Phylogeny and mitochondrial gene order variation in Lophotrochozoa in the light of new mitogenomic data from Nemertea

<p>Abstract</p> <p>Background</p> <p>The new animal phylogeny established several taxa which were not identified by morphological analyses, most prominently the Ecdysozoa (arthropods, roundworms, priapulids and others) and Lophotrochozoa (molluscs, annelids, brachiopods and others). Lophotrochozoan interrelationships are under discussion, e.g. regarding the position of Nemertea (ribbon worms), which were discussed to be sister group to e.g. Mollusca, Brachiozoa or Platyhelminthes. Mitochondrial genomes contributed well with sequence data and gene order characters to the deep metazoan phylogeny debate.</p> <p>Results</p> <p>In this study we present the first complete mitochondrial genome record for a member of the Nemertea, <it>Lineus viridis</it>. Except two <it>trnP </it>and <it>trnT</it>, all genes are located on the same strand. While gene order is most similar to that of the brachiopod <it>Terebratulina retusa</it>, sequence based analyses of mitochondrial genes place nemerteans close to molluscs, phoronids and entoprocts without clear preference for one of these taxa as sister group.</p> <p>Conclusion</p> <p>Almost all recent analyses with large datasets show good support for a taxon comprising Annelida, Mollusca, Brachiopoda, Phoronida and Nemertea. But the relationships among these taxa vary between different studies. The analysis of gene order differences gives evidence for a multiple independent occurrence of a large inversion in the mitochondrial genome of Lophotrochozoa and a re-inversion of the same part in gastropods. We hypothesize that some regions of the genome have a higher chance for intramolecular recombination than others and gene order data have to be analysed carefully to detect convergent rearrangement events.</p

Nemertean taxonomy- Implementing changes in the higher ranks, dismissing Anopla and Enopla

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comparative investigations of reproduction and development

1 Einleitung 1.1 Anatomie der Nemertea 1.2 Systematik der Nemertea 1.3 Phylogenetische Stellung der Nemertea und Arbeitshypothesen 1.4 Aufgaben und Ziele der Arbeit 2 Material und Methoden 2.1 Untersuchte Arten 2.2 Zucht und Hälterung 2.3 Fixierungen 2.4 Konfokale Laserscan-Mikroskopie (cLSM) 2.5 Transmissionselektronenmikroskopie (TEM) 2.6 Aufbau der Dissertation 3 Veröffentlichte oder zur Publikation eingereichte Ergebnisse 3.1 Die Reproduktion von Lineus viridis (Heteronemertea) weist auf pheromon- vermitteltes Paarungsverhalten hin 3.2 Ultrastruktur der Spermien und männlichen Gonaden von Lineus viridis (Heternonemertea) 3.3 Das Schicksal der larvalen Epidermis der intrakapsulären Larve von Lineus viridis (Heteronemertea, Nemertea) 3.4 Ultrastruktur und Entwicklung der rhabdomerischen Augen von Lineus viridis (Heteronemertea, Nemertea) 3.5 Vergleichende Ultrastruktur der Spermien von Nemertinen 3.6 Larvalentwicklung mit transitorischer Epidermis von Paranemertes peregrina und anderen Hoplonemertinen (Nemertea) 3.7 Kopfnieren der planuliformen Larven von Procephalothrix filiformis und Carinoma mutabilis (Nemertea) 4 Unveröffentlichte Ergebnisse 4.1 Entwicklung der Muskulatur 4.1.1 Procephalothrix oestrymnicus Junoy & Gibson, 1991 („Palaeonemertea“) 4.1.2 Lineus viridis (Müller, 1774) (Heteronemertea) 4.1.3 Paranemertes peregrina Coe, 1901 (Hoplonemertea) 4.1.4 Carinoma mutabilis Griffin, 1898 („Palaeonemertea“) 4.1.5 Bewertung der Befunde 4.2 Ultrastruktur der Augen 4.2.1 Carinoma mutabilis Griffin, 1898 („Palaeonemertea“) 4.2.2 Procephalothrix oestrymnicus Junoy & Gibson, 1991 („Palaeonemertea“) 4.2.3 Paranemertes peregrina Coe, 1901 (Hoplonemertea) 4.2.4 Bewertung der Befunde 5 Diskussion 5.1 Lineus viridis (Heteronemertea) 5.2 Vergleichende Spermienultrastruktur – Bedeutung für die Nemertinen-Phylogenie 5.3 Merkmale der larvalen Organisation 5.3.1 Metamorphose 5.3.2 Planuliforme Larve 5.3.3 Muskeldifferenzierung 5.3.4 Augen 5.3.5 Protonephridiale Kopfnieren 5.4 Phylogenetische Stellung der Nemertea 5.4.1 Nemertea als Schwestergruppe der Plathelminthes 5.4.2 Nemertea: eine Teilgruppe der Trochozoa oder deren Schwestergruppe? 6 Zusammenfassung 7 Summary 8 Literaturverzeichnis AnhangNemertea (Schnurwürmer) sind unsegmentierte, wurmförmige Spiralier, deren Monophylie aufgrund einer Reihe von Autapomorphien sicher begründet ist. Sie besitzen einen ausstülpbaren Rüssel, der in Ruhe in einem flüssigkeitsgefülltem Hohlraum liegt, ein ringförmiges Cerebralganglion, das sich statt um den Schlund um die Insertion des Rüssels legt, und ein mit einem Epithel ausgekleidetes laterales Gefäßsystem. Abgesehen davon gelten sie als merkmalsarm, was dazu geführt hat, dass sie als basale Spiralier gewertet und in Verwandtschaft mit den Plathelminthes gestellt wurden. Neuere Hypothesen sehen die Nemertea an der Basis der Trochozoa oder platzieren sie innerhalb der Trochozoa. Jede der formulierten Hypothesen lässt eine spezifische Merkmalskombination in der Ausbildung des Hautmuskelschlauches, der Nierenorgane und der pigmentierten Augen erwarten. Daten zu diesen Organsystemen sind bei Nemertinen, im Gegensatz zu den möglichen Schwestergruppen, nicht ausreichend vorhanden, um die genannten Verwandtschafts-hypothesen zu bewerten. Insbesondere für die basalen Teilgruppen der Nemertea sind diese Daten nicht bekannt, wären aber für eine Evaluation der Merkmalspolarität von besonderer Relevanz. Zur Klärung der phylogenetischen Stellung der Nemertea innerhalb der Spiralia wurden die Entwicklung der pigmentierten Augen und der Muskulatur sowie die Ultrastruktur der Nierenorgane in Larven und Jungtieren von Vertretern der meisten hochrangigen Teiltaxa der Nemertea vergleichend untersucht. Im Zuge dieser Arbeit wurden die Kenntnisse zur Biologie einer der häufigsten europäischen Nemertinen-Arten, Lineus viridis, erweitert und eine vergleichende Untersuchung zur Spermienmorphologie zur Klärung der Innengruppen-verhältnisse bei den Nemertea durchgeführt. Deren Ergebnisse bestätigen aktuelle Hypothesen über die Verwandtschaftsverhältnisse innerhalb der Nemertea und erlauben eine Bewertung der konkurrierenden Verwandtschaftshypothesen zu anderen Spiralia. Aufgrund der neuen Daten kann ein Schwestergruppenverhältnis mit den Plathelminthes ausgeschlossen werden. Ob die Nemertea an die Basis der Spiralia, als Schwestergruppe der übrigen Trochozoa oder ein Teiltaxon der Trochozoa darstellen, ist in weiteren Untersuchungen zu klären. Die bisherigen Befunde lassen sich am sparsamsten durch ein Schwestergruppen-verhältnis zwischen den Nemertea und den Trochozoa erklären.Nemertea are unsegmented spiralian worms whose monophyly is undisputed due to various autapomorphic traits. They possess an eversible proboscis, a ring- shaped cerebral ganglion that surrounds the proboscis insertion instead of the pharynx, and an epithelialized vascular system. In other respects they are character-poor and thus have formerly been regarded as basal Spiralia with closest affinities to Platyhelminthes. More recent hypotheses place the Nemertea as sister taxon to or among the Trochozoa, putatively with closest relationship to annelids. Each of these hypotheses implies a distinctive combination of character traits concerning the formation of the body wall musculature, larval nephridia, and pigmented eyes. In contrast to other spiralian taxa, data on these character systems are largely missing in nemerteans, which hampered an evaluation of the phylogenetic hypotheses thus far. Especially in the basally branching "palaeonemertean" subtaxa, which are essential for determining character polarities, the relevant data are unknown. In order to clarify the phylogenetic position of Nemertea within the Spiralia, comparative studies on the above mentioned organ systems were conducted for most of the high-ranking subtaxa of the Nemertea. Additional studies on the reproductive biology of one of the most common European nemertean species, Lineus viridis, were complemented by a comprehensive survey on nemertean sperm ultrastructure. The latter contributed to a clarification of the ingroup relations of the Nemertea with new morphological support for topologies that yet were merely based on analyses of molecular data. Data obtained permit a discussion of the currently competing phylogenetic hypotheses on the phylogenetic position of the Nemertea. A sister group relationship to Platyhelminthes is umambiguously rejected by the new data. Whether nemerteans form the sister group to remaining Trochozoa or are more closely related to annelids still requires further studies. Presently the most parsimonious explanation for the observed character states in Nemertea is a sister group relationship to Trochozoa

Z-Projections of immuno-stained specimens of <i>Carinina ochracea</i>.

<p>Orientation indicated by compass (d, dorsal; l, left; r, right; v, ventral); apical is up. A and B: serotonin-like immunoreactivity (<i>5HT-lir</i>). A: Larva at four days post-fertilization (<i>4-dpf</i>) (γ: 0.9). The number of neurites and neurons of the brain, displaying 5HT-lir, have increased both ventrally (<i>bv</i>) and dorsally (<i>bd</i>). From the ventral portion of the brain-ring neurite bundle (<i>br</i>) the first, weak 5HT-lir neurites of the lateral nerve cords (<i>nc</i>) are detectable. 5HT-lir signals of the peripheral plexus (<i>pp</i>) are visible as well as numerous 5HT-lir apical signals (<i>as</i>). Note the weak unspecific signals of epidermal mucus gland cells (<i>gc</i>) B: Larva at <i>7-dpf</i> (γ: 0.69). The neurites of the brain-ring (<i>br</i>) and the associated brain neurons (<i>bv</i> and <i>bd</i>) have further increased in number. The 5HT-lir dorsal neurite signals (<i>dn</i>) and neurite signals of the lateral nerve cords (<i>nc</i>) are extended posteriorly. More anteriorly located neurons (<i>ln</i>), associated with the lateral nerve cords, display 5HT-lir. 5HT-lir is also visible in the peripheral plexus (<i>pp</i>), the apical signals (<i>as</i>), and, albeit weak and unspecific, in some epidermal mucus gland cells (<i>gc</i>). C and D: FMRFamide-like immunoreactivity (RFa-lir). C: Larva at <i>2-dpf</i> (γ: 0.96). The first RFa-lir signal detectable is a neurite-like mass (<i>nm</i>) located dorso-medially, anterior of the mouth opening. D: Larva at <i>3-dpf</i> (γ: 1). The first pair of large, dorsal, RFa-lir neuron signals (<i>bd</i>) is visible. Their neurite signals project into the dorsal neurite-like mass that is continuous with the first RFa-lir neurites of the developing brain-ring (<i>br</i>). Note the unspecific signals of epidermal mucus gland cells (<i>gc</i>).</p

Development of the Nervous System of <i>Carinina ochracea</i> (Palaeonemer-tea, Nemertea)

<div><p>The various clades of Lophotrochozoa possess highly disparate adult morphologies. Most of them, including Nemertea (ribbon worms), are postulated to develop via a pelagic larva of the trochophora type, which is regarded as plesiomorphic in Lophotrochozoa. With respect to the nervous system, the trochophora larva displays a set of stereotypic features, including an apical organ and trochal neurites, both of which are lost at the onset of metamorphosis. In the investigated larvae of Nemertea, the nervous system is somewhat divergent from the postulated hypothetical trochophore-like pattern. Moreover, no detailed data is available for the “hidden” trochophore larva, the hypothetical ancestral larval type of palaeonemertean species. Therefore, the development of the nervous system in the larva of <i>Carinina ochracea</i>, a basally branching palaeonemertean species, was studied by means of immunofluorescence and confocal laserscanning microscopy. Like in the other investigated nemertean larvae, the prospective adult central nervous system in <i>C</i>. <i>ochracea</i> develops in an anterior to posterior direction, as an anterior brain with paired longitudinal nerve cords. Thus, development of the adult nervous system in Nemertea is largely congruent with currently accepted hypotheses of nervous system development in Spiralia. In early development, transitory apical, serotonin-like immunoreactive flask-shaped cells are initially present, but the trochal neurites that have been considered as pivotal to lophotrochozoan development, are absent. In the light of the above stated hypothesis, trochal neurites have to be interpreted as reduced in Nemertea. On the other hand, due to the unsettled systematic status of Palaeonemertea, more comparative data are desirable to answer the remaining questions regarding the evolution of nervous system development in Nemertea.</p></div

Schematic representation of nervous system development of <i>Carinina ochracea</i>.

<p>Serotonin-like (<i>5HT-lir</i>) and FMRFamide-like (<i>RFa-lir</i>) immunoreactivity are color coded in magenta and green respectively; the general nervous system architecture as outlined by synapsin-like immunoreactivity (<i>syn-lir</i>) is shown in grey. All larval stages are drawn to scale with apical tuft (<i>at</i>), epidermal cilia, fronto-lateral epidermal invaginations (<i>fi</i>), midgut (<i>mg</i>), mouth opening (<i>mo</i>), and the pigmented larval eye (<i>pe</i>) serving as landmarks (cilia of the frontal epidermal invaginations, the stomodaeum, the midgut, and epidermal cilia in the aspect of view omitted), apical is up. For clarity, the peripheral plexus is not shown. A and B: from left to right: lateral view from right, ventral view, dorsal view, lateral view from left. Since the nervous system is roughly symmetrical only one side is shown (left: 5HT-lir, right: RFa-lir). A: Larva at five days post-fertilization (<i>5-dpf</i>). B: Larva at <i>7-dpf</i>. Further explanations, see text.–<i>bd</i>, dorsal brain neuron; <i>br</i>, brain-ring; <i>bv</i>, vental brain neuron; <i>ln</i>, lateral nerve cord neuron; <i>dl</i>, dorso-lateral neuron; <i>dn</i>, dorsal nerve; <i>nc</i>, lateral nerve cord; <i>or</i>, oral ring neurite bundle; <i>po</i>, pre-oral neurite bundle; <i>so</i>, sub-oral neurite; <i>vl</i>, ventrolateral neuron.</p

Schematic representation of nervous system development of <i>Carinina ochracea</i>.

<p>Serotonin-like (<i>5HT-lir</i>) and FMRFamide-like (<i>RFa-lir</i>) immunoreactivity are color coded in magenta and green respectively; the general nervous system architecture as outlined by synapsin-like immunoreactivity (<i>syn-lir</i>) is shown in grey. All larval stages are drawn to scale with apical tuft (<i>at</i>), epidermal cilia, fronto-lateral epidermal invaginations (<i>fi</i>), midgut (<i>mg</i>), mouth opening (<i>mo</i>), and the pigmented larval eye (<i>pe</i>) serving as landmarks (cilia of the frontal epidermal invaginations, the stomodaeum, the midgut, and epidermal cilia in the aspect of view omitted), apical is up. A and B: left: lateral view from right, right: ventral view. Transitory neuronal/neuritic elements are indicated by stippling/dotted lines. A: Post-gastrula stage larva at one day post-fertilization (<i>1-dpf</i>). B: Larva at <i>2-dpf</i>. C: Larva at <i>3-dpf</i>. From left to right: lateral view from right, ventral view, dorsal view, lateral view from left. Since the nervous system is roughly symmetrical, only one side is shown (left: 5HT-lir, right: RFa-lir). For clarity, the peripheral plexus is not shown. Further explanations, see text.–<i>bd</i>, dorsal brain neuron; <i>br</i>, brain-ring; <i>bv</i>, vental brain neuron; <i>an1</i>, 1^st apical median (transitory) neuron; <i>an2</i>, 2^nd apical medio-ventral (transitory) neuron; <i>nc</i>, lateral nerve cord; <i>cn1</i>, 1^st caudal median (transitory) neuron; <i>cn2</i>, 2^nd caudal medio-dorsal (transitory) neuron; <i>nm</i>, dorso-median neurite mass.</p

Development of nervous system specific immunoreactivity in larvae of <i>Carinina ochracea</i>.

<p>Development of nervous system specific immunoreactivity in larvae of <i>Carinina ochracea</i>.</p

A simple method for long-term vital-staining of ciliated epidermal cells in aquatic larvae

Observing the process of growth and differentiation of tissues and organs is of crucial importance for the understanding of the evolution of organs in animals. Unfortunately, it is notoriously difficult to continuously monitor developmental processes due to the extended time they take. Long-term labeling of the tissues of interest represents a promising alternative to raise these pivotal data. In the case of the prototroch, a band of ciliated cells typical of marine, planktotrophic trochophora larvae, we were able to apply a long-term fluorescent vital-staining to the prototroch cells that remains detectable throughout further larval life. We were able to stain ciliated cells of planktonic larvae from different spiralian clades by using long-chain dialkylcarbocyanine dyes that are detectable in different fluorescent emission spectra in combination with a non-ionic surfactant. The larvae survived and developed normally, their ciliated cells retaining the originally applied fluorescent labels. Combined with additional fluorescent staining of the larvae after fixation, we provide an easy, versatile, and broadly applicable method to investigate the processes of the differentiation of epidermal organs in various aquatic larvae