionequal or even superior to that of characters traditionally usedin morphological phylogenetic studies. This article sets outto summarize the current state of the discussion on arthropodphylogeny and briefly evaluates the morphological charactersthat have been used as arguments in favor of the traditionalTracheata hypothesis. Then, a thorough overview is given ofcharacters derived from structure and development of the arthropodbrain and the ventral nerve cord from the cellular level tothe level of larger neuropil systems. These characters supportthe new Tetraconata hypothesis suggested by Dohle and provideevidence for a clade that unites malacostracan and remipedecrustaceans with the Hexapoda.     

Higher-level crustacean phylogeny: Consensus and conflicting hypotheses

This paper presents an overview of current hypotheses of higher-level crustacean phylogeny in order to assist and help focus further research. It concentrates on hypotheses proposed or debated in the recent literature based on morphological, molecular and combined evidence phylogenetic analyses. It can be concluded that crustacean phylogeny remains essentially unresolved. Conflict is rife, irrespective of whether one compares different morphological studies, molecular studies, or both. Using the number of recently proposed alternative sister group hypotheses for each of the major tetraconatan taxa as a rough estimate of phylogenetic uncertainty, it can be concluded that the phylogenetic position of Malacostraca remains the most problematic, closely followed by Branchiopoda, Cephalocarida, Remipedia, Ostracoda, Branchiura, Copepoda and Hexapoda. Future progress will depend upon a broader taxon sampling in molecular analyses, and the further exploration of new molecular phylogenetic markers. However, the need for continued revision and expansion of morphological datasets remains undiminished given the conspicuous lack of agreement between molecules and morphology for positioning several taxa. In view of the unparalleled morphological diversity of Crustacea, and the likely nesting of Hexapoda somewhere within Crustacea, working out a detailed phylogeny of Tetraconata is a crucial step towards understanding arthropod body plan evolution.     

Evolution of identified arthropod neurons: the serotonergic system in relation to engrailed-expressing cells in the embryonic ventral nerve cord of the american lobster homarus americanus milne edwards, 1873 (malacostraca,pleocyemata, homarida)

One of the long-standing questions in zoology is that on the phylogenetic relationships within the Arthropoda. Comparative studies on structure and development of the nervous system can contribute important arguments to this discussion. In the present report, the arrangement of serotonin- and engrailed-expressing cells was examined in the embryonic ventral nerve cord of the American lobster Homarus americanus Milne Edwards, 1873 (Malacostraca, Pleocyemata, Homarida), and the spatial relationship of these two cell classes was explored by a double-labelling approach. The goal of this study was to determine whether the lobster serotonergic neurons are homologous to similar cells present in representatives of the Hexapoda and other Arthropoda. The results indicate that, in fact, these neurons in the lobster ventral nerve cord have corresponding counterparts in many other mandibulate taxa. Based on the finding of these homologies, the arrangement of serotonergic neurons in a model trunk ganglion of the mandibulate ground pattern was reconstructed as comprising an anterior and a posterior pair of serotonergic neurons per hemiganglion, each cell with both an ipsilateral and a contralateral neurite. Starting from this ground pattern, the evolutionary diversification of this class of neurons within the Mandibulata is discussed.     

From variable to constant cell numbers: cellular characteristics of the arthropod nervous system argue against a sister-group relationship of Chelicerata and “Myriapoda” but favour the Mandibulata concept

In the new debate on arthropod phylogeny, structure and development of the nervous system provide important arguments. The architecture of the brain of Hexapoda, Crustacea and Chelicerata in recent years has been thoroughly compared against an evolutionary background. However, comparative aspects of the nervous systems in these taxa at the cellular level have been examined in only a few studies. This review sets out to summarize these aspects and to analyse the existing data with respect to the concept of individually identifiable neurons. In particular, mechanisms of neurogenesis, the morphology of serotonergic interneurons, the number of motoneurons, and cellular features and development of the lateral eyes are discussed. We conclude that in comparison to the Mandibulata, in Chelicerata the numbers of neurons in the different classes examined are much higher and in many cases are not fixed but variable. The cell numbers in Mandibulata are lower and the majority of neurons are individually identifiable. The characters explored in this review are mapped onto an existing phylogram, as derived from brain architecture in which the Hexapoda are an in-group of the Crustacea, and there is not any conflict of the current data with such a phylogenetic position of the Hexapoda. Nevertheless, these characters argue against a sister-group relationship of Myriapoda and Chelicerata as has been recently suggested in several molecular studies, but instead provide strong evidence in favour of the Mandibulata concept.Edited by D. Tautz     

Mitochondrial genomes suggest that hexapods and crustaceans are mutually paraphyletic

For over a century the relationships between the four major groups of the phylum Arthropoda (Chelicerata, Crustacea, Hexapoda and Myriapoda) have been debated. Recent molecular evidence has confirmed a close relationship between the Crustacea and the Hexapoda, and has included the suggestion of a paraphyletic Hexapoda. To test this hypothesis we have sequenced the complete or near-complete mitochondrial genomes of three crustaceans (Parhyale hawaiensis, Squilla mantis and Triops longicaudatus), two collembolans (Onychiurus orientalis and Podura aquatica) and the insect Thermobia domestica. We observed rearrangement of transfer RNA genes only in O. orientalis, P. aquatica and P. hawaiensis. Of these, only the rearrangement in O. orientalis, an apparent autapomorphy for the collembolan family Onychiuridae, was phylogenetically informative.We aligned the nuclear and amino acid sequences from the mitochondrial protein-encoding genes of these taxa with their homologues from other arthropod taxa for phylogenetic analysis. Our dataset contains many more Crustacea than previous molecular phylogenetic analyses of the arthropods. Neighbour-joining, maximum-likelihood and Bayesian posterior probabilities all suggest that crustaceans and hexapods are mutually paraphyletic. A crustacean clade of Malacostraca and Branchiopoda emerges as sister to the Insecta sensu stricto and the Collembola group with the maxillopod crustaceans. Some, but not all, analyses strongly support this mutual paraphyly but statistical tests do not reject the null hypotheses of a monophyletic Hexapoda or a monophyletic Crustacea. The dual monophyly of the Hexapoda and Crustacea has rarely been questioned in recent years but the idea of both groups' paraphyly dates back to the nineteenth century. We suggest that the mutual paraphyly of both groups should seriously be considered.     

Ultrastructural analysis of the ovary and oogenesis in Spinicaudata and Laevicaudata (Branchiopoda) and its phylogenetic implications

Recent molecular studies have indicated a close relationship between Crustacea and Hexapoda and postulated their unification into the Pancrustacea/Tetraconata clade. Certain molecular analyses have also suggested that the crustacean lineage, which includes the Branchiopoda, might be the sister group of Hexapoda. We test this hypothesis by analyzing the structure of the ovary and the ultrastructural features of oogenesis in two branchiopod species, Cyzicus tetracerus and Lynceus brachyurus, representing two separate orders, Spinicaudata and Laevicaudata, respectively. The female gonads of these species have not been investigated before. Here, we demonstrate that in both studied species the ovarian follicles develop inside characteristic ovarian protrusions and comprise a germline cyst surrounded by a simple somatic (follicular) epithelium, supported by a thin basal lamina. Each germline cyst consists of one oocyte and three supporting nurse cells, and the oocyte differentiates relatively late during ovarian follicle development. The synthesis of oocyte reserve materials involves rough endoplasmic reticulum and Golgi complexes. The follicular cells are penetrated by a complex canal system and there is no external epithelial sheath covering the ovarian follicles. The structure of the ovary and the ultrastructural characteristics of oogenesis are not only remarkably similar in both Cyzicus and Lynceus, but also share morphological similarities with Notostraca as well as the basal hexapods Campodeina and Collembola. Possible phylogenetic implications of these findings are discussed.     

Ontogeny of the ventral nerve cord in malacostracan crustaceans: a common plan for neuronal development in Crustacea, Hexapoda and other Arthropoda?

This review sets out to summarize our current knowledge on the structural layout of the embryonic ventral nerve cord in decapod crustaceans and its development from stem cell to the mature structure. In Decapoda, neuronal stem cells, the neuroblasts, mostly originate from ectodermal stem cells, the ectoteloblast, via a defined lineage. The neuroblasts undergo repeated asymmetric division and generate ganglion mother cells. The ganglion mother cells later divide again to give birth to ganglion cells (neurons) and there is increasing evidence now that ganglion mother cells divide again not only once but repeatedly. Various other aspects of neuroblast proliferation such as their temporal patterns of mitotic activity and spatial arrangement as well as the relation of neurogenesis to the development of the segmental appendages and maturation of motor behaviors are described. The link between cell lineage and cell differentiation in Decapoda so far has only been established for the midline neuroblast. However, there are several other identified early differentiating neurons, the outgrowing neurites of which pioneer the axonal scaffold within the neuromeres of the ventral nerve cord. The maturation of identified neurons as examined by immunohistochemistry against their neurotransmitters or engrailed, is briefly described. These processes are compared to other Arthropoda (including Onychophora, Chelicerata, Diplopoda and Hexapoda) in order to shed light on variations and conserved motifs of the theme 'neurogenesis'. The question of a 'common plan for neuronal development' in the ventral nerve cords of Hexapoda and Crustacea is critically evaluated and the possibility of homologous neurons arising through divergent developmental pathways is discussed.     

Arthropod phylogeny: An overview from the perspectives of morphology,molecular data and the fossil record

Monophyly of Arthropoda is emphatically supported from both morphological and molecular perspectives. Recent work finds Onychophora rather than Tardigrada to be the closest relatives of arthropods. The status of tardigrades as panarthropods (rather than cycloneuralians) is contentious from the perspective of phylogenomic data. A grade of Cambrian taxa in the arthropod stem group includes gilled lobopodians, dinocaridids (e.g., anomalocaridids), fuxianhuiids and canadaspidids that inform on character acquisition between Onychophora and the arthropod crown group. A sister group relationship between Crustacea (itself likely paraphyletic) and Hexapoda is retrieved by diverse kinds of molecular data and is well supported by neuroanatomy. This clade, Tetraconata, can be dated to the early Cambrian by crown group-type mandibles. The rival Atelocerata hypothesis (Myriapoda + Hexapoda) has no molecular support. The basal node in the arthropod crown group is embroiled in a controversy over whether myriapods unite with chelicerates (Paradoxopoda or Myriochelata) or with crustaceans and hexapods (Mandibulata). Both groups find some molecular and morphological support, though Mandibulata is presently the stronger morphological hypothesis. Either hypothesis forces an unsampled ghost lineage for Myriapoda from the Cambrian to the mid Silurian.     

Phylogenetic relationships of basal hexapods among the mandibulate arthropods: a cladistic analysis based on comparative morphological characters

In this paper we propose a reappraisal of the relationships between the basal hexapod lineages (the former 'apterygote' insects) and the other major groups of mandibulate arthropods. It results from a cladistic analysis including 72 characters based on external morphology, internal anatomy and development. Detailed comments are provided on the various characters used and the scoring of their states. The 35 terminal taxa include 12 hexapods (9 of which are basal 'apterygote' representatives), 7 myriapods, 13 crustaceans, and 3 chelicerates taken as outgroups. The results of our analyses are discussed in detail for each of the taxonomic groupings, and compared with those recently obtained by other authors using different approaches based on morphological, palaeontological, developmental or molecular sequence data. Our results support the monophyly of the Mandibulata, Crustacea, Atelocerata (Tracheata) and Hexapoda, but the assemblage of Myriapoda appears poorly supported. A close relationship between Crustacea and Hexapoda, as hypothesized by several authors, is not found in any of our analyses. Within Hexapoda, the Protura and the Collembola appear as independent clades, whereas the two unresolved dipluran taxa are grouped with the monophyletic Ectognatha (Archaeognatha, Zygentoma and Pterygota).     

An immunohistochemical study of structure and development of the nervous system in the brine shrimp Artemia salina Linnaeus, 1758 (Branchiopoda, Anostraca) with remarks on the evolution of the arthropod brain

Brain morphology is an important character in the discussion of arthropod relationships. While a large body of literature is available on the brains of Hexapoda and Malacostraca, the structure of the brain has been rarely studied in representatives of the Entomostraca. This account examines the morphology and development of the nervous system in the brine shrimp Artemia salina Linnaeus, 1758 (Crustacea, Branchiopoda, Anostraca) by classical histology and immunohistochemistry against synaptic proteins (synapsins), and the neurotransmitters serotonin and histamine. The results indicate that the shape of the developing larval brain in A. salina (a circumstomodeal ring of neuropil) closely resembles that in malacostracan embryos. Furthermore, the organization of the central complex as well as the tritocerebral innervation pattern of the labrum is homologous in this species and in Malacostraca. Nevertheless, differences exist in the layout of the deutocerebrum, specifically in the absence of olfactory glomeruli in A. salina while the glomerular organization of the olfactory lobe is a character in the ground pattern of Malacostraca. These findings are compared to the brain structure in other Euarthropoda and possible phylogenetic implications are discussed.     

The Engrailed-expressing secondary head spots in the embryonic crayfish brain: examples for a group of homologous neurons in Crustacea and Hexapoda?

Hexapoda have been traditionally seen as the closest relatives of the Myriapoda (Tracheata hypothesis) but molecular studies have challenged this hypothesis and rather have suggested a close relationship of hexapods and crustaceans (Tetraconata hypothesis). In this new debate, data on the structure and development of the arthropod nervous system contribute important new data ("neurophylogeny"). Neurophylogenetic studies have already provided several examples for individually identifiably neurons in the ventral nerve cord that are homologous between insects and crustaceans. In the present report, we have analysed the emergence of Engrailed-expressing cells in the embryonic brain of a parthenogenetic crayfish, the marbled crayfish (Marmorkrebs), and have compared our findings to the pattern previously reported from insects. Our data suggest that a group of six Engrailed-expressing neurons in the optic anlagen, the so-called secondary head spot cells can be homologised between crayfish and the grasshopper. In the grasshopper, these cells are supposed to be involved in establishing the primary axon scaffold of the brain. Our data provide the first example for a cluster of brain neurons that can be homologised between insects and crustaceans and show that even at the level of certain cell groups, brain structures are evolutionary conserved in these two groups.     

Phylogenetic analysis of arthropods using two nuclear protein-encoding genes supports a crustacean + hexapod clade

Recent phylogenetic analyses using molecular data suggest that hexapods are more closely related to crustaceans than to myriapods, a result that conflicts with long-held morphology-based hypotheses. Here we contribute additional information to this debate by conducting phylogenetic analyses on two nuclear protein-encoding genes, elongation factor-1 alpha (EF-1 alpha) and the largest subunit of RNA polymerase II (Pol II), from an extensive sample of arthropod taxa. Results were obtained from two data sets. One data set comprised 1092 nucleotides (364 amino acids) of EF-1 alpha and 372 nucleotides (124 amino acids) of Pol II from 30 arthropods and three lobopods. The other data set contained the same EF-1 alpha fragment and an expanded 1038-nucleotide (346-amino-acid) sample of Pol II from 17 arthropod taxa. Results from maximum-parsimony and maximum-likelihood analyses strongly supported the existence of a Crustacea + Hexapoda clade (Pancrustacea) over a Myriapoda + Hexapoda clade (Atelocerata). The apparent incompatibility between the molecule-based Pancrustacea hypothesis and morphology-based Atelocerata hypothesis is discussed.     

Molecular phylogeny of the major arthropod groups indicates polyphyly of crustaceans and a new hypothesis for the origin of hexapods

A phylogeny of the arthropods was inferred from analyses of amino acid sequences derived from the nuclear genes encoding elongation factor-1 alpha and the largest subunit of RNA polymerase II using maximum- parsimony, neighbor-joining, and maximum-likelihood methods. Analyses of elongation factor-1 alpha from 17 arthropods and 4 outgroup taxa recovered many arthropod clades supported by previous morphological studies, including Diplopoda, Myriapoda, Insecta, Hexapoda, Branchiopoda (Crustacea), Araneae, Tetrapulmonata, Arachnida, Chelicerata, and Malacostraca (Crustacea). However, counter to previous studies, elongation factor-1 alpha placed Malacostraca as sister group to the other arthropods. Branchiopod crustaceans were found to be more closely related to hexapods and myriapods than to malacostracan crustaceans. Sequences for RNA polymerase II were obtained from 11 arthropod taxa and were analyzed separately and in combination with elongation factor-1 alpha. Results from these analyses were concordant with those derived from elongation factor-1 alpha alone and provided support for a Hexapoda/Branchiopoda clade, thus arguing against the monophyly of the traditionally defined Atelocerata (Hexapoda + Myriapoda).      

Pancrustacean phylogeny: hexapods are terrestrial crustaceans and maxillopods are not monophyletic

Recent molecular analyses indicate that crustaceans and hexapods form a clade (Pancrustacea or Tetraconata), but relationships among its constituent lineages, including monophyly of crustaceans, are controversial. Our phylogenetic analysis of three protein-coding nuclear genes from 62 arthropods and lobopods (Onychophora and Tardigrada) demonstrates that Hexapoda is most closely related to the crustaceans Branchiopoda (fairy shrimp, water fleas, etc.) and Cephalocarida + Remipedia, thereby making hexapods terrestrial crustaceans and the traditionally defined Crustacea paraphyletic. Additional findings are that Malacostraca (crabs, isopods, etc.) unites with Cirripedia (barnacles, etc.) and they, in turn, with Copepoda, making the traditional crustacean class Maxillopoda paraphyletic. Ostracoda (seed shrimp)--either all or a subgroup--is associated with Branchiura (fish lice) and likely to be basal to all other pancrustaceans. A Bayesian statistical (non-clock) estimate of divergence times suggests a Precambrian origin for Pancrustacea (600 Myr ago or more), which precedes the first unambiguous arthropod fossils by over 60 Myr.