Stefan Koenemann

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.

Crustacea and Arthropod Relationships

INTRODUCTION Gould, Schram, and the paleontological perspective in evolutionary biology C. Baron & J.T. Hoeg PALEOZOOLOGY Decapod crustaceans, the K/P event, and Palaeocene recovery C.E. Schweitzer & R.M. Feldmann Oelandocaris oelandica and the stem lineage of Crustacea M. Stein, D. Waloszek & A. Maas Early Palaeozoic non-lamellipedian arthropods J. Bergstrom & X.-G. Hou Comparative morphology and relationships of the Agnostida T.J. Cotton & R.A. Fortey DEVELOPMENT AND EVOLUTION Heads, Hox and the phylogenetic position of trilobites G. Scholtz & G.D. Edgecombe Resolving arthropod relationships: Present and future insights from evo-devo studies S. Hrycaj & A. Popadi COMPARATIVE MORPHOLOGY Evolution of eye structure and arthropod phylogeny C. Bitsch & J. Bitsch Appendage loss and regeneration in arthropods: A comparative view D. Maruzzo, L. Bonato, C. Brena, G. Fusco & A. Minelli ARTHROPOD PHYLOGENETICS What are Ostracoda? A cladistic analysis of the extant superfamilies of the subclasses Myodocopa and Podocopa (Crustacea: Ostracoda) D.J. Horne, I. Schon, R.J. Smith & K. Martens Relationships within the Pancrustacea: Examining the influence of additional Malacostracan 18S and 28S rDNA C.C. Babbit & N.H. Patel Relationships between hexapods and crustaceans based on four mitochondrial genes A. Carapelli, F. Nardi, R. Dallai, J.L. Boore, P. Lio & F. Frati The position of crustaceans within the Arthropoda - Evidence from nine molecular loci and morphology G. Giribet, S. Richter, G.D. Edgecombe & W.C. Wheeler METAZOAN PHYLOGENETICS Playing another round of metazoan phylogenetics: Historical epistemology, sensitivity analysis, and the position of Arthropoda within the Metazoa on the basis of morphology R.A. Jenner & G. Scholtz Appendices: Publications of Frederick R. Schram Taxa erected by or in collaboration with F.R. Schram Color insert

Arthropod phylogeny revisited, with a focus on crustacean relationships.

Higher-level arthropod phylogenetics is an intensely active field of research, not least as a result of the hegemony of molecular data. However, not all areas of arthropod phylogenetics have so far received equal attention. The application of molecular data to infer a comprehensive phylogeny of Crustacea is still in its infancy, and several emerging results are conspicuously at odds with morphology-based studies. In this study, we present a series of molecular phylogenetic analyses of 88 arthropods, including 57 crustaceans, representing all the major lineages, with Onychophora and Tardigrada as outgroups. Our analyses are based on published and new sequences for two mitochondrial markers, 16S rDNA and cytochrome c oxidase subunit I (COI), and the nuclear ribosomal gene 18S rDNA. We designed our phylogenetic analyses to assess the effects of different strategies of sequence alignment, alignment masking, nucleotide coding, and model settings. Our comparisons show that alignment optimization of ribosomal markers based on secondary structure information can have a radical impact on phylogenetic reconstruction. Trees based on optimized alignments recover monophyletic Arthropoda (excluding Onychophora), Pancrustacea, Malacostraca, Insecta, Myriapoda and Chelicerata, while Maxillopoda and Hexapoda emerge as paraphyletic groups. Our results are unable to resolve the highest-level relationships within Arthropoda, and none of our trees supports the monophyly of Myriochelata or Mandibulata. We discuss our results in the context of both the methodological variations between different analyses, and of recently proposed phylogenetic hypotheses. This article offers a preliminary attempt to incorporate the large diversity of crustaceans into a single molecular phylogenetic analysis, assessing the robustness of phylogenetic relationships under varying analysis parameters. It throws into sharp relief the relative strengths and shortcomings of the combined molecular data for assessing this challenging phylogenetic problem, and thereby provides useful pointers for future studies.

Global Biodiversity and Phylogenetic Evaluation of Remipedia (Crustacea)

Remipedia is one of the most recently discovered classes of crustaceans, first described in 1981 from anchialine caves in the Bahamas Archipelago. The class is divided into the order Enantiopoda, represented by two fossil species, and Nectiopoda, which contains all known extant remipedes. Since their discovery, the number of nectiopodan species has increased to 24, half of which were described during the last decade. Nectiopoda exhibit a disjunct global distribution pattern, with the highest abundance and diversity in the Caribbean region, and isolated species in the Canary Islands and in Western Australia. Our review of Remipedia provides an overview of their ecological characteristics, including a detailed list of all anchialine marine caves, from which species have been recorded. We discuss alternative hypotheses of the phylogenetic position of Remipedia within Arthropoda, and present first results of an ongoing molecular-phylogenetic analysis that do not support the monophyly of several nectiopodan taxa. We believe that a taxonomic revision of Remipedia is absolutely essential, and that a comprehensive revision should include a reappraisal of the fossil record.

Post‐embryonic development of remipede crustaceans

SUMMARY During diving explorations of anchialine cave systems on Abaco Island, Bahamas, we collected five larvae that represent different developmental stages of remipede crustaceans. Based on four early naupliar stages and a post‐naupliar larva, it is possible for the first time to reconstruct the postembryonic development of Remipedia some 25 years after their discovery. These specimens begin to fill in some critical gaps in our knowledge of this important group of crustaceans.

Phylogenetic Analysis and Systematic Revision of Remipedia (Nectiopoda) From Bayesian Analysis of Molecular Data

We performed a phylogenetic analysis of the crustacean class Remipedia. For this purpose, we generated sequences of three different molecular markers, 16S rRNA (16S), histone 3 (H3), and cytochrome c oxidase subunit I (COI). The analyses included sequences from 20 of the 27 recent species of Remipedia, plus four still-undescribed species. The data matrix was complemented with sequences from online databases (The European Molecular Biology Laboratory and GenBank®). Campodea tillyardi (Diplura), Hutchinsoniella macracantha (Cephalocarida), Penaeus monodon (Malacostraca) and Branchinella occidentalis (Branchiopoda) served as out-groups. In addition to the classic computer-based alignment methods used for protein-coding markers (H3 and COI), an alternative approach combining structural alignment and manual optimization was used for 16S. The results of our analyses uncovered several inconsistencies with the current taxonomic classification of Remipedia. Godzilliidae and the genera Speleonectes and Lasionectes are polyphyletic, while Speleonectidae emerges as a paraphyletic group. We discuss current taxonomic diagnoses based on morphologic characters, and suggest a taxonomic revision that accords with the topologies of the phylogenetic analyses. Three new families (Kumongidae, Pleomothridae, and Cryptocorynetidae) as well as three new genera (Kumonga, Angirasu, and Xibalbanus) are erected. The family Morlockiidae and the genus Morlockia are removed from synonymy and returned to separate status.

Behavior of Remipedia in the laboratory, with supporting field observations

Abstract We observed in the laboratory the behavior of six individuals of an as yet undescribed species of Speleonectes (Remipedia) over a period of 76 days. The live specimens were collected from an anchialine cave on the Yucatan Peninsula and maintained in separate aquaria at the Zoological Museum Amsterdam. In addition, field observations were conducted in the same cave to compare the laboratory results with naturally occurring behaviors. We found a variety of complex behavioral traits that include several new and unexpected findings. For example, our observations suggest that remipedes are not obligatory, but rather facultative carnivores, and that filtering particles might be the predominant mode of feeding. A digital video with examples of various behavioral traits can be downloaded at http://www.tiho-hannover.de/einricht/botanik/research.htm.

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon

BackgroundRemipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated α-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa.ResultsThe dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes.ConclusionsThe brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.

A new, disjunct species of Speleonectes (Remipedia, Crustacea) from the Canary Islands

We describe Speleonectes atlantida n. sp. as the third species of Remipedia that was found outside the main distribution area of this group in the Caribbean region. S. atlantida was collected by cave divers equipped with closed circuit rebreathers from the far interior of the Túnel de la Atlántida, an anchialine volcanic lava tube, on the Canarian island of Lanzarote. The new species occurs in sympatry with S. ondinae, to which it is morphologically closely related. S. atlantida can be distinguished from S. ondinae by a more slender habitus and smaller pleurotergites in the posterior trunk. The valid status of S. atlantida as a new species of Remipedia could be corroborated by intra- and interspecific comparisons of 16S rDNA and CO1 sequence data.

Serotonin-immunoreactive neurons in the ventral nerve cord of Remipedia (Crustacea): support for a sister group relationship of Remipedia and Hexapoda?

BackgroundRemipedia were initially seen as a primitive taxon within Pancrustacea based on characters considered ancestral, such as the homonomously segmented trunk. Meanwhile, several morphological and molecular studies proposed a more derived position of Remipedia within Pancrustacea, including a sister group relationship to Hexapoda. Because of these conflicting hypotheses, fresh data are crucial to contribute new insights into euarthropod phylogeny. The architecture of individually identifiable serotonin-immunoreactive neurons has successfully been used for phylogenetic considerations in Euarthropoda. Here, we identified neurons in three species of Remipedia with an antiserum against serotonin and compared our findings to reconstructed ground patterns in other euarthropod taxa. Additionally, we traced neurite connectivity and neuropil outlines using antisera against acetylated α-tubulin and synapsin.ResultsThe ventral nerve cord of Remipedia displays a typical rope-ladder-like arrangement of separate metameric ganglia linked by paired longitudinally projecting connectives. The peripheral projections comprise an intersegmental nerve, consisting of two branches that fuse shortly after exiting the connectives, and the segmental anterior and posterior nerve. The distribution and morphology of serotonin-immunoreactive interneurons in the trunk segments is highly conserved within the remipede species we analyzed, which allows for the reconstruction of a ground pattern: two posterior and one anterior pair of serotonin-immunoreactive neurons that possess a single contralateral projection. Additionally, three pairs of immunoreactive neurons are found in the medial part of each hemiganglion. In one species (Cryptocorynetes haptodiscus), the anterior pair of immunoreactive neurons is missing.ConclusionsThe anatomy of the remipede ventral nerve cord with its separate metameric ganglia mirrors the external morphology of the animal’s trunk. The rope-ladder-like structure and principal architecture of the segmental ganglia in Remipedia corresponds closely to that of other Euarthropoda. A comparison of the serotonin-immunoreactive cell arrangement of Remipedia to reconstructed ground patterns of major euarthropod taxa supports a homology of the anterior and posterior neurons in Pancrustacea. These neurons in Remipedia possess unbranched projections across the midline, pointing towards similarities to the hexapod pattern. Our findings are in line with a growing number of phylogenetic investigations proposing Remipedia to be a rather derived crustacean lineage that perhaps has close affinities to Hexapoda.