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.     

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.     

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.     

Phylogenetic analysis of mitochondrial protein coding genes confirms the reciprocal paraphyly of Hexapoda and Crustacea

Background

The phylogeny of Arthropoda is still a matter of harsh debate among systematists, and significant disagreement exists between morphological and molecular studies. In particular, while the taxon joining hexapods and crustaceans (the Pancrustacea) is now widely accepted among zoologists, the relationships among its basal lineages, and particularly the supposed reciprocal paraphyly of Crustacea and Hexapoda, continues to represent a challenge. Several genes, as well as different molecular markers, have been used to tackle this problem in molecular phylogenetic studies, with the mitochondrial DNA being one of the molecules of choice. In this study, we have assembled the largest data set available so far for Pancrustacea, consisting of 100 complete (or almost complete) sequences of mitochondrial genomes. After removal of unalignable sequence regions and highly rearranged genomes, we used nucleotide and inferred amino acid sequences of the 13 protein coding genes to reconstruct the phylogenetic relationships among major lineages of Pancrustacea. The analysis was performed with Bayesian inference, and for the amino acid sequences a new, Pancrustacea-specific, matrix of amino acid replacement was developed and used in this study.

Results

Two largely congruent trees were obtained from the analysis of nucleotide and amino acid datasets. In particular, the best tree obtained based on the new matrix of amino acid replacement (MtPan) was preferred over those obtained using previously available matrices (MtArt and MtRev) because of its higher likelihood score. The most remarkable result is the reciprocal paraphyly of Hexapoda and Crustacea, with some lineages of crustaceans (namely the Malacostraca, Cephalocarida and, possibly, the Branchiopoda) being more closely related to the Insecta s.s. (Ectognatha) than two orders of basal hexapods, Collembola and Diplura. Our results confirm that the mitochondrial genome, unlike analyses based on morphological data or nuclear genes, consistently supports the non monophyly of Hexapoda.

Conclusion

The finding of the reciprocal paraphyly of Hexapoda and Crustacea suggests an evolutionary scenario in which the acquisition of the hexapod condition may have occurred several times independently in lineages descending from different crustacean-like ancestors, possibly as a consequence of the process of terrestrialization. If this hypothesis was confirmed, we should therefore re-think our interpretation of the evolution of the Arthropoda, where terrestrialization may have led to the acquisition of similar anatomical features by convergence. At the same time, the disagreement between reconstructions based on morphological, nuclear and mitochondrial data sets seems to remain, despite the use of larger data sets and more powerful analytical methods.

     

Testing for misleading effects in the phylogenetic reconstruction of ancient lineages of hexapods: influence of character dependence and character choice in analyses of 28S rRNA sequences

The present analyses employ the almost complete sequence of the 28S rRNA gene to investigate phylogenetic relationships among Pancrustacea, placing special emphasis on the position of basal hexapod lineages. This study utilizes a greater number of characters and taxa of Protura, Collembola and Diplura than previous analyses to focus on conflicts in the reconstruction of the early steps in hexapod evolution. Phylogenetic trees are mainly based on Bayesian approaches, but likewise include analyses with Maximum Likelihood and Maximum Parsimony. Different analyses, including the application of a mixed DNA/RNA substitution model, were performed to narrow possible misleading effects of non-stationarity of nucleotide frequencies, saturation and character independence down to a minimum. This is the first time that a mixed DNA/RNA model is applied to analyse 28S rRNA sequences of basal hexapods. All methods yielded strong support for the monophyly of Collembola, Diplura, Dicondylia and Insecta s.str. , as well as for a cluster composed of Diplura and Protura ('Nonoculata-hypothesis'). However, the last cluster may be an artifact caused by a shared GC bias of the 28S sequences between these orders, in combination with a long branch effect. The instability of the position of the 'Nonoculata' within Pancrustacea further bears out the misleading effect of non-stationarity of nucleotide frequencies. Protura and Diplura either form the sister-group to Collembola (Entognatha) or cluster with branchiopod crustaceans. Overall, the phylogenetic signal of the complete sequences of the 28S rRNA gene favours monophyly of Hexapoda over paraphyly. However, further corroboration from independent data is needed to rule out the competing hypothesis of mutually paraphyletic Crustacea and Hexapoda.     

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.     

Brain organization in Collembola (springtails)

Arthropoda is comprised of four major taxa: Hexapoda, Crustacea, Myriapoda and Chelicerata. Although this classification is widely accepted, there is still some debate about the internal relationships of these groups. In particular, the phylogenetic position of Collembola remains enigmatic. Some molecular studies place Collembola into a close relationship to Protura and Diplura within the monophyletic Hexapoda, but this placement is not universally accepted, as Collembola is also regarded as either the sister group to Branchiopoda (a crustacean taxon) or to Pancrustacea (crustaceans + hexapods). To contribute to the current debate on the phylogenetic position of Collembola, we examined the brains in three collembolan species: Folsomia candida, Protaphorura armata and Tetrodontophora bielanensis, using antennal backfills, series of semi-thin sections, and immunostaining technique with several antisera, in conjunction with confocal laser scanning microscopy and three-dimensional reconstructions. We identified several neuroanatomical structures in the collembolan brain, including a fan-shaped central body showing a columnar organization, a protocerebral bridge, one pair of antennal lobes with 20-30 spheroidal glomeruli each, and a structure, which we interpret as a simply organized mushroom body. The results of our neuroanatomical study are consistent with the phylogenetic position of Collembola within the Hexapoda and do not contradict the hypothesis of a close relationship of Collembola, Protura and Diplura.     

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).      

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).     

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.     

First molecular evidence for the existence of a Tardigrada + Arthropoda clade

The complete 18S rDNA gene sequence of Macrobiotus group hufelandi (Tardigrada) was obtained and aligned with 18S rDNA and rRNA gene sequences of 24 metazoans (mainly protostomes). Discrete character (maximum-parsimony) and distance (neighbor-joining) methods were used to infer their phylogeny. The evolution of bootstrap proportions with sequence length (pattern of resolved nodes, PRN) was studied to test the resolution of the nodes in neighbor-joining trees. The results show that arthropods are monophyletic. Tardigrades represent the sister group of arthropods (in parsimony analyses) or they are related with crustaceans (distance analysis and PRN). Arthropoda are divided into two main evolutionary lines, the Hexapoda + Crustacea line (weakly supported), and the Myriapoda + Chelicerata line. The Hexapoda + Crustacea line includes Pentastomida, but the internal resolution is far from clear. The Insecta (Ectognatha) are monophyletic, but no evidence for the monophyly of Hexapoda is found. The Chelicerata are a monophyletic group and the Myriapoda cluster close to Arachnida. Overall, the results obtained represent the first molecular evidence for a Tardigrada + Arthropoda clade. In addition, the congruence between molecular phylogenies of the Arthropoda from other authors and this obtained here indicates the need to review those obtained solely on morphological characters.      

The complete sequence of the mitochondrial genome of the crustacean Penaeus monodon: are malacostracan crustaceans more closely related to insects than to branchiopods?

The complete sequence of the mitochondrial genome of the giant tiger prawn, Penaeus monodon (Arthropoda, Crustacea, Malacostraca), is presented. The gene content and gene order are identical to those observed in Drosophila yakuba. The overall AT composition is lower than that observed in the known insect mitochondrial genomes, but higher than that observed in the other two crustaceans for which complete mitochondrial sequence is available. Analysis of the effect of nucleotide bias on codon composition across the Arthropoda reveals a trend with the crustaceans represented showing the lowest proportion of AT-rich codons in mitochondrial protein genes. Phylogenetic analysis among arthropods using concatenated protein-coding sequences provides further support for the possibility that Crustacea are paraphyletic. Furthermore, in contrast to data from the nuclear gene EF1alpha, the first complete sequence of a malacostracan mitochondrial genome supports the possibility that Malacostraca are more closely related to Insecta than to Branchiopoda.     

Ecdysozoan phylogeny and Bayesian inference: first use of nearly complete 28S and 18S rRNA gene sequences to classify the arthropods and their kin

Relationships among the ecdysozoans, or molting animals, have been difficult to resolve. Here, we use nearly complete 28S+18S ribosomal RNA gene sequences to estimate the relations of 35 ecdysozoan taxa, including newly obtained 28S sequences from 25 of these. The tree-building algorithms were likelihood-based Bayesian inference and minimum-evolution analysis of LogDet-transformed distances, and hypotheses were tested wth parametric bootstrapping. Better taxonomic resolution and recovery of established taxa were obtained here, especially with Bayesian inference, than in previous parsimony-based studies that used 18S rRNA sequences (or 18S plus small parts of 28S). In our gene trees, priapulan worms represent the basal ecdysozoans, followed by nematomorphs, or nematomorphs plus nematodes, followed by Panarthropoda. Panarthropoda was monophyletic with high support, although the relationships among its three phyla (arthropods, onychophorans, tardigrades) remain uncertain. The four groups of arthropods-hexapods (insects and related forms), crustaceans, chelicerates (spiders, scorpions, horseshoe crabs), and myriapods (centipedes, millipedes, and relatives)-formed two well-supported clades: Hexapoda in a paraphyletic crustacea (Pancrustacea), and 'Chelicerata+Myriapoda' (a clade that we name 'Paradoxopoda'). Pycnogonids (sea spiders) were either chelicerates or part of the 'chelicerate+myriapod' clade, but not basal arthropods. Certain clades derived from morphological taxonomy, such as Mandibulata, Atelocerata, Schizoramia, Maxillopoda and Cycloneuralia, are inconsistent with these rRNA data. The 28S gene contained more signal than the 18S gene, and contributed to the improved phylogenetic resolution. Our findings are similar to those obtained from mitochondrial and nuclear (e.g., elongation factor, RNA polymerase, Hox) protein-encoding genes, and should revive interest in using rRNA genes to study arthropod and ecdysozoan relationships.     

Genealogical portraits of speciation in montane grasshoppers (genus Melanoplus) from the sky islands of the Rocky Mountains

Grasshoppers in the genus Melanoplus have undergone a radiation in the 'sky islands' of western North America, with many species originating during the Pleistocene. Despite their recent origins, phylogenetic analyses indicate that all the species exhibit monophyletic or paraphyletic gene trees. The objectives of this study were to determine whether the monophyletic genealogies are the result of a bottleneck at speciation and to investigate the extent to which the different phylogenetic states of eight species (i.e. monophyletic versus paraphyletic gene trees) can be ascribed to the effects of speciation. A coalescent simulation was used to test for a bottleneck at speciation in each species. The effective population sizes and demographic histories of species were compared across taxa to evaluate the possibility that the paraphyly versus monophyly of the species reflects differential rates of lineage loss rather than speciation mode. While coalescent analyses indicate that the monophyly of Melanoplus species might not be indicative of bottlenecks at speciation, the results suggest that the paraphyletic gene trees may reflect the demography of speciation, involving localized divergences in the ancestral species. With respect to different models of Pleistocene divergence, the data do not support a model of founder-effect speciation but are compatible with divergence in allopatric refugia.     

The complete mitochondrial genome of the black mud crab, Scylla serrata (Crustacea: Brachyura: Portunidae) and its phylogenetic position among (pan)crustaceans

The black mud crab, Scylla serrata (Forsk?l 1775), is the most economically important edible crab in South-East Asia. In the present study, the complete mitochondrial genome of black mud crab, S.?serrata, was determined with the sequential polymerase chain reaction and primer walking sequencing. The complete mitochondrial genome was 15,721?bp in length with an A+T content of 69.2?% and contained 37 mitochondrial genes (13 protein coding genes (PCGs), 2 ribosomal RNA genes and 22 transfer RNA genes) and a control region (CR). The analysis of the CR sequence shows that it contains a multitude of repetitive fragments which can fold into hairpin-like or secondary structures and conserved elements as in other arthropods. The gene order of S. serrata mainly retains as the pancrustacean ground pattern, except for a single translocation of trnH. The gene arrangement of S. serrata appears to be a typical feature of portunid crabs. Phylogenetic analyses with concatenated amino acid sequences of 12 PCGs establishes that S. serrata in a well-supported monophyletic Portunidae and is consistent with previous morphological classification. Moreover, the phylogenomic results strongly support monophyletic Pancrustacea (Hexapoda plus “Crustaceans”). Within Pancrustacea, this study identifies Malacostraca?+?Entomostraca and Branchiopoda as the sister group to Hexapoda, which confirms that “Crustacea” is not monophyletic. Cirripedia?+?Remipedia appear to be a basal lineage of Pancrustacea. The present study also provides considerable data for the application of both population and phylogenetic studies of other crab species.     

The complete mitochondrial genome of Pollicipes mitella (Crustacea, Maxillopoda, Cirripedia): non-monophylies of maxillopoda and crustacea

The whole mitochondrial genome (14,915 nt) of Pollicipes mitella (Crustacea, Maxillopoda, Cirripedia, Thoracica) was sequenced and characterized. It is the shortest of the 31 completely sequenced crustacean mitochondrial genomes, with the exception of a copepod Tigriopus japonicus (14,628 nt). It consists of the usual 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 relatively short non-coding region (294 nt). The thoracican cirripeds apart from Megabalanus volcano have the same arrangement of protein-coding genes as Limulus polypemus, but there are frequent tRNA gene translocations (at least 8). Some interesting translocation features that may be specific to the thoracican cirriped lineage are as follows: 1) trnK-trnQ lies between the control region and trnI, 2) trnA-trnE lies between trnN and trnS1, 3) trnP lies between ND4L and trnT, and 4) trnY-trnC lies between trnS2 and ND1. In P. mitella there are two trnL genes (L1 and L2) in the typical crustacean positions (ND1-L1-LrRNA and CO1-L2-CO2). The present result is compared and discussed with the other three cirriped mitochondrial genomes from one pedunculate (Pollicipes polymerus) and two sessiles (Tetraclita japonica and M. volcano) published so far. Mitochondrial protein phylogenies reconstructed by the BI and ML algorithms show that the thoracican Cirripedia is monophyletic (BPP 100/BP 100) and associated with Remipedia (BPP 98/BP 35). In addition, Oligostraca, including Ostracoda, Branchiura, and Pentastomida, is a monophyletic group (BPP 99/BP 68), and is basal to all the other examined arthropods. Remipedia + Cirripedia appears as an independent lineage within Arthropoda, apart from Thoracopoda (Malacostraca, Branchiopda, and Cephalocarida). The Thoracopoda is paraphyletic to Hexapoda. The present result suggests that the monophylies of Crustacea and Maxillopoda should be reconsidered.     

The causes of mitochondrial DNA gene tree paraphyly in birds

Gene tree paraphyly is a potentially serious problem because many phylogenetic and phylogeographic studies assume species are monophyletic. Funk and Omland (Funk, D.J., Omland, K.E., 2003. Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu. Rev. Ecol. Evol. Syst. 34, 397–423) found that a seemingly high proportion of bird species (16.7%) were paraphyletic in their mtDNA gene trees. This could imply that mtDNA is an unreliable or even misleading marker for delimiting species. We expand on Funk and Omland’s survey and identify the causes of species-level paraphyly in birds. We find that in most cases paraphyly is caused by incorrect taxonomy. In such cases, mtDNA serves systematics by exposing and clarifying taxonomic errors. We find the next most common cause of paraphyly to be incomplete lineage sorting due to recent speciation. Here mtDNA gives a consistent picture of evolution, given the timeframe, but it is not useful for delimiting species and other criteria must be employed. There were relatively few clear instances of paraphyly due to hybridization, though there were more cases where incomplete lineage sorting and hybridization could not be distinguished. We ultimately conclude that, far from a hindrance, mtDNA is generally a useful tool that should continue to facilitate delimitation of avian species.     

Pancrustacean phylogeny in the light of new phylogenomic data: support for Remipedia as the possible sister group of Hexapoda

Remipedes are a small and enigmatic group of crustaceans, first described only 30 years ago. Analyses of both morphological and molecular data have recently suggested a close relationship between Remipedia and Hexapoda. If true, the remipedes occupy an important position in pancrustacean evolution and may be pivotal for understanding the evolutionary history of crustaceans and hexapods. However, it is important to test this hypothesis using new data and new types of analytical approaches. Here, we assembled a phylogenomic data set of 131 taxa, incorporating newly generated 454 expressed sequence tag (EST) data from six species of crustaceans, representing five lineages (Remipedia, Laevicaudata, Spinicaudata, Ostracoda, and Malacostraca). This data set includes all crustacean species for which EST data are available (46 species), and our largest alignment encompasses 866,479 amino acid positions and 1,886 genes. A series of phylogenomic analyses was performed to evaluate pancrustacean relationships. We significantly improved the quality of our data for predicting putative orthologous genes and for generating data subsets by matrix reduction procedures, thereby improving the signal to noise ratio in the data. Eight different data sets were constructed, representing various combinations of orthologous genes, data subsets, and taxa. Our results demonstrate that the different ways to compile an initial data set of core orthologs and the selection of data subsets by matrix reduction can have marked effects on the reconstructed phylogenetic trees. Nonetheless, all eight data sets strongly support Pancrustacea with Remipedia as the sister group to Hexapoda. This is the first time that a sister group relationship of Remipedia and Hexapoda has been inferred using a comprehensive phylogenomic data set that is based on EST data. We also show that selecting data subsets with increased overall signal can help to identify and prevent artifacts in phylogenetic analyses.