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Evolution der Muskelentwicklung der (Crustacea) – Vergleichende Untersuchung ausgewählter Vertreter vor dem Hintergrund embryonaler und larvaler Transformationen

Kumulative Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Am Lehrstuhl für Allgemeine und Spezielle Zoologie des Instituts für Biowissenschaften an der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Rostock

vorgelegt von Günther Joseph Jirikowski, geboren am 24.11.1983 in Ulm / Baden-Württemberg Rostock, Februar 2015

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Gutachter:

1. Gutachter: Prof. Dr. Stefan Richter, Allgemeine und Spezielle Zoologie, Institut für Biowissenschaften, Universität Rostock

2. Gutachter: Prof. Dr. Gerhard Scholtz, Vergleichende Zoologie, Institut für Biologie, Humboldt-Universität zu Berlin,

Datum der Einreichung: 20. Februar 2015

Datum der Verteidigung: 17. April 2015

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4 Inhaltsverzeichnis

1 Einleitende Zusammenfassung ...... 7 1.1 Übersicht ...... 7 1.2 Die Malacostraca ...... 9 1.3 Einauplius und kryptische Larvalentwicklung ...... 10 1.4 Entwicklungsmodi der Malacostraca ...... 12 1.4.1 Larvalentwicklung ...... 12 1.4.1.1 Entwicklung über eine Naupliuslarve ...... 12 1.4.1.2 Entwicklung über eine Zoea-ähnliche Larve ...... 12 1.4.2 Pseudodirektentwicklung ...... 13 1.4.3 Direktentwicklung ...... 14 1.5 Heterochronie und Entwicklungsmodus...... 14 1.6 Embryogenese der Malacostraca und Entwicklung des Mesoderms ...... 16 1.7 Myogenese ...... 18 1.8 Ziele der Dissertation ...... 19 1.9 Ergebnisse ...... 20 1.9.1 Untersuchte Arten ...... 20 1.9.2 Beschreibung der Myogenese ...... 20 1.9.3 Topographie embryonaler Muskulatur ...... 21 1.9.4 Vergleichende Analyse des Entwicklungsverlaufes...... 23 1.9.4.1 Kodierung von Entwicklungssequenzen ...... 23 1.9.4.2 Heterochronieanalyse ...... 24 1.9.5 Ergebnisse der Heterochronienalyse ...... 25 1.10 Evolution der Entwicklung der Malacostraca ...... 27 1.10.1 Evolution des Entwicklungsmodus ...... 27 1.10.2 Evolution der Naupliuslarve und des Einaupliusstadiums ...... 33 1.10.3 Die evolutionäre Bedeutung von Mesoderm und Muskulatur ...... 34 1.11 Alternative phylogenetische Hypothesen ...... 35 1.12 Schlussfolgerungen ...... 35 1.13 Literaturverzeichnis der einleitenden Zusammenfassung ...... 37 2 Erklärung über den Eigenanteil an den Manuskripten ...... 43 3 Selbstständigkeitserklärung ...... 45 4 Danksagung ...... 46 5 5 Jirikowski GJ, Kreissl S, Richter S, Wolff C. 2010. Muscle development in the marbled - insights from an emerging model organism (Crustacea, Malacostraca, ). Development, genes and Evolution, 220:89-105...... 47 6 Jirikowski GJ, Richter S, Wolff C. 2013. Myogenesis of Malacostraca – the “egg-nauplius” concept revisited; Frontiers in Zoology, 10:76-103...... 67 7 Jirikowski GJ, Wolff C, Richter S. 2015. Evolution of Eumalacostracan development – new insights into loss and reacquisition of larval stages revealed by heterochrony analysis, Evo Devo, 6:4...... 97

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Abbildung 1. Kernfärbung an einem Metanauplius von Artemia franciscana (, ) (a) und dem Einaupliusstadium von heteropoda (Malacostraca, Decapoda, ) (b). Maßstabsbalken: 100µm.

1 Einleitende Zusammenfassung

1.1 Übersicht Die Malacostraca (Crustacea) beeindrucken neben der enormen Artenvielfalt und Disparität in Bezug auf adulte Tiere auch durch eine immense Diversität von Entwicklungsmodi, die sich in der Ausbildung unterschiedlichster embryonaler Morphologien oder Larvenformen zeigt. Die phylogenetischen Beziehungen innerhalb der Malacostraca konnten mit den Methoden der Molekularsystematik, trotz der rasanten technischen Fortschritte, bis zum heutigen Tage nicht mit ausreichender Zuverlässigkeit aufgeklärt werden (Jenner et al., 2009). Die gängigen Verwandtschaftshypothesen begründen sich auf morphologischen Merkmalen (Richter & Scholtz, 2001; Wirkner & Richter, 2010; Watling et al., 2000) und wurden wiederholt angezweifelt. Der ursprüngliche Entwicklungsmodus der Gruppe spielt dabei eine zentrale Rolle. Die Mehrzahl der 7 `Crustacea´ entwickelt sich über eine planktische Larve mit drei Extremitätenpaaren (erste Antenne, zweite Antenne und Mandibel), die als Nauplius bezeichnet wird (Abbildung 1a) (Walossek & Müller, 1990; Anderson, 1973; Lauterbach, 1986). Es ist unklar, ob die Naupliuslarve Teil des Grundmusters der Tetraconata () ist (Richter, 2002). Innerhalb der Malacostraca besitzen nur zwei Teilgruppen eine Naupliuslarve: die (Decapoda) (Martin et al., 2014) und die Euphausiacea (Martin & Gomez-Gutierrez, 2014). Die Ergebnisse neuerer phylogenetischer Analysen (Edgecombe et al., 2000; Regier et al., 2010; Reumont et al., 2012) deuten darauf hin, dass die Naupliuslarve in der Stammlinie der Malacostraca verlorengegangen ist. Nach der von Richter & Scholtz vorgeschlagenen Phylogenie der Malacostraca (Richter & Scholtz, 2001; Wirkner & Richter, 2010) ist die Naupliuslarve beider Gruppen sekundär innerhalb der Malacostraca entstanden. Scholtz (Scholtz, 2000) diskutiert in diesem Zusammenhang die mögliche Bedeutung des Einauplius (Abbildung 1b), eines Embryonalstadiums, das für das Grundmuster der Malacostraca anzunehmenden ist. Der Einauplius spiegelt die Morphologie der Naupliuslarve insofern wider, dass er nur Anlagen der ersten Antenne, der zweiten Antenne und der Mandibel besitzt. Scholtz (Scholtz, 2000) argumentiert, dass der Einauplius auf ein plesiomorphes Entwicklungsprogramm zurückzuführen ist, das von Vorfahren mit Naupliuslarve übernommen wurde. Zudem führt er aus, dass der Einauplius die Entstehung einer sekundären Naupliuslarve ermöglicht hat und dass für diese Transformation Heterochronien – Veränderungen im zeitlichen Verlauf der Entwicklung – eine fundamentale Rolle gespielt haben.

Die vorliegende Arbeit hat das Ziel, die Bedeutung des Einauplius für die Evolution des Entwicklungsmodus der Malacostraca zu prüfen. Dazu soll der Entwicklungsprozess als Ganzes und unter Einbeziehung der zugrundeliegenden zellulären Prozesse der Morphogenese einer vergleichenden Analyse unterzogen werden. Die Entwicklung des Mesoderms, insbesondere der Muskulatur, ist dabei von besonderem Interesse, da sie in der Diskussion des Einauplius (Scholtz, 2000) nicht ausreichend berücksichtigt wird. Der Schwerpunkt der vorliegenden Arbeit liegt deshalb darin, die Muskelentwicklung der Malacostraca zu beschreiben und in eine vergleichende Analyse einzubringen. Die Evolution des Entwicklungsprozesses innerhalb der Malacostraca soll mit computergestützten Methoden der Heterochronieanalyse rekonstruiert werden, um die Bedeutung von Heterochronie im Zusammenhang mit evolutiven Veränderungen des Entwicklungsmodus und der sekundären Evolution der Naupliuslarve zu klären.

8 1.2 Die Malacostraca `Crustacea´. Die Gruppe wurde 1802 von Latreille eingeführt und 1888 von Claus um die Gruppe erweitert (Claus, 1888). Der bisher älteste fossile Vertreter der Malacostraca ist Cinerocaris magnifica (Malacostraca, ) aus dem Silur (Briggs et al., 2004). Die Grundlage für eine morphologisch-phylogenetische Hypothese der Malacostraca wurde von Hansen geschaffen (Hansen, 1893). Er stellte die Leptostraca allen übrigen Malacostraca () als Schwestergruppe gegenüber. Sein System wurde in Calmans Klassifikation aufgenommen (Calman, 1909). Letzterer unterteilte die Eumalacostraca in (Stomatopoda),

Abbildung 2. Die phylogenetischen Beziehungen der Malacostraca, vereinfacht nach Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter 2010). Nicht gezeigte Taxa: (Decapoda), (), (Peracarida), (Peracarida) und (Peracarida). Aufgrund des Fehlens erscheinen und , bzw. Caridea und Reptantia in dieser Darstellung als Schwestergruppen.

(Euphausiacea und Decapoda) und Peracarida (, Cumacea, Tanaidacea, Isopoda und Amphipoda). Ich folge in der vorliegenden Arbeit den von Richter & Scholtz vorgeschlagenen phylogenetischen Beziehungen (Richter & Scholtz, 2001; Wirkner & Richter, 2010) (Abbildung 2).

Die Monophylie der Malacostraca wird u.a. durch die einheitliche Tagmatisierung begründet (Gruner, 1993; Richter & Scholtz, 2001; McLaughlin, 1980). Der Bauplan der Malacostraca beinhaltet den Kopf mit fünf, den Thorax mit acht und das Pleon mit sechs extremitätentragenden Segmenten. Dazu 9 kommt eine spezielle Bildungsweise des postnauplialen Keimstreifs durch teloblastische Proliferation von präanal gelegenen stammzellähnlichen Zellen, den Teloblasten. Weitere mögliche Apomorphien sind die konstante Lage der Geschlechtsöffnungen und die Bildung von drei optischen Neuropilen, die durch Chiasmata verknüpft sind.

Nach Richter & Scholtz (Richter & Scholtz, 2001; Richter & Wirkner 2010) sind die Leptostraca einem Taxon Eumalacostraca als Schwestergruppe gegenübergestellt (Abbildung 2). Innerhalb der Eumalacostraca bilden die Stomatopoda die Schwestergruppe der Caridoida. Letztere besitzen als Autapomorphie einen charakteristisch gestalteten pleonalen Muskelapparat und den spezialisierten caridoiden Fluchtreflex. Die Caridoida, wiederum werden in Decapoda und Xenommacarida unterteilt. Eine alternative Hypothese geht von einem Monophylum aus Euphausiacea, Decapoda und Amphionidacea aus, das als Eucarida bezeichnet wird (Calman, 1909; Spears et al., 2005; Watling et al., 2000; Wills, 1998). Als Apomorphie dieser Gruppe wird die Verschmelzung des Carapax mit allen acht Thoraxsegmenten genannt. Innerhalb der Decapoda bilden die Dendrobranchiata nach Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter 2010) die Schwestergruppe der , die sich wiederum aus Caridea und einem Monophylum aus Stenopodidea und Reptantia zusammensetzen. Die Xenommacarida umfassen die Gruppen , Euphausiacea, Pancarida () und Peracarida. Dabei bilden die Syncarida die Schwestergruppe eines Monophylums aus Euphausiacea und Neocarida (Pancarida + Peracarida). Die Peracarida besitzen als Autapomorphie einen ventralen Brutraum (Marsupium), der bei weiblichen Tieren gebildet wird und der Brutpflege dient.

1.3 Einauplius und kryptische Larvalentwicklung Bei Leptostraca (Manton, 1934; Olesen & Walossek, 2000; Pabst & Scholtz, 2009), Stomatopoda (Fischer & Scholtz, 2010; Shiino, 1942), Pleocyemata (Alwes & Scholtz, 2006; Öishi, 1960; Öishi, 1959; Scheidegger, 1976; Scholtz, 1992; Weygoldt, 1961), Syncarida (Hickman, 1936; Jakobi, 1958; Jakobi, 1954) und Thermosbaenacea (Zilch, 1974) tritt in der Embryonalentwicklung ein Einauplius auf (Scholtz, 2000). Dieses frühe Keimstreifstadium enthält die Anlagen der vordersten drei Extremitätenpaare (naupliale Extremitäten), die posterior folgenden (postnauplialen) Extremitätenanlagen fehlen jedoch oder stellen bestenfalls kleine epidermale Ausstülpungen dar (Abbildung 1). Die Gestalt des Einauplius kann zwischen verschiedenen Vertretern der Malacostraca hinsichtlich der Größe und Ausprägung der optischen Loben, des Labrums und der einzelnen Extremitätenanlagen sowie in der Form der postnauplial gelegenen Kaudalpapille variieren. In der vorliegenden Arbeit wird der Begriff Einaupliusstadium verwendet, um einen Einauplius im weiteren Sinne zu beschreiben. Dabei werden auch Embryonalstadien von Vertretern mit Naupliuslarve eingeschlossen (rot markierte Objekte in Abbildung 3). Der Einauplius sensu Scholtz (Scholtz, 2000)

10 ist dagegen als Alternative zu einer freischwimmenden Naupliuslarve definiert (rot markierte Objekte in Abbildung 3a,-b,-d,-e).

Dahms (Dahms, 2000) und Williams (Williams, 1994) stellen die These auf, dass die Crustacea ein phylotypisches Stadium durchlaufen, das sich in der Naupliuslarve und dem Einauplius gleichermaßen widerspiegelt. Diese Ansicht muss schon alleine deshalb kritisch betrachtet werden, weil ein Einauplius bei der Mehrzahl der Peracarida fehlt. Scholtz (Scholtz, 2000) interpretiert den Einauplius als `Rekapitulation´ der Naupliuslarve, jedoch nicht in einem Müller´schen/Haeckel´schen Sinne (Haeckel, 1905; Müller, 1864), sondern in einem modernen Sinne nach dem die Information zur Bildung einer Naupliuslarve in diesem Embryonalstadium noch aktiv ist, obwohl es nicht mehr zur Ausbildung der Larve kommt. Scholtz (Scholtz, 2000) führt aus, dass ausgehend von einem

Abbildung 3. Übersicht über die Entwicklungsmodi der Malacostraca, gezeigt an ausgewählten Vertretern in Form von vertikal dargestellten Stadienserien. a. bipes (Manton, 1934), b. Gonodactylus falcatus (Shanboghue, 1978), c. monodon (Motoh, 1981), d. varians (Weygoldt, 1961), e. Procambarus fallax forma virginalis (Alwes & Scholtz 2006), f. Hemimysis lamornae (Manton, 1928), g. Parhyale hawaiensis (Browne et al., 2005). Die Querreihen implizieren keine Homologie von Entwicklungsstadien zwischen Arten. Embryonalstadien sind durch einen Kreis gekennzeichnet, der die Eihülle symbolisiert. Bei Arten mit interner Brutpflege sind diejenigen Stadien, die sich im Brutraum befinden, mit einem schematisierten Adulttier gekennzeichnet. Das Einaupliusstadium ist rot markiert.

11 Entwicklungsmodus in dem ein Einauplius-Stadium durchlaufen wird nur relativ wenige evolutionäre Veränderungen notwendig wären, um einen Entwicklungsmodus mit Naupliuslarve herbeizuführen und dass diese Veränderungen zum großen Teil Heterochronien darstellen.

1.4 Entwicklungsmodi der Malacostraca Die Malacostraca zeigen ein breites Spektrum an Entwicklungsmodi. Diese beinhalten verschiedene Ausprägungen von Brutpflege sowie das Durchlaufen spezialisierter planktischer Larvalstadien (Abbildung 4). Die Naupliuslarve ist dabei nur eine von vielen vorkommenden Larvenformen. Auch das Fehlen von Larven (Direktentwicklung) ist weit verbreitet. Im Hinblick auf die Zielstellung der vorliegenden Arbeit ist es wichtig zu zeigen, in welcher Weise in den verschiedenen Gruppen der Malacostraca die Bildung der Adultgestalt bewerkstelligt wird und welche larvalen Morphologien dabei auftreten können.

1.4.1 Larvalentwicklung

1.4.1.1 Entwicklung über eine Naupliuslarve Innerhalb der Malacostraca besitzen die Dendrobranchiata (Decapoda) (Martin et al., 2014) und die Euphausiacea eine freischwimmende Naupliuslarve (Orthonauplius) (Martin & Gomez-Gutierrez, 2014). Diese Larvenform ist durch das Vorhandensein von drei Extremitätenpaaren gekennzeichnet, den ersten Antennen, zweiten Antennen und Mandibeln. Alle drei Extremitätenpaare führen rhythmische Ruderbewegungen aus und bewerkstelligen somit die Fortbewegung in der Wassersäule, wobei die zweiten Antennen die Rolle der Hauptantriebsorgane einnehmen (Williams, 1994). Bei Euphasusiacea und Dendrobranchiata werden mehrere Naupliusstadien durchlaufen, die sich durch zusätzliche posteriore Extremitätenpaare und Segmentanlagen auszeichnen (Williamsson, 1969). In anschließenden Stadien kommen weitere Segmente hinzu, es werden die Facettenaugen angelegt und der Carapax gebildet (Protozoea bei Dendrobranchiata, Calyptopis bei Euphausiacea) (Anderson et al., 1949; Dobkin, 1963; Dobkin, 1961; Knight, 1978; Knight, 1976; Mauchline & Fisher, 1969; Williamson, 1969; Zimmer, 1956). Darauf folgen Larvalstadien, in denen die Exopoditen der Thoracopoden die Aufgabe der Lokomotion übernehmen, funktionstüchtige Pleopoden aber noch fehlen ( bei Dendrobranchiata, Furcilia bei Euphausiacea). In den letzten Stadien (Megalopa oder Postlarve bei Dendrobranchiata, Calyptopis bei Euphausiacea) treten bewegliche Pleopoden auf.

1.4.1.2 Entwicklung über eine Zoea-ähnliche Larve Bei einem Großteil der Malacostraca schlüpfen Larven, die sich durch die vollständige oder nahezu vollständige Zahl von Rumpfsegmenten auszeichnen sowie durch einen beweglichen Rumpf mit einer

12 paddelförmigen Telsonplatte oder einem Schwanzfächer. Diese Larven werden hier als Zoea-ähnliche Larven zusammengefasst.

Bei den Pleocyemata (Decapoda) entsprechen die Zoea-ähnlichen Larven in ihren Grundzügen den Mysis- oder Megalopa- Stadien der Dendrobranchiata. Sie besitzen Komplexaugen, mindestens ein funktionales Thorakopodenpaar und mitunter Uropoden (Williamson & Rice, 1996). Bei verschiedenen Gruppen treten thorakale Dornen (Gurney, 1936; Heegard, 1969; Manning & Provenzano, 1963) sowie teilweise stark abgewandelte Körpergestalten auf, wie bei der Phyllosoma- Larve der Palinuridae und Scyllaridae (Kittaka et al., 1997; Robertson, 1965). Die Larven der Amphionidacea (Heegard, 1969), die nach der Phylogenie von Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter, 2010) die Schwestergruppe der restlichen Decapoden darstellen und eine stark abweichende Adultmorphologie zeigen, ähneln sehr stark der Zoea-Larve der Caridea. Bei den Stomatopoda, mit Ausnahme der Lysiosquillidae, schlüpft eine Pseudozoea genannte Larve. Diese trägt nur am ersten und zweiten Thoraxsegment Extremitäten und verfügt über funktionstüchtige Pleopoden. (Manning & Provenzano, 1963; Provenzano & Manning, 1978; Shanbhogue, 1978). Die Lysiosquillidae schlüpfen als Antizoea mit lediglich fünf paar Thoracopoden (Gruner, 1993b) und einer extremitätenlosen Pleonanlage.

Die schlüpfen als Larven mit funktionstüchtigen Kopfextremitäten (erste und zweite Antenne, Mandibel, erste und zweite Maxille), dem ersten Thorakopodenpaar, aber einer unvollständigen Zahl von Pleonsegmenten (Jakobi, 1958; Jakobi, 1954). Die Tiere durchlaufen eine Parazoea-Phase in der die zweiten und dritten Thorakopoden in Erscheinung treten, die Antennen aber immer noch als Hauptfortbewegungsorgan dienen. Darauf folgt die Bathynellid-Phase, in der die restlichen Thorakopoden und die Uropoden erscheinen und die Antennen ihre Aufgabe bei der Fortbewegung verlieren. Manchen Arten fehlt eine Parazoea-Phase. Bei ihnen beginnt die Larvalentwicklung mit dem Bathynellid. Die Bathynellacea behalten auch im Stadium der Geschlechtsreife eine Larven-ähnliche Morphologie (Gruner, 1993c).

1.4.2 Pseudodirektentwicklung Bei den Cladoceromorpha (Branchiopoda) tritt ein besonderer Entwicklungsmodus auf für den der Begriff Pseudodirektentwicklung eingeführt wurde (Fritsch et al., 2013). Hierbei schlüpfen embryoähnliche Larven aus Subitaneiern. Sie zeichnen sich durch fehlende oder nur geringe Beweglichkeit aus und verbleiben im Brutraum bis sie durch weitere Häutungen das Juvenilstadium erreichen. Pseudodirektentwicklung findet sich auch bei den Malacostraca. Bei den schlüpft bereits früh in der Entwicklung die unbewegliche Nauplioid-Larve, die eine eigene Cuticula und verlängerte Anlagen der ersten und zweiten Antennen besitzt, aber sonst einen größtenteils undifferenzierten Keimstreif darstellt (Scholtz, 1984; Wittmann, 1981; Wortham-Neal & Price, 2002). 13 Unter der Nauplioid-Cuticula differenzieren sich alle Segmente und Extremitäten des Adultus. Durch eine weitere Häutung vollzieht sich ein Übergang zu dem Postnauplioid-Stadium, das Beweglichkeit erlangt und das Marsupium verlässt. Ähnliche Entwicklungsmodi finden sich auch bei den Leptostraca (Manton, 1934) und Themosbaenacea (Barker, 1962; Zilch, 1974).

1.4.3 Direktentwicklung Die Direktentwicklung beschreibt einen Entwicklungsmodus, bei dem alle morphogenetischen Prozesse zur Gestaltung der Adultmorphologie während der Embryogenese stattfinden und ein juveniles Tier aus dem Ei schlüpft. Dieser Entwicklungsmodus tritt in der Regel zusammen mit ausgeprägter Brutpflege durch das Muttertier, einem erhöhten Dottergehalt der Eier sowie einer geringeren Gelegegröße (Zahl abgelegter Eier) auf. Direktentwicklung kommt innerhalb der Malacostraca bei süßwasserbewohnenden Decapoden, wie den (Vogt, 2013), den (Hickman, 1936) und den Thermosbaenacea vor (Barker, 1962). Folgt man der Phylogenie von Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter 2010), so ist dieser Entwicklungsmodus auch für das Grundmuster der Neocarida (Thermosbaenacea und Peracarida) anzunehmen.

1.5 Heterochronie und Entwicklungsmodus Der Begriff Heterochronie und die zugrundeliegenden theoretischen Konzepte haben im Laufe der Geschichte einen dynamischen Wandel erfahren (Bininda-Emonds et al., 2002; McNamara & McKinney, 2005). Das Konzept geht in seinem Ursprung auf Ernst Haeckel (Haeckel, 1905) zurück, der mit „Heterochronismus“ eine Situation beschreibt, in der die Ontogenie eines Organismus nicht die Phylogenie rekapituliert und die somit eine Ausnahmen des biogenetischen Grundgesetzes darstellt. De Beer (De Beer, 1930) dagegen definierte Heterochronie in einem allgemeineren Sinne, nämlich als eine Abweichung im zeitlichen Auftreten einer Struktur in der Ontogenese eines Organismus relativ zur Ontogenese seiner Vorfahren. Steven J. Gould argumentiert in seinem einflussreichen Buch „Ontogeny and Phylogeny“ (Gould, 1977) für ein Heterochroniekonzept, in dem Heterochronie ein sehr spezifischer Vorgang ist, der Parallelen zwischen Ontogenie und Phylogenie erzeugt. Alberch (Alberch et al., 1979) entwickelt wiederum ein umfassenderes Heterochroniekonzept, das Veränderungen im Zeitpunkt von Entwicklungsereignissen und in der Rate von Entwicklungsprozessen gleichermaßen einschließt. Evolutionäre Neuerungen können, so argumentiert er, in vielen Fällen als Effekt von Heterochronie auf den Phänotyp erklärt werden. Ein wesentlicher Aspekt von Alberchs Heterochroniekonzept ist, dass die zeitliche Veränderung nur auf bestimmte Teile des Organismus wirkt. Dagegen sprechen McKinney & McNamara (McKinney & McNamara, 1991) eher von Heterochronie als einem global wirkenden Phänomen und argumentieren darüber hinaus für eine hierarchische Struktur der Heterochronie, da 14 Entwicklungsprozesse von der Zellebene auf die Organismusebene wirken würden. In der vorliegenden Arbeit wird das Konzept der Sequenzheterochronie (Smith, 2001) als operationales Heterochroniekonzept benutzt. Nach diesem Konzept werden Entwicklungsprozesse als Abfolge diskreter Entwicklungsereignisse (Events) beschrieben. Damit wird auch Alberchs (Alberch et al., 1979) Heterochroniekonzept aufgegriffen, da nur Teilaspekte des Individuums (Larve oder Embryo) als Event beschrieben werden. Heterochronien in diesem Sinne stellen Veränderungen in der zeitlichen Relation von Entwicklungsereignissen zueinander dar.

Nur wenige Arbeiten haben bisher die Bedeutung von Heterochronie für die Evolution des Entwicklungsmodus mithilfe systematischer Heterochronieanalysen untersucht. Harrington et al. konnten zeigen, dass die Evolution der Direktentwicklung bei den Anura mit einer vorzeitigen Verknöcherung mehrerer Elemente des kranialen Skelettes einhergeht (Harrington et al., 2013). Fritsch et al. (Fritsch et al., 2013) zeigten, dass der Verlust der Naupliuslarve innerhalb der Branchiopoda mit einer Verzögerung in der Differenzierung nauplialer Strukturen gegenüber postnauplialen Strukturen einherging sowie einer zeitlichen Vorverlagerung in der Entwicklung der anterioren Rumpfsegmente. Es liegt in der Natur des Entwicklungsprozesses, dass Entwicklungsstadien in hohem Maße durch die vorausgegangen Stadien bedingt werden. Die Gestalt z.B. einer Naupliuslarve wird nicht zuletzt durch die morphogenetischen Prozesse der Embryogenese hervorgerufen, die ihr vorausgehen (Abbildung 3c). Eine Veränderung des Entwicklungsmodus, sei es durch evolutionären Verlust oder Entstehung einer Naupliuslarve, ist daher mit einer heterochronen Veränderung in der Bildung postnauplialer Strukturen gegenüber dem Schlupfereignis verbunden. Im Zusammenhang mit der sekundären Evolution des Nauplius bei Malacostraca sind somit Heterochronien zu erwarten. Ein Ziel der vorliegenden Arbeit ist es, herauszufinden, welche Aspekte der Entwicklung im Zusammenhang mit dem evolutiven Wandel des Entwicklungsmodus durch Heterochronie verändert wurden und welche Aspekte unverändert geblieben sind. Je nachdem wie stark ein plesiomorphes naupliales Entwicklungsmuster ausgeprägt ist, sind für die sekundäre Evolution der Naupliuslarve zwei Szenarien vorstellbar (A und B), die je ein Ende eines Spektrums möglicher Zwischenformen darstellen:

A. Das plesiomorphe naupliale Entwicklungsmuster betrifft alle embryonalen Gewebe des Mesoderms und des Ektoderms gleichermaßen. Bei der Evolution der Naupliuslarve bleibt das naupliale Entwicklungsmuster unverändert. Danach jedoch wird die Differenzierung der nauplialen Anlagen fortgesetzt bis hin zur lebensfähigen Larve, während die Bildung der postnauplialen Segmente durch Heterochronie in die Larvalphase verschoben wird.

B. Das plesiomorphe naupliale Entwicklungsmuster betrifft nur diejenigen embryonalen Gewebe, die vorrangig bei der Beschreibung des Einauplius sensu Scholtz (Scholtz, 2000)

15 berücksichtigt wurden, also die externe Morphologie und die Bildung epidermaler Strukturen. Bei der Evolution der Naupliuslarve kommt es in diesem Szenario zu einer heterochronen Vorverlagerung der verbleibenden Entwicklungsprozesse, wie der Bildung der nauplialen Muskulatur und des nauplialen Nervensystems, sodass alle Gewebe, die eine lebensfähige Naupliuslarve braucht, vor dem Schlupf ausdifferenziert sind. Auch in diesem Szenario kommt es zu einer Verzögerung der Bildung postnauplialer Segmente in die Larvalphase.

1.6 Embryogenese der Malacostraca und Entwicklung des Mesoderms Die Zusammensetzung des plesiomorphen nauplialen Entwicklungsmusters und seine Bedeutung für die Evolution der Naupliuslarve bei Malacostraca soll in der vorliegenden Arbeit untersucht werden. Deshalb sind die zellulären Prozesse, insbesondere in der bisher wenig erforschten Entwicklung des Mesoderms, von besonderem Interesse.

Seit Beginn der embryologischen Forschung an Krebstieren im neunzehnten Jahrhundert wurden einige klassisch histologische Arbeiten mit teilweise beachtlichem Detailreichtum an verschiedenen Vertretern der Malacostraca durchgeführt. Darunter sind Untersuchungen an Leptostraca (Manton, 1934), Stomatopoda (Shiino, 1942), Decapoda (Huxley, 1879; Rathke, 1829; Reichenbach, 1886; Scheidegger, 1976; Weygoldt, 1961), Anaspidacea (Hickman, 1936), Euphausiacea (Zimmer, 1956), Mysidacea (Manton, 1928), Amphipoda (Weygoldt, 1958). Unter Verwendung von elektronenmikroskopischen und histochemischen Methoden wurden auch neuere Untersuchungen der Embryogenese der Malacostraca durchgeführt (Alwes & Scholtz, 2006; Alwes & Scholtz, 2004; Biffis et al., 2009; Browne et al., 2005; Fischer & Scholtz, 2010; Kiernan & Hertzler, 2006; Wolff, 2009). Außerdem erfolgten zellgenealogische Studien an ausgewählten Arten (Alwes & Scholtz, 2004; Gerberding et al., 2002; Hertzler & Clark, 1992; Hunnekuhl & Wolff, 2012; Wolff, 2002; Wolff et al., 2008). Genexpressionsstudien und Knock-out Versuche wurden ebenfalls an einigen wenigen Arten durchgeführt (Abzhanov & Kaufman, 2000; Brena et al., 2005; Browne et al., 2006; Liubicich et al.,

16 Abbildung 4. Schematische Übersicht über die verschiedenen mesodermalen Zellpopulationen, die in der Embryogenese gebildet werden. Die verschiedenen Zellpopulationen sowie die Mesoteloblasten und der Dotter sind farblich kodiert. a. Ventralansicht eines schematisierten Malakostrakenembryo. Die optischen Loben sowie die sechs vordersten Paare der Extremitätenanlagen sind angedeutet. Der posteriore Bereich des Embryos, der die Kaudalpapille bildet, ist in dieser Ansicht nach hinten geklappt. b. Lateralansicht desselben Embryo mit der Ventralseite nach links gerichtet. Die Kaudalpapille ist nach anterior geklappt. Abkürzungen: Ol optischer Lobus, A1 Anlage der ersten Antenne, A2 Anlage der zweiten Antenne, Md Anlage der Mandibel, Mx1 Anlage der ersten Maxille, Mx2 Anlage der zweiten Maxille, T1 Anlage des ersten Thorakopoden.

2009; Prpic & Telford, 2008; Schmid, 2011). Aus den oben aufgeführten Untersuchungen lässt sich ein allgemeines Bild von den Prozessen der Embryogenese im Hinblick auf die Morphogenese nauplialer und postnauplialer Strukturen ableiten. Der Furchungsmodus ist bei der Mehrzahl der Malacostraca superfiziell. Bei ihnen kondensieren bereits vor der Gastrulation einige Blastodermzellen zu einer Keimscheibe, die ventral einer großen Dottermasse liegt (Abbildung 4b). Aus der Keimscheibe gehen die optischen Loben und vordersten drei Körpersegmente (naupliale Segmente) hervor. Während der Gastrulation gelangen Blastodermzellen durch Immigration oder Invagination unter die Keimscheibe und bilden das naupliale Mesoderm (Abbildung 4a, b). Durch Zellimmigration im anterioren Bereich des Blastoderms wird das präantennale Mesoderm gebildet (Manton, 1934; Manton, 1928; Weygoldt, 1961). Posterior der nauplialen Mesodermpopulation wird die Wachstumszone etabliert, die 8 Mesoteloblasten und 19 Ektoteloblasten (im Grundmuster der Malacostraca) beinhaltet. Die Mesoteloblasten und Ektoteloblasten generieren durch wiederholte asymmetrische Zellteilung die mesodermalen Anlagen der Segmente und die ektodermalen Anlagen der Parasegmente posterior der ersten Maxille (Abbildung 4a, -b). Die Teilungsprodukte der Mesoteloblasten und Ektoteloblasten sind zunächst in Querreihen angeordnet, wobei im Mesoderm genau 4 Tochterzellen pro Hemisegment gebildet werden (Dohle, 1976; Dohle, 1972; Dohle, 1970; Hahnenkamp, 1974; Scholtz, 1992). Im Keimstreif kommt es auf diese Weise zur sukzessiven Ausbildung der embryonalen Segment- und Extremitätenanlagen. Bei den Malacostraca findet sich neben dem preantennalen, dem nauplialen und dem teloblastischen Mesoderm noch eine weitere mesodermale Zellpopulation, das Telsonmesoderm, das posterior der Teloblasten liegt (Alwes & Scholtz, 2006; Manton, 1928; Shiino, 1942) (Abbildung 4a,-b).

Bei den Leptostraca, Stomatopoda, Decapoda, Euphausiacea, Thermosbaenacea, Bathynellacea und Anaspidacea wird eine Kaudalpapille ausgebildet, eine posterior gelegene, aber nach anterior gerichtete Struktur. Diese enthält Segmentanlagen, die Teloblasten und die Telsonanlage sowie einen Teil des Keimstreifs (Abbildung 4b). Im gesamten Keimstreif treten Extremitätenanlagen als wachsende segmentale Ausstülpungen des Ektoderms in Erscheinung. Die Ausstülpungen gehen auch mit der Proliferation darunterliegender Mesodermzellen einher. Unabhängig vom jeweiligen

17 Entwicklungsmodus setzt zum Ende der Embryonalentwicklung die Differenzierung der im Schlupfstadium vorhandenen und funktionalen Strukturen ein.

1.7 Myogenese Die Entwicklung der Muskulatur der Malacostraca ist für das Verständnis der Evolution der Naupliuslarve und des Einaupliusstadiums von großem Interesse. Eine lebensfähige Naupliuslarve benötigt funktionale Muskulatur für die Lokomotion und die Nahrungsaufnahme. Dies macht die Muskelentwicklung zu einem entscheidenden Element des nauplialen Entwicklungsprogrammes. Bisher ist nur wenig über die Struktur und Topographie der Muskelvorläufer bekannt, ebenso darüber welchen Veränderungen Struktur und Topographie im Verlauf der Entwicklung unterliegen. Die Untersuchung der Myogenese stellt deshalb den Kern der vorliegenden Arbeit dar. Die zellulären Prozesse der Myogenese sind innerhalb der Arthropoden am besten bei den Insekten untersucht (Bate, 1990; Baylies & Michelson, 2001; Steffens et al., 1995; Tixier et al., 2010; Xie et al., 1994). An der Taufliege wurde das founder cell- Modell der Myogenese entwickelt (Bate, 1990). Nach diesem Modell beginnt die Bildung eines Muskels mit der Spezifikation einer Pioniermuskelzelle (engl. muscle founder cell) im Mesoderm (Abbildung 5). Pioniermuskelzellen werden durch asymmetrische Zellteilung einer mesodermalen Vorläuferzelle gebildet und durchlaufen selbst keine weiteren Zellteilungen (Haralalka & Abmayr, 2010; Tixier et al., 2010). Sie exprimieren bereits muskelfaserspezifische Proteine (Xie et al., 1994), wodurch sie mit spezifischen immunhistochemischen Färbetechniken lokalisiert werden können. Pioniermuskelzellen sind von spindelförmiger Gestalt, wobei die Orientierung der Zelle die Orientierung des späteren Muskels vorgibt. Durch Zellfusion der Pioniermuskelzellen mit umliegenden fusionskompetenten Myoblasten (FCMs) entsteht ein synzyzialer Muskelvorläufer (Abbildung 5a-e) (Abmayr & Pavlath, 2012; Bate, 1990; Haralalka & Abmayr, 2010; Richardson et al., 2008). In diesem Muskelvorläufer differenzieren sich die Myofibrillen und bilden Sarkomere aus, wodurch die zelluläre Struktur der kontraktionsfähigen Muskelfaser entsteht. Arbeiten an Malacostraca (Harzsch & Kreissl, 2010; Kreissl et al., 2008) haben gezeigt, dass das founder cell- Modell höchst wahrscheinlich auch für die Pancrustacea/Tetraconata gilt.

Das Muskelsystem der Malacostraca kann, aufbauend auf Hesslers vergleichender Darstellung der Skelettmuskulatur verschiedener Crustacea (Hessler, 1964), in folgende Elemente unterteilt werden:

Somatische Muskulatur Rumpfmuskulatur Dorsale Rumpfmuskulatur Ventrale Rumpfmuskulatur

18 Extremitätenmuskulatur Extrinsische Extremitätenmuskulatur Mediane extrinsische Muskeln Laterale extrinsische Muskeln Intrinsische Extremitätenmuskulatur Viszerale Muskulatur Darm-Ringmuskulatur Darm-Dilatatoren Mitteldarmdrüsenmuskulatur Gefäßmuskulatur Herzmuskulatur

Diese Klassifikation richtet sich in erster Linie nach der mechanischen Funktion der jeweiligen Muskelgruppe im Organismus. Einige der aufgeführten Muskelgruppen lassen sich jedoch bestimmten mesodermalen Zellpopulationen des frühen Keimstreifs (Abbildung 3) zuordnen. Somit stellt die viszerale Muskulatur im Bereich des Pharynx wahrscheinlich ein Derivat des präantennalen Mesoderms dar. Die viszerale Muskulatur im Bereich des Hinterdarms wird vom Telsonmesoderm gebildet (Shiino, 1942; Weygoldt, 1960). Die Extremitätenmuskulatur und Rumpfmuskulatur aller Segmente posterior der ersten Maxille sind Derivate der Mesoteloblasten. Hunnekuhl & Wolff (Hunnekuhl & Wolff, 2012) haben an Orchestia cavimana gezeigt, dass aus jeder der anfänglichen 4 je Hemisegment von den Mesoteloblasten gebildeten Tochterzellen eine bestimmte Komponente des Muskelapparates gebildet wird. Die ventrale Rumpfmuskulatur, Extremitätenmuskulatur, dorsale Rumpfmuskulatur und die Herzmuskulatur gehen auf jeweils eine der 4 Tochterzellen zurück. Für den vergleichenden Teil der vorliegenden Arbeit wird ein Schwerpunkt auf die ventrale Rumpfmuskulatur, die extrinsische Extremitätenmuskulatur sowie Ring- und Dilatator-Muskeln des Oesophagus gelegt.

1.8 Ziele der Dissertation i. Die Struktur des sich entwickelnden Muskelsystems soll mithilfe histochemischer und immuhistochemischer Färbetechniken sowie konfokaler Laserscanningmikroskopie und computergestützter 3D-Rekontruktion an unterschiedlichen Vertretern der Malacostraca beschrieben werden. Der Verlauf der Muskelentwicklung wird dabei durch Analyse unterschiedlicher Entwicklungsstadien dokumentiert. ii. Die Evolution des Entwicklungsprozesses, einschließlich der Muskelentwicklung, innerhalb der Malacostraca soll rekonstruiert und die aufgetretenen Heterochronien ermittelt werden. Dazu ist es notwendig, den Entwicklungsprozess für jeden der untersuchten Vertreter als 19 Sequenz von Entwicklungsereignissen zu kodieren. Anhand dieser Entwicklungssequenzen soll dann eine Rekonstruktion des evolutionären Szenarios durch computerbasierte Heterochronieanalyse vorgenommen werden. Die von Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter, 2010) für die Malacostraca vorgeschlagenen Verwandtschaftsbeziehungen werden dafür zugrunde gelegt. iii. Auf Grundlage der rekonstruierten Sequenzevolution sollen Aussagen darüber getroffen werden, welche Heterochronien für die Veränderungen des Entwicklungsmodus in der Evolution der Malacostraca ausschlaggebend waren und welche Rolle sie bei der sekundären Evolution der Naupliuslarve gespielt haben.

1.9 Ergebnisse

1.9.1 Untersuchte Arten In der vorliegenden Arbeit wurde die Muskelentwicklung von fünf Vertretern der Malacostraca untersucht: Gonodactylaceus falcatus (Stomatopoda), Neocaridina heteropoda (Decapoda, Caridea), Procambarus fallax forma virginalis (Decapoda, Reptantia), integer (Peracarida) und Parhyale hawaiensis (Peracarida). Für den Vergleich wurden die publizierten Daten der Muskelentwicklung von Sicionia ingentis (Decapoda, Dendrobranchiata) hinzugezogen. Mehrere von Dr. Phillip Hertzler freundlicherweise zur Verfügung gestellte Originaldatensätze wurden nachuntersucht. Als Außengruppe wurde Artemia franciscana (Branchiopoda, Anostraca) gewählt. Euphausiacea konnten nicht untersucht werden.

1.9.2 Beschreibung der Myogenese Die umfassendste Beschreibung der Muskelentwicklung wurde für P. fallax f. virg. vorgenommen (Jirikowski et al., 2010: Kapitel 5). Die im Rahmen der vorliegenden Arbeit bei G. falcatus, N. heteropoda, N. integer und P. hawaiensis beschriebenen Muskelvorläufer und Pioniermuskelzellen zeigen deutliche strukturelle Übereinstimmungen mit denen von P. fallax f. virg. Der für die Pioniermuskelzellen dokumentierte Entwicklungsverlauf stimmt mit dem founder cell Modell und den für weitere Vertreter der Malacostraca publizierten Beobachtungen (Harzsch & Kreissl, 2010; Kreissl et al., 2008) in vielen Punkten überein (Abbildung 5a´- e´). Zwar ermöglichen die hier verwendeten Methoden keine direkte Beobachtung der Zellfusion, doch es wird deutlich, dass eine Zunahme an Zellkernen im Muskelvorläufer der Bildung quergestreifter Fasern in der Regel vorausgeht. Auf die Pioniermuskelzelle (Abbildung 5a´) folgt ein mehrkerniger Muskelvorläufer, der jedoch noch keine zytoplasmatischen Filamente zeigt (Abbildung 5b´). Erst danach werden F-Actin- und Myosin-positive, filamentöse Strukturen im Zytoplasma erkennbar (Abbildung 5c´). Mit fortschreitender Entwicklung nimmt die Zahl der Filamente und das Volumen des Muskelvorläufers

20 zu (Abbildung 5d´). Zuletzt zeigt sich eine enge Aneinanderlagerung der Fasern innerhalb des Muskels (Abbildung 5e´). Pioniermuskelzellen wurden bei P. fallax f. virg. im Bereich der somatischen Muskulatur für intrinsische- und extrinsische Extremitätenmuskulatur, Rumpfmuskulatur sowie im Bereich der viszeralen Muskulatur für die Ringmuskulatur des Darmes (Abbildung 5f) und die Muskulatur der Mitteldarmdrüse (Abbildung 5g) gefunden. Letztere unterscheiden sich von den übrigen Pioniermuskelzellen durch den Besitz mehrerer radial angeordneter Zellfortsätze, die den Zellen eine sternähnliche Form geben. In allen Bereichen der Muskulatur kommt es innerhalb der mehrkernigen Muskelvorläufer zur Ausbildung von Myofibrillen mit Sarkomerstruktur, die mehrheitlich Querstreifen zeigen. Glatte Muskulatur wird fast ausschließlich als Vorläufer quergestreifter Muskulatur beobachtet. Die Ringmuskulatur des Darmes (Abbildung 5i) und das Myokard (Abbildung 5h, -m) zeigen in fortgeschrittenen Entwicklungsstadien die charakteristischen Querstreifen. Lediglich für die Muskulatur der Mitteldarmdrüse konnten keine Querstreifen festgestellt werden. Möglicherweise handelt es sich hierbei tatsächlich um ein glattes Muskelgewebe. Es ist jedoch nicht auszuschließen, dass später in der Entwicklung noch Sarkomere in der Mitteldarmdrüsenmuskulatur ausgebildet werden. Zwischen den Arten lassen sich Unterschiede im Zeitpunkt der Sarkomerbildung beobachten. N. heteropoda besitzt einzellige Vorläufer von extrinsischen Extremitätenmuskeln, die bereits Querstreifen zeigen (Jirikowski et al., 2013: Kapitel 6) (Abbildung 5j). Bei P. hawaiensis werden dagegen Muskelvorläufer beobachtet, die eine große Anzahl von Kernen besitzen bevor es zur Ausbildung von Sarkomeren kommt (Jirikowski et al., 2013: Kapitel 6). Kontraktionen von Muskelvorläufern im lebenden Tier können bei P. fallax f. virg. schon vor der Ausbildung der Sarkomerstruktur beobachtet werden, so z.B. für die Herzanlage in Stadium 6 oder die Muskulatur der Mitteldarmdrüse in Stadium 8.

1.9.3 Topographie embryonaler Muskulatur Steffens et al. (Steffens et al., 1995) beschreiben für die Entwicklung der ventralen Körperwandmuskulatur von Schistocerca gregariia ein Muster sich segmental wiederholender Muskelvorläufer mit charakteristischer Anordnung aus dem im Verlauf der Entwicklung, teils durch Verlust einzelner Vorläufer, teils durch Zuwachs und Differenzierung, Unterschiede in der Muskelmorphologie zwischen den Segmenten herausgebildet werden. Ein vergleichbares Muster wird für die untersuchten Vertreter der Malacostraca beobachtet (Jirikowski et al., 2013: Kapitel 6) (Abbildung 6). Alle Segmente des Rumpfes sowie das Mandibel- und das zweite Antennensegment zeigen im Verlauf der Entwicklung transversal ausgerichtete Muskelvorläufer lateral und medial der Extremitätenknospen. Diese Muskelvorläufer treten in der Regel paarweise auf. Dies wurde jedoch niemals für den medianen extrinsischen Muskelvorläufer der Mandibel beobachtet. Die Rumpfsegmente, einschließlich des ersten Maxillensegments, zeigen zudem zwei longitudinal

21 ausgerichtete Muskelvorläufer. Ersterer liegt proximal der Beinknospe, letzterer liegt dorsolateral des Ersten. Sie stellen die ventralen bzw. dorsalen Rumpfmuskeln dar und bilden bereits früh in der Entwicklung kontinuierliche Muskelstränge. Bei den untersuchten Vertretern der Decapoda und Stomatopoda wurde beobachtet, dass sich ein posteriorer Teil der ventralen Längsmuskelstränge bis in die Telsonanlage posterior der Wachstumszone erstreckt (lmp-post in Abbildung 6). Im Bereich des Stomodeum bildet sich ein Muskelvorläufer, der beginnt das Stomodeum ringförmig zu umschließen. Dazu entstehen Vorläufer, die nach anterior und lateral ziehen und später Dilatatoren des Ösophagus bilden.

In der vorliegenden Arbeit wird erstmalig ein konserviertes topographisches Muster von Muskelvorläufern bei Vertretern der Crustacea beschrieben. Dazu wurde für die Benennung individueller Muskelvorläufer ein eigenes Vokabular entwickelt (Jirikowski et al., 2013: Kapitel 6, Jirikowski et al., 2015: Kapitel 7) (Abbildung 6b). Pioniermuskelzellen konnten für die untersuchten Vertreter in nahezu allen Bereichen der somatischen und viszeralen Muskulatur identifiziert werden. Im Bereich der somatischen Muskulatur jedoch wird die mögliche Rolle der frühen Muskelvorläufer für die Bildung eines Gerüstes (Steffens et al., 1995; Xie et al., 1994), das die Position und Orientierung der sich entwickelnden Muskeln vorgibt, besonders deutlich. Dem posterioren Longitudinalmuskelvorläufer (lmp-post in Abbildung 6), der hier für Stomatopoda und Decapoda beschrieben wird, kommt vor dem Hintergrund des Entwicklungsmodus eine besondere Rolle zu. Der ventrale embryonale Längsmuskelstrang zeigt nicht den anteroposterioren Entwicklungsgradienten, der für die Derivate des teloblastischen Mesoderms erwartet wird, sondern erstreckt sich bis in die Telsonanlage, wo er hinter den Mesoteloblasten endet. Dieses Muster lässt darauf schließen, dass es sich bei dem posterioren Teil des Längsmuskelstranges um einen Muskelvorläufer der Telsonanlage handelt, der, analog zu den posterioren Pionierneuronen (Fischer & Scholtz, 2010) (PPN in Abbildung 6), zelluläre Ausläufer nach anterior entsendet. Dort schließen sie zu den segmentalen

Abbildung 6. Schematische Übersicht über die Strukturen, deren Erscheinen für die vergleichende Analyse als Entwicklungsereignisse kodiert wird. Die optischen Loben sowie die sechs vordersten Paare von Extremitätenanlagen sind angedeutet, ebenso das sechste Pleonsegent, die sechste Pleopodenanlage und die Telsonanlage. Auf der rechten Seite sind die Nummern und Abkürzungen für alle kodierten Entwicklungsereignisse wie sie in Jirikowski et al (Jirikowski et al., 2015: Kapitel 7) verwendet werden, gelistet. Für die verschiedenen Strukturen und die entsprechenden Entwicklungsereignisse wird ein Farbcode verwendet, um die Unterscheidung nauplialer und postnauplialer Entwicklungsereignisse zu erleichtern. Verwendete Abkürzungen: Ol optischer Lobus, A1 Anlage der ersten Antenne, A2 Anlage der zweiten Antenne, Md Anlage der Mandibel, Mx1 Anlage der ersten Maxille, Mx2 Anlage der zweiten Maxille, T1 Anlage des ersten Thorakopoden, P6 Anlage des sechsten Pleopoden, T2-P5 Segmente vom zweiten Thoraxsegment bis zum fünften Pleonsegment, T Telsonanlage, pc Protocerebrum, dc Deutocerebrum, tc Tritocerebrum.

22

Longitudinalmuskelvorläufern auf, die von den Mesoteloblasten gebildet wurden. Weiterhin werden Vorläufer von Ringmuskeln im Bereich des Proctodeum beobachtet, die sich ebenfalls hinter den Teloblasten befinden und vermutlich dem Telsonmesoderm zuzuschreiben sind. Dies stimmt mit den Beobachtungen von Weygoldt und Shiino (Shiino, 1942; Weygoldt, 1961) überein.

1.9.4 Vergleichende Analyse des Entwicklungsverlaufes

1.9.4.1 Kodierung von Entwicklungssequenzen Die zu vergleichenden Entwicklungsprozesse werden in dieser Arbeit als Abfolge diskreter Entwicklungsereignisse (Events) (Smith, 2001) beschrieben. Ein Entwicklungsereignis in diesem Sinne ist das Auftreten einer bestimmten beobachtbaren und zwischen Individuen vergleichbaren Eigenschaft in der Ontogenese.

Für die Beschreibung der Entwicklungsprozesse der untersuchten Arten wurde eine Auswahl von Entwicklungsereignissen benannt, die für die vorliegende Fragestellung potentiell informativ sind. Dabei galt es, die Muskelentwicklung ausreichend zu berücksichtigen und zugleich die Entwicklung der ektodermalen Gewebe einzubeziehen. Abbildung 6 zeigt eine schematische Übersicht über die

23 Strukturen, deren Bildung als Entwicklungsereignisse kodiert wurde. Diese Entwicklungsereignisse umfassen das erstmalige Auftreten von Muskelvorläufern, Ganglienanlagen und Extremitätenknospen in den nauplialen Segmenten, dem ersten und zweiten Maxillensegment, dem ersten Thoraxsegment und dem sechsten Pleonsegment (Jirikowski et al., 2015: Kapitel 7). Weiterhin wurde das Erscheinen der stomodealen Muskeln und der posterioren Longitudinalmuskelvorläufer des Telson, des Naupliusauges und der posterioren Pionierneuronen kodiert. Der Abschluss der Segmentbildung im Mesoderm wurde ebenfalls als Ereignis kodiert. Hierbei handelt es sich um den Zeitpunkt zu dem alle segmentalen Derivate der Mesoteloblasten vorhanden sind. Auch der Schlupf aus der Eihülle wird als Ereignis kodiert. Da die Methode der Heterochronieanalyse mit der Software PGI (siehe 1.9.4.2 Heterochronieanalyse) sehr rechenintensiv ist, wurde von einer Einbeziehung weiterer Ereignisse abgesehen. Die Rechenzeit konnte dadurch auf unter 2 Monate reduziert werden. Eine Übersicht über die Entwicklungssequenzen der untersuchten Arten ist in Abbildung 7 gezeigt.

Um die Interpretation von Entwicklungssequenzen im Hinblick auf ein plesiomorphes naupliales Entwicklungsmuster zu veranschaulichen, wird hier der Begriff der Einaupliusphase eingeführt. Die Einaupliusphase stellt denjenigen Teil des Entwicklungsprozesses dar in dem ausschließlich in den Naupliussegmenten Entwicklungsereignisse auftreten. Bei Vertretern mit freischwimmender Naupliuslarve enthält die Einaupliusphase alle Ereignisse, die zur Bildung einer lebensfähigen Larve notwendig sind, also Muskelvorläufer, Ganglien und neuronale Anlagen sowie Extremitätenanlagen der Naupliussegmente (Sequenzen von A. salina und S. ingentis in Abbildung 7). Bei diesen Vertretern folgt auf die Einaupliusphase das Schlupfereignis. Bei Vertretern ohne freischwimmende Naupliuslarve ist die Einaupliusphase auf eine geringere Zahl von Ereignissen in den nauplialen Segmenten beschränkt und das Schlupfereignis erfolgt erst nach der Bildung postnauplialer Strukturen (übrige Sequenzen in Abbildung 7).

1.9.4.2 Heterochronieanalyse

In den letzten zwei Jahrzehnten wurden Methoden entwickelt, die es erlauben, die Evolution von Entwicklungssequenzen anhand vorgegebener phylogenetischer Hypothesen zu rekonstruieren, ohne dass eine Zuordnung von Entwicklungsstadien über die Artgrenzen hinweg vorausgesetzt wird. Dazu gehören die event-pair Methode (Smith, 2001; Velhagen, 1997), event pair cracking (Jeffery et al., 2002), parsimov (Jeffery et al., 2005), search based character optimization (Schulmeister & Wheeler, 2004) und PGi (parsimov based genetic inference) (Harrisson & Larsson, 2008). Alle diese Methoden benutzen das relative Verhältnis von einzelnen Entwicklungsereignissen zueinander um die Sequenzevolution anhand einer vorgegebenen Phylogenie zu rekonstruieren.

24 In der vorliegenden Arbeit wird PGi (Harrisson & Larsson, 2008) für die Rekonstruktion der anzestralen Entwicklungssequenzen und die Bestimmung von Heterochronien benutzt. Die Phylogenie der Malacostraca von Richter und Scholtz (Richter & Scholtz, 2001; Wirkner & Richter, 2010) wird für die Rekonstruktion des evolutionären Verlaufs zugrunde gelegt. PGi behandelt die vollständige Entwicklungssequenz als einzelnes komplexes Merkmal. Dadurch wird ein Problem der event-pair-Methode umgangen, die, da sie jede mögliche paarweise Kombination von Ereignissen als unabhängiges Merkmal behandelt, zur Rekonstruktion widersprüchlicher Ereignispositionen in der anzestralen Sequenz führen kann (Schulmeister & Wheeler, 2004). PGi führt mehrere Einzelanalysen durch, wobei es einen heuristischen Algorithmus verwendet. Über eine festgelegte Zahl von Wiederholungen werden für jeden Knoten der vorgegebenen Phylogenie hypothetische Entwicklungssequenzen generiert und schrittweise optimiert. Dabei wird die event-pair basierte Methode parsimov (Jeffery et al., 2005) benutzt, um alternative evolutionäre Szenarien zu bewerten. Als Ergebnis einer Einzelanalyse wird ein sogenannter Pseudokonsensus-Baum erstellt, der nach dem 50%-Majoritätskriterium aus den sparsamsten Szenarien berechnet wird. Die Analyse selbst wurde dreimal wiederholt und die Ergebnisse zu einem sogenannten Superkonsensusbaum (Harrisson & Larsson, 2008) (Anhang 3 in Jirikowski et al., 2015: Kapitel 7) zusammengeführt. Die Entwicklungssequenzen der einzelnen Knoten des Superkonsensbaumes werden bestimmt, indem für jedes Entwicklungsereignis der Rang, also die Position an der das Ereignis in der Entwicklungssequenz steht, gemittelt wird. Dies führt dazu, dass bei Datensätzen mit hoher Variabilität die rekonstruierten Ereignispositionen geringfügig von den rekonstruierten Heterochronien abweichen können. Für jede der ermittelten Heterochronien berechnet PGi einen Bootstrapwert. Die Bootstrapwerte wurden verwendet, um für jedes Ereignis eine durchschnittliche Heterochronierate zu berechnen. Dies geschah durch Multiplikation des mittleren Bootstrapwertes mit der mittleren Anzahl der im Superkonsensusbaum für das jeweilige Ereignis auftretenden Heterochronien. Die durchschnittliche Heterochronierate gibt Auskunft darüber, wie häufig ein bestimmtes Ereignis in der Evolution der Malacostraca eine heterochrone Verschiebung erfahren hat.

1.9.5 Ergebnisse der Heterochronienalyse Das mit PGi rekonstruierte evolutionäre Szenario weist mit etwa sieben heterochronen Verschiebungen pro Ast ein hohes Maß an Heterochronie auf. Zudem zeigt sich, dass die mittlere Heterochronierate zwischen den unterschiedlichen Gewebetypen stark divergiert. Die mittlere Heterochronierate bei der Bildung der Muskelvorläufer beträgt 1,72, bei der Bildung neuronaler Anlagen 1,32 und bei der Bildung von epidermalen Extremitätenknospen 0,91. Damit wird deutlich, dass in der Evolution der Malacostraca die Entwicklung der Muskulatur einer wesentlich stärkeren Veränderung durch Heterochronie unterlag als die Entwicklung der ektodermalen Gewebe.

25 Die mithilfe von PGi rekonstruierten anzestralen Sequenzen sind aufgrund der starken Variation im Datensatz nicht vollständig deckungsgleich mit den ermittelten Heterochronien. Es muss also bei der Interpretation berücksichtigt werden, dass die Position eines Ereignisses in einer anzestralen Sequenz um ein bis zwei Positionen verschoben sein kann, ohne dass dies sich in den rekonstruierten Heterochronien wiederspiegelt. Im folgenden Abschnitt werden rekonstruierte Entwicklungssequenzen und aufgetretene Heterochronien für die letzten gemeinsamen Vorfahren der verschiedenen Malakostrakengruppen sowie die Heterochronien die in der Linie zu S. ingentis (Dendrobranchiata) führt, kurz beschrieben. Abbildungen 8 und -9 zeigen die entsprechenden Ausschnitte aus dem Superkonsensusbaum.

Letzter gemeinsamer Vorfahr von Branchiopoda und Malacostraca

Die rekonstruierte Entwicklungssequenz zeigt eine Einaupliusphase, in der die epidermalen und neuronalen Anlagen in den nauplialen Segmenten gebildet werden, aber ein großer Teil der nauplialen Muskelvorläufer fehlt (Abbildung 8). Das Schlupfereignis tritt erst nach der Bildung von postnauplialen Strukturen auf.

Letzter gemeinsamer Vorfahr der Eumalacostraca

In der Stammlinie der Eumalacostraca treten mehrere heterochrone Verschiebungen in der Myogenese der nauplialen Segmente auf, wodurch nur noch die nauplialen Extremitätenknospen und die nauplialen Ganglienanlagen in der Einaupliusphase gebildet werden (Abbildung 8). Die Bildung des Naupliusauges und der posterioren Pionierneurone findet unmittelbar nach der

Einaupliusphase statt. Mehrere postnaupliale Entwicklungsereignisse erfahren in der Linie zu den Eumalacostraca eine Verschiebung an eine frühere Position. Das Schlupfereignis wird an eine spätere Position verschoben.

Letzter gemeinsamer Vorfahr der Caridoida

In der Linie zu den Caridoida treten Verschiebungen einiger postnauplialer Entwicklungsereignisse an spätere Positionen auf, darunter die Bildung der Extremitätenknospen und der longitudinalen Muskelvorläufer im sechsten Pleonsegment (Abbildung 8). Innerhalb der Einaupliusphase treten keine Heterochronien auf.

Letzter gemeinsamer Vorfahr der Decapoda

In der Stammlinie der Decapoda wird die Bildung von zwei nauplialen Muskelvorläufern an eine frühere Position verschoben (Abbildung 8). Die Sequenz zeigt eine Einaupliusphase, in der

26 Extremitätenknospen, Ganglienanlagen und einige Muskelvorläufer der nauplialen Segmente gebildet werden. Zudem wird erstmalig der posteriore Longitudinalmuskelvorläufer gebildet.

Letzter gemeinsamer Vorfahr der Pleocyemata

In der Stammlinie der Pleocyemata verschieben sich die Bildung der nauplialen Ganglien und die Bildung eines nauplialen Muskelvorläufers innerhalb der Einaupliusphase an eine spätere Position (Abbildung 8). Die Bildung des Naupliusauges wird aus der Einaupliusphase in die postnaupliale Phase verlagert. Zudem werden der Abschluss der Segmentbildung und die Bildung des Longitudinalmuskelvorläufers im sechsten Pleonsegment innerhalb der postnauplialen Phase zeitlich vorverlagert.

S. ingentis (Dendrobranchiata)

In der Linie, die zu S. ingentis führt, kommt es zur Verschiebung der Bildung von zwei nauplialen Muskelvorläufern in die Einaupliusphase (Abbildung 9a). Die Bildung der stomodealen Muskeln wird aus der Einaupliusphase heraus und das Schlupfereignis an eine frühere Position verschoben, sodass es sich unmittelbar an die Einaupliusphase anschließt. Die Bildung des ersten thorakalen Ganglions wird innerhalb der postnauplialen Entwicklungsphase an eine spätere Position verschoben.

Letzter gemeinsamer Vorfahr der Peracarida

In der Linie zu den Peracarida kommt es zum Verlust des Naupliusauges und der posterioren Pionierneuronen (Abbildung 9b). Die Bildung von zwei nauplialen Muskelvorläufern wird innerhalb der postnauplialen Phase an einen späteren Zeitpunkt verschoben. Die Bildung eines postnauplialen Muskels und der Extremitätenknospen des sechsten Pleonsegmentes verschieben sich innerhalb der postnauplialen Phase nach vorne.

1.10 Evolution der Entwicklung der Malacostraca

1.10.1 Evolution des Entwicklungsmodus In der mit Hilfe von PGi rekonstruierten Entwicklungssequenz des letzten gemeinsamen Vorfahren der Eumalacostraca findet ein wesentlicher Teil der postnauplialen Ereignisse vor dem Schlupf statt und ist somit Teil der Embryogenese. Dies bestätigt die Annahme, dass im Grundmuster der Eumalacostraca keine Naupliuslarve gebildet wurde. Heterochronien in der zu den Eumalacostraca führenden Linie beinhalten die späte Verschiebung des Schlupfereignisses und die frühe

27 Abbildung 7. Übersicht über die für A. franciscana, G. falcatus, P. fallax f. virg., N. heteropoda, S. ingentis, N. integer und P. hawaiensis kodierten Entwicklungssequenzen. Die Phylogenie nach Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter, 2010) (Kladogramm links) ist auf die hier untersuchten Arten reduziert. Daher erscheinen Decapoda und Peracarida als Schwestergruppen. Eine schematische Naupliuslarve kennzeichnet die Gruppen, die als Nauplius schlüpfen. Der Farbcode aus Abbildung 6 wird für die Ereignisnummern in den Sequenzen übernommen. Die Abfolge von Nummern einer Zeile stellt die Entwicklungssequenz dar. Simultan auftretende Ereignisse sind durch einen Querstrich verbunden, nacheinander auftretende Ereignisse sind durch ein Komma getrennt. Die Einaupliusphase ist durch einen länglichen Balken gekennzeichnet. Abkürzungen:

28

Abbildung 8. Anzestrale Sequenzen der Pleocyemata, Decapoda, Caridoida, Eumalacostraca und des letzten gemeinsamen Vorfahren von Branchiopoda und Eumalacostraca. Die Sequenzen sind das Ergebnis der Heterochronieanalyse mit PGi (Darstellungsweise wie in Abbildung 7). Farbige Pfeile zeigen die rekonstruierten Heterochronien. Der Farbcode aus Abbildung 6 wird für die Ereignisnummern in den Sequenzen und die Pfeile übernommen. Dreiecke mit +/- zeigen evolutionären Neuerwerb oder Verlust von Ereignissen. Abkürzungen: ENP Einaupliusphase.

29

30 Abbildung 9. Weitere Ergebnisse der Heterochronieanalyse mit PGi (Darstellung wie in Abbildung 8). a. Sekundäre Evolution der Naupiuslarve der Dendrobranchiata. b. Evolution des Entwicklungsmodus der Peracarida und von P. hawaiensis. c. Evolution des Entwicklungsmodus von N. integer. Abkürzungen: ENP Einaupliusphase.

Verschiebung von postnauplialen Muskelvorläufern, Ganglienanlagen und Beinknospen. Dieses stimmt mit einem Verlust der Naupliuslarve in der Linie der Malacostraca überein. Weiterhin deutet die Entwicklungssequenz darauf hin, dass es sich bei dem Entwicklungsmodus im Grundmuster der Eumalacostraca um Direktentwicklung handelte und nicht um die Entwicklung über eine Zoea- ähnliche Larve. Die frühe Bildung der longitudinalen Muskelvorläufer und Extremitätenknospen des sechsten Pleonsegmentes lässt darauf schließen, dass das sechste Pleonsegment bereits vor dem Schlupf ausdifferenziert wurde. Da die Pseudozoea-Larven der Stomatopoda Pleopoden besitzen, kann aus der anzestralen Entwicklungssequenz der Eumalacostraca nur die Abwesenheit einer Larvenform wie sie bei den Decapoda vorkommt, abgeleitet werden. Der rekonstruierten Entwicklungssequenz der Eumalacostraca fehlt der posteriore Längsmuskelvorläufer des Telson, der für die Bildung einer Zoea-ähnlichen Larve möglicherweise von Bedeutung ist. Unter dieser Voraussetzung kann eine Direktentwicklung im Grundmuster der Eumalacostraca als wahrscheinlicher angenommen werden.

Das mit PGi rekonstruierte evolutionäre Szenario spricht dafür, dass sich in den Linien zu den Stomatopoda und Decapoda unabhängig voneinander ein Wandel des Entwicklungsmodus von Direktentwicklung hin zur Bildung einer planktischen Zoea-ähnlichen Larve vollzogen hat. Die posterioren Longitudinalmuskelvorläufer entstehen laut der Rekonstruktion konvergent in diesen beiden Linien. In der Linie zu den Decapoda wird zudem die Bildung der Extremitätenknospen und der Longitudinalmuskelvorläufer des sechsten Pleomers zeitlich nach hinten verschoben. Die übrigen postnauplialen Entwicklungsereignisse bleiben weitestgehend unverändert. Die Einaupliusphase wird in der Stammlinie der Decapoda um die Bildung des Naupliusauges, der posterioren Pionierneuronen sowie einiger nauplialer Muskelvorläufer erweitert. In der Linie zu den Dendrobranchiata zeigt sich der Wechsel zu einem Entwicklungsmodus mit Naupliuslarve durch die Vervollständigung der Einaupliusphase und die Verschiebung des Schlupfereignisses an eine frühere Position. Die Bildung der stomodealen Muskelvorläufer wird jedoch aus der Einaupliusphase heraus verschoben. Das Fehlen dieser für die Ernährung wichtigen Strukturen ist laut der Rekonstruktion als abgeleitetes Merkmal der Dendrobranchiata zu interpretieren, deren Naupliuslarve keine Nahrung aufnimmt. In der Linie zu den Pleocyemata zeigt die Rekonstruktion Vorverschiebungen, die einen Wechsel zur Direktentwicklung als Entwicklungsmodus andeuten. Jedoch muss dies als Artefakt bewertet werden, das durch die Auswahl untersuchter Arten bedingt ist. Die beiden hier untersuchten Vertreter (N.

31 heteropoda und P. fallax f. virg.) zeigen Direktentwicklung, die mehrfach konvergent innerhalb der Decapoda entstanden ist.

Das rekonstruierte Szenario könnte auf eine Veränderung des Entwicklungsmodus in der Linie von den Caridoida zu den Peracarida hindeuten. Die Entwicklungssequenz der Caridoida zeigt eine späte Position von pleonalen Entwicklungsereignissen, welche für einen Entwicklungsmodus mit Zoea- ähnlicher Larve spricht. Jedoch fehlt der Sequenz der posteriore Longitudinalmuskelvorläufer. In der Linie zu den Peracarida kommt es zu einer Vorverlagerung der Bildung der sechsten Pleopodenanlagen sowie zur Verschiebung des Erscheinens einzelner nauplialer Muskelanlagen nach hinten sowie zum Verlust des posterioren Longitudinalmuskelvorläufers, des Naupliusauges und der posterioren Pionierneuronen. Ob diese Veränderungen auf die Entstehung eines direkten Entwicklungsmodus hinweisen oder ob die Direktentwicklung für die Peracarida als plesiomorph gedeutet werden kann, muss offen bleiben. Für die Beantwortung dieser Frage müssten die Schlüsseltaxa Anaspidacea, Bathynallacea und Euphausiacea in die Analyse einbezogen werden.

Der pseudodirekte Entwicklungsmodus der Mysidacea ist vor dem Hintergrund der Entwicklungssequenz schwer zu deuten. Das Schlupfereignis wird in der Linie zu N. integer vorverlagert, jedoch bleiben weitere naupliale Entwicklungsereignisse aus (Abbildung 9c). Es werden einige Heterochronien für die zu den Mysidacea führende Linie rekonstruiert. Diese stellen zwar zeitliche Vorverlagerungen der Muskelentwicklung dar, doch handelt es sich dabei ausschließlich um postnaupliale Muskelvorläufer. Dies spricht dagegen, dass das Nauplioid-Stadium der Mysidacea das Relikt eines nauplialen Entwicklungsprogrammes im Sinne eines Einauplius ist. Unter der Bedingung der spezialisierten Brutpflege der Peracarida ist davon auszugehen, dass innerhalb dieser Gruppe Entwicklungsmuster reduziert werden, die Relikte von larvalen Entwicklungsmodi darstellen. Die Reduktion zeigt sich besonders deutlich in der Entwicklungssequenz von P. hawaiensis (Abbildung 9b). In der zu P. hawaiensis führenden Linie kommt es zur zeitlichen Vorverlagerung von postnauplialen neuronalen Entwicklungsereignissen und zur Verschiebung von mehreren Ereignissen der Myogenese an eine spätere Position. Dadurch zeigt sich in der Entwicklungssequenz eine beschleunigte Morphogenese, bei der die Extremitätenknospen und Ganglienanlagen in schneller Abfolge gebildet werden. Die Myogenese beginnt erst nachdem die Segmentbildung durch die Teloblasten abgeschlossen und die vollständige Zahl von Extremitätenknospen vorhanden ist. Diese Veränderungen der Entwicklungssequenz können als Adaptation an eine möglichst effiziente Bildung der Adultmorphologie gedeutet werden.

32 1.10.2 Evolution der Naupliuslarve und des Einaupliusstadiums Es ist bekannt, dass bei der Mehrzahl der Arthropoda eine Gruppe anterior gelegener Segmente in anderer Weise gebildet wird als die posterior folgenden (Scholtz & Wolff, 2013). Dies kann somit auch für das Grundmuster angenommen werden. Die Bildung einer Naupliuslarve bzw. das Auftreten eines Einaupliusstadiums ist eine spezielle Ausprägung diese Entwicklungsmusters. Unklar ist jedoch, inwieweit es sich dabei auch um ein zeitliches Entwicklungsmuster innerhalb der anterioren Segmente handelt. Die Entwicklungsereignisse der Einaupliusphase zeigen eine starke Variation hinsichtlich der Ereignisreihenfolge. Bei Vertretern, die keine Naupliuslarve, jedoch ein Einaupliusstadium besitzen, zeigt sich zudem eine starke Variation in der Zusammensetzung der Einaupliusphase. Der Vergleich zwischen der Außengruppe A. franciscana und S. ingentis (Abildung 7), den einzigen Vertretern mit einer freischwimmenden Naupliuslarve, zeigt trotz des übereinstimmenden Entwicklungsmodus starke Abweichungen im Verlauf der Entwicklungssequenzen. Die Sequenzen stimmen jedoch darin überein, dass das Schlupfereignis erst nach der Bildung der nauplialen Muskeln, Ganglien, Extremitäten und des Naupliusauges stattfindet. Bei S. ingentis wird, im Gegensatz zu A. franciscana, die Muskulatur des Stomodeum erst nach der Einaupliusphase gebildet. Diese Beobachtungen legen den Schluss nahe, dass sich das naupliale Entwicklungsprogramm nicht in einer spezifischen Abfolge von Entwicklungsereignissen innerhalb der Einaupliusphase darstellt, sondern primär in der Abundanz nauplialer Entwicklungsereignisse am Anfang der Entwicklungssequenz.

Die Einaupliusphase, der für die Eumalacostraca rekonstruierten Entwicklungssequenz, beinhaltet lediglich die Bildung der nauplialen Extremitätenknospen und der nauplialen Ganglien. Demnach beschränkt sich das plesiomorphe naupliale Entwicklungsprogramm, also der Einauplius sensu Scholtz (Scholtz, 2000), auf Derivate des Ektoderms. Dabei geht die Bildung nauplialer Extremitätenknospen der Bildung nauplialer Ganglien voraus. In den für die Caridoida und Decapoda rekonstruierten Sequenzen beginnt die Entwicklung stets mit der Bildung der nauplialen Extremitätenknospen. Die Bildung nauplialer Ganglien, des Naupliusauges und der posterioren Pionierneuronen tritt stets in der frühen Phase der Entwicklungssequenz auf und erfährt keine heterochrone Verschiebung. In der Linie die zu den Decapoda führt, wird die Einaupliusphase um einige naupliale Muskelvorläufer erweitert. Die sekundäre Evolution der Naupliuslarve in der Linie zu S. ingentis (Dendrobranchiata) wird durch eine relativ geringe Zahl von Heterochronien herbeigeführt. Diese sind die zeitliche Vorverlagerung von zwei Myogeneseereignissen und des Schlupfereignisses. Die Hypothese von Scholtz (Scholtz, 2000), nach welcher der Einauplius eine Prädisposition für die sekundäre Bildung der Naupliuslarve der Dendrobranchiata darstellt, wird durch diese Beobachtungen bestätigt. Zusätzlich muss jedoch darauf hingewiesen werden, dass angesichts des rekonstruierten Szenarios die sekundäre Evolution des Nauplius das Ergebnis eines 33 schrittweisen Prozesses ist. In der Stammlinie der Decapoda wurde die auf ektodermale Entwicklungsereignisse beschränkte Einaupliusphase um die Bildung von Muskelvorläufern erweitert. Darüber hinaus muss betont werden, dass die für die sekundäre Evolution des Nauplius notwendigen Heterochronien vorrangig die Bildung nauplialer Muskelanlagen betrafen. Somit ist diese evolutionäre Transformation in hohem Maße das Resultat von Heterochronie zwischen mesodermalen und ektodermalen Geweben.

1.10.3 Die evolutionäre Bedeutung von Mesoderm und Muskulatur Die Arbeit an Arthropoden-Modellorganismen hat gezeigt, dass die Entwicklung des Mesoderms zu einem erheblichen Grad einer Kontrolle durch das Ektoderm unterliegt (Hannibal et al., 2012; Rao et al., 1991). Der Vergleich der durchschnittlichen Heterochronieraten, die für jedes Ereignis ermittelt wurden, zeigt, dass die Myogenese in deutlich stärkerem Maße von heterochronen Veränderungen betroffen war als die Neurogenese und die Bildung der Extremitätenknospen (Tabelle 4 und Abbildung 10a in Jirikowski et al., 2015: Kapitel 7). Vergleicht man die segmentspezifischen Heterochronieraten für die drei untersuchten Gewebetypen, so fällt auf, dass insbesondere in den nauplialen Segmenten die Myogenese einer stärkeren Dynamik unterliegt als die Neurogenese oder die Bildung der Extremitätenknospen (Abbildung 10b in Jirikowski et al., 2015: Kapitel 7). Vor dem Hintergrund einer besonderen instruktiven Rolle des Ektoderms gegenüber dem Mesoderm im Entwicklungsprogramm ist es plausibel, dass Veränderungen im Verlauf der ektodermalen Morphogenese gravierendere Auswirkungen auf die Fitness haben und demnach in stärkerem Maße der Selektion unterliegen. Die Heterochronieanalyse zeigt somit, dass das charakteristische Embryonalstadium, das als Einauplius sensu Scholtz (Scholtz, 2000) bekannt geworden ist und von manchen Forschern als phylotypisches Stadium interpretiert wurde (Williams, 1994; Dahms, 2000), wahrscheinlich auf eine spezifische modulare Organisation des Entwicklungsprogrammes der Eumalacostraca zurückgeführt werden kann.

Das Mesoderm der Malacostraca setzt sich aus mehreren Zellpopulationen zusammen, die sich hinsichtlich ihrer ontogenetischen Bildungsweise unterscheiden, wie das präantennale Mesoderm, das naupliale Mesoderm, das Mesoderm des ersten Maxillensegmentes, das teloblastische Mesoderm und das Telsonmesoderm (Abbildung 3). Die Muskelvorläufer, die auf diese verschiedenen mesodermalen Zellpopulationen zurückgehen, zeigen jedoch keine deutlichen Unterschiede in ihrer Heterochronierate. Der posteriore Longitudinalmuskelvorläufer (Telsonmesoderm) sowie die Extremitätenmuskeln des ersten Thoraxsegmentes (teloblastisches Mesoderm) zeichnen sich durch das Fehlen von Heterochronie aus. Ob dies jedoch als Effekte einer besonderen modularen Organisation zu interpretieren ist, kann anhand der verwendeten Daten nicht plausibel gemacht werden. 34 1.11 Alternative phylogenetische Hypothesen Die Rekonstruktion des evolutionären Verlaufes geschieht hier auf der Grundlage der phylogenetischen Beziehungen aus Richter & Scholtz (Richter & Scholtz, 2001; Wirkner & Richter, 2010). Andere Untersuchungen unterstützen ein Monophylum aus Decapoda und Euphausiacea, das Eucarida genannt wird (Spears et al., 2005; Watling et al., 2000; Wills, 1998). Nach diesen Szenarien wäre die Naupliuslarve nur einmal innerhalb der Malacostraca entstanden. Für die in der vorliegenden Arbeit durchgeführten Untersuchungen war jedoch kein Vertreter der Euphausiacea verfügbar und eine Rekonstruktion der Sequenzevolution unter der Eucarida-Hypothese damit nicht möglich. Eine Bewertung der beiden alternativen Szenarien mithilfe der Heterochronieanalyse war daher nicht möglich. Die sekundäre Evolution der Naupliuslarve konnte nur für die Dendrobranchiata untersucht werden.

Jenner et al. (Jenner et al., 2009) haben in großem Umfang morphologische und molekulare Daten aus verschiedenen Publikationen zusammengetragen und verschiedene Analysemethoden und Datenpartitionen auf ihre phylogenetische Aussagekraft hin untersucht. Sie kommen zu dem Schluss, dass eine zuverlässige Phylogenie der Malacostraca aus der aktuellen Datenlage nicht abgeleitet werden kann. Dies wird zum Teil darauf zurückgeführt, dass sich die phylogenetischen Signale der verwendeten molekularen Marker gegenseitig widersprechen. In der Mehrzahl der Kladogramme bilden jedoch die Leptostraca die Schwestergruppe aller anderen Malacostraca. Die morphologischen phylogenetischen Analysen von Jenner et al. (Jenner et al., 2009), für die eine Auswahl von publizierten Datensätzen kombiniert wurde (Pires, 1987; Poore, 2005; Richter & Scholtz, 2001; Schram & Hof, 1998; Wills, 1998), stellen zudem die Bathynellacea an die Basis der Eumalacostraca. Innerhalb der Schwestergruppe der Bathynellacea stehen wiederum die Stomatopoda den übrigen Taxa als Schwestergruppe gegenüber. Da den Leptostraca, Bathynellacea und Stomatopoda eine Naupliuslarve fehlt, wäre dies auch nach den Ergebnissen von Jenner et al. (Jenner et al., 2009) für den Entwicklungsmodus im Grundmuster der Malacostraca anzunehmen. Die Naupliuslarve wäre ebenfalls zweimal unabhängig innerhalb der Malacostraca entstanden.

1.12 Schlussfolgerungen Die Untersuchung der Muskelentwicklung hat bestätigt, dass die beteiligten zellulären Prozesse bei den Malacostraca mit denen der Insekten stark übereinstimmen. Zudem konnte gezeigt werden, dass ein konserviertes segmentales Muster von Muskelvorläufern gebildet wird, für das, wie auch bei Insekten, eine Gerüstfunktion für die weitere Muskelbildung angenommen werden kann.

Die Rekonstruktion der Sequenzevolution und die Heterochronieanalyse haben gezeigt, dass heterochrone Veränderungen der Muskelentwicklung für larval-embryonale Übergänge in der

35 Evolution der Malacostraca von großer Bedeutung sind. Zudem unterstützen die Ergebnisse der Heterochronieanalyse die Theorie, dass viele Aspekte der ektodermalen Gewebe hinsichtlich der zugrundeliegenden regulatorischen Mechanismen ein Entwicklungsmodul darstellen, welches der Myogenese hierarchisch übergeordnet ist. Die mit dem Einaupliusstadium im Grundmuster der Eumalacostraca zusammenhängende Entwicklungssequenz beschränkt sich auf die Bildung ektodermaler Strukturen und ging bei den Peracarida im Zusammenhang mit dem spezialisierten direkten Entwicklungsmodus verloren. Die Ergebnisse sprechen weiterhin dafür, dass ein plesiomorphes naupliales Entwicklungsprogramm als Prädisposition für die sekundäre Evolution der Naupliuslarve bei den Dendrobranchiata diente.

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42 2 Erklärung über den Eigenanteil an den Manuskripten

Methoden

Ich wurde von Dr. Martin Fritsch und Dr. Ole Möller (WMi, A & S Zoologie) in die Methoden der Präparation, Immunhistochemie und Mikroskopietechnik eingewiesen, die in jedem der drei Manuskripte zum Einsatz kamen. Der Umgang mit der 3D-Rekonstruktionssoftware Imaris (Bitplane) wurde mir von Dr. Martin Stegner und Dr. Jonas Keiler (WMi, A & S Zoologie) vermittelt.

Jirikowski GJ, Kreissl S, Richter S, Wolff C. 2010. Muscle development in the marbled crayfish - insights from an emerging model organism (Crustacea, Malacostraca, Decapoda). Development, genes and Evolution. 220:89-105.

Das für dieses Manuskript untersuchte Eimaterial wurde von Tieren aus der Laborkultur der A & S Zoologie gesammelt. Präparation, Fixierung und Immunhistochemie wurden von mir eigenständig geplant und durchgeführt, ebenso die mikroskopische Dokumentation, konfokale Laserscanningmikroskopie und 3D- Bildbearbeitung mit Imaris (Bitplane). Die verwendeten monoklonalen Myosin-Antikörper 16C6 und 17C5 wurden von Dr. Sabine Kreissl im Rahmen eines anderen Forschungsprojektes generiert und für die Arbeit an der Marmorkrebsentwicklung zur Verfügung gestellt. Der im Rahmen der gemeinsamen Arbeit erstmals publizierte Antikörper 17C5 wurde von Dr. Sabine Kreissl durch Westernblot charakterisiert. Sie erstellte die Abbildung 1 für das Manuskript. Die Bildtafeln und die erste Version des Manuskriptes wurden von mir erstellt. Das endgültige Manuskript wurde von Prof. Dr. Stefan Richter, Dr. Sabine Kreissl, Dr. Carsten Wolff und mir selbst gemeinsam verfasst. Eine Englisch-Korrektur wurde von Lucy Cathrow vorgenommen.

Jirikowski GJ, Richter S, Wolff C. 2013. Myogenesis of Malacostraca – the “egg-nauplius” concept revisited. Frontiers in Zoology. 10:76-103.

Das Konzept für dieses Manuskript wurde von mir und Prof. Dr. Richter erstellt. Die Planung und Durchführung der Experimente wurde von mir vorgenommen. Die von Dr. Sabine Kreissl bereitgestellten Antikörper wurden für dieses Manuskript weiterverwendet. Eimaterial von Gonodactylaceus falcatus wurde von Prof. Dr. Stefan Richter und Dr. Carsten Wolff bei einer Sammelreise nach Coconut Island, Hawaii gesammelt. Ich erstellte eine vorläufige Version des Manuskriptes, einschließlich der Bildtafeln, anhand derer die endgültige Fassung geschrieben wurde, ebenfalls in gemeinsamer Arbeit von Prof. Dr. Stefan Richter, Dr. Carsten Wolff und mir. Dr. Joanna Wolfe führte eine English-Korrektur des Textes durch.

Jirikowski GJ, Wolff C, Richter S. 2015. Evolution of Eumalacostracan development – new insights into loss and reacquisition of larval stages revealed by heterochrony analysis. Evo Devo. 6:4. 43 Das Konzept für die vergleichende Arbeit unter Zuhilfenahme computerbasierter Heterochronieanalyse wurde von Prof. Dr. Stefan Richter erstellt. Für die Kodierung von Entwicklungssequenzen wurden größtenteils die Ergebnisse des zweiten Manuskriptes verwendet. Ich führte jedoch auch weitere immunhistochemische Färbungen durch. Außerdem wurden für die Art Originale von bereits publizierten Bilddaten nachuntersucht, die von Dr. Phillipp Hertzler bereitgestellt wurden. Die Kodierung von Entwicklungsserien wurde von mir in Eigenregie durchgeführt. Die Benutzung der Software PGi, für die Heterochronieanalyse in der freien Programmierumgebung „R“, habe Ich mir unter der Anleitung von Dr. Luke Harrisson angeeignet. Die Durchführung der Analysen und die Erstellung des vorläufigen Manuskriptes lagen ebenfalls in meiner Verantwortung. Prof. Dr. Stefan Richter, Dr. Carsten Wolff und ich verfassten gemeinsam die endgültige Version des Manuskriptes.

Unterschriften

Günther Jirikoski Prof. Dr. Stefan Richter (Betreuer)

44 3 Selbstständigkeitserklärung

Ich versichere hiermit an Eides statt, dass ich die vorliegende Arbeit selbstständig und ohne fremde Hilfe angefertigt habe. Ich erkläre, dass keine Hilfsmittel und Quellen zum Einsatz kamen, außer den von mir angegebenen. Weiterhin lege ich hiermit Zeugnis darüber ab, dass weder Textabschnitte noch Abbildungen von Dritten übernommen wurden, sofern dies nicht ausdrücklich gekennzeichnet ist.

Diese Arbeit wurde keiner anderen Prüfungsbehörde vorgelegt.

Berlin, den 15.02.2015

Günther Jirikowski

45 4 Danksagung Mein besonderer Dank gilt meinem Betreuer Prof. Dr. Stefan Richter. Ich danke Ihm für die Einführung in das faszinierende Reich der Krebse, vor allem aber für die jahrelange tatkräftige Unterstützung, bei der wissenschaftlichen Arbeit und seinem Engagement bei der Organisation derselben. Ebenso dafür, dass er es mir ermöglicht hat an weiteren, teils nicht-biologischen akademischen Projekten mitzuwirken.

Mein Ko-Betreuer Dr. Carsten Wolff hat mich in die Entwicklungsbiologie der Krebse eingeführt und mich im Umgang mit ihren zahllosen Tücken und praktischen Herausforderungen vertraut gemacht. Ich danke ihm für seine Hilfsbereitschaft und seine Geduld.

Dr. Christian Wirkner danke ich für die Vermittlung seiner strengen, aber dafür erstklassigen Schule der Graphik und Präsentation sowie für das gelegentliche Singen im Nachbarzimmer.

Den Zimmerkollegen Dr. Martin Stegner und Dr. Martin Schwentner danke ich für die tolle Zeit in der Bürogemeinschaft und die akademischen Ausschweifungen.

Besonders Jonas Keiler, Martin Fritsch und Susi Böx danke ich für die wunderbare gemeinsame Zeit an der Rostocker Zoologie sowie allen anderen Kollegen, die mich in diesen Jahren begleitet haben.

Meiner Freundin Lucie danke ich für das beständige Aufheitern und den Rückhalt, den sie mir gibt. Natürlich auch für die Karikaturen meiner hochwissenschaftlichen Schemazeichnungen.

Zuletzt danke ich meinen Eltern die mich stets bei allem, was ich tat, ermutigt haben. Ohne sie wäre diese Arbeit nicht möglich gewesen.

46 5 Jirikowski GJ, Kreissl S, Richter S, Wolff C. 2010. Muscle development in the marbled crayfish - insights from an emerging model organism (Crustacea, Malacostraca, Decapoda). Development, genes and Evolution. 220:89-105.

47

48 Dev Genes Evol (2010) 220:89–105 DOI 10.1007/s00427-010-0331-7

ORIGINAL ARTICLE

Muscle development in the marbled crayfish—insights from an emerging model organism (Crustacea, Malacostraca, Decapoda)

Günther Jirikowski & Sabine Kreissl & Stefan Richter & Carsten Wolff

Received: 6 April 2010 /Accepted: 16 June 2010 /Published online: 14 August 2010 # Springer-Verlag 2010

Abstract The development of the crustacean muscular development of the muscular heart tissue from myogenic system is still poorly understood. We present a structural precursors and the formation and differentiation of visceral analysis of muscle development in an emerging model musculature. organism, the marbled crayfish—a representative of the Cambaridae. The development and differentiation of mus- Keywords Marmorkrebs . Development . cle tissue and its relation to the mesoderm-forming cells are Muscle precursor . Myogenesis . Evolution described using fluorescent and non-fluorescent imaging tools. We combined immunohistochemical staining for early isoforms of myosin heavy chain with phallotoxin Introduction staining of F-actin, which distinguishes early and more differentiated myocytes. We were thus able to identify Malacostracans exhibit a diverse set of developmental single muscle precursor cells that serve as starting points modes. Adult individuals can arise from a free-swimming for developing muscular units. Our investigations show a nauplius larva that carries only three appendage-bearing significant developmental advance in head appendage body segments and passes through a series of further larval muscles and in the posterior end of the longitudinal trunk stages. This is the case in Dendrobranchiata and Euphau- muscle strands compared to other forming muscle tissues. siacea. The hatching of more advanced larvae, zoea- or These findings are considered evolutionary relics of larval zoea-like larvae, is observed in Stomatopoda, Bathynella- developmental features. Furthermore, we document the cea, Amphionidacea, Caridea, Stenopodidea, and Reptantia (Richter and Scholtz 2001). Peracarida, freshwater crayfish, and Leptostraca share the feature of direct development, in Communicated by S. Roth which juveniles hatch as a premature version of the adult. G. Jirikowski (*) : S. Richter The development of the muscle system can be assumed Universität Rostock, Institut für Biowissenschaften/Allgemeine to reflect this diversity, due not least to its importance in und Spezielle Zoologie, larval locomotion. At the same time, the mesodermal tissue Universitätsplatz 2, from which postnaupliar muscles are formed originates in 18055 Rostock, Germany e-mail: [email protected] the same way in all malacostracan representatives: post- naupliar body segments are generated by teloblastic C. Wolff proliferation from specific posterior cells, the mesotelo- Humboldt-Universität zu Berlin, Institut für Biologie, blasts. Segmental primordia of embryonic mesoderm are AG Vergleichende Zoologie, Philippstr.13, laid down in a process of repeated asymmetrical division of 10115 Berlin, Germany these eight cells. The cells are arranged in a transverse row, as in the Peracarida (Dohle 1970; Dohle 1972; Scholtz S. Kreissl 1990) or in a ring around the gut primordium as seen, for Universität Konstanz, Fachbereich Biologie, example, in Decapoda (Scholtz 2002). General aspects of 78457 Konstanz, Germany mesoderm formation have been described in the decapods 90 Dev Genes Evol (2010) 220:89–105

Palaemonetes varians (Weygoldt 1961), Heptacarpus cells already show muscle-specific protein expression and rectirostris (Oishi 1959), Pagurus samuelis, Hemigrapsus have the potential to recruit undifferentiated mesodermal sanguineus (Oishi 1960), Cherax destructor (Scholtz cells, termed “fusion-competent myoblasts”.Fusionof 1992), and the amphipods Gammarus pulex (Scholtz muscle pioneers and fusion-competent myoblasts give rise 1990; Weygoldt 1958) and more recently in Orchestia to syncytial muscle precursors, which eventually differen- cavimana and Parhyale hawaiensis (Wolff and Scholtz tiate into functional myotubes (Baylies et al. 1998; Paululat 2002; Gerberding et al. 2002). et al. 1999; Campos-Ortega and Hartenstein 1997). In this This particular mode of mesoderm formation leads to a paper, we hope to clarify if certain aspects of gradient of developmental progress along the AP axis. myogenesis also hold true for the crayfish. Because the segmental mesoderm primordia each represent High resolution analysis techniques such as confocal genealogical entities that are serially homologous (Scholtz laser scanning microscopy and computer aided 3D visual- 2002), different developmental stages can be compared, ization and editing make it possible to look at muscular even within one specimen. This way, valuable data is added development on a cellular level and greatly facilitates the to the strictly temporal tracing of development and presentation of the results. The conserved segment polarity differentiation. factor Engrailed is a general marker for segment boundaries There are few published studies on the morphogenesis of in embryonic development and can be visualized with the the muscle system. Recent work on the amphipod P. help of the cross reactive antibody Mab4D9 (Developmen- hawaiensis has revealed that orthologs of muscle- tal Studies Hybridoma Bank, University of Iowa) (Abzha- determining genes are expressed in a segmental fashion as nov and Kaufman 2000). Since somites of the the germ band elongates (Price and Patel 2008). This malacostracan trunk are initially formed as single rows of pattern is expected, if muscle progenitors are considered cells and exhibit conserved stereotypic cell division patterns derivatives of the mesoteloblast cells. Phallotoxin staining in the ectoderm (Scholtz 1992), we use this marker to of dendrobranchiate embryos (Kiernan and Hertzler 2006) assign developing muscle components to their precise revealed that development of the muscle apparatus in this location within the segment. species is dominated by the formation of the nauplius locomotor musculature. We wanted to describe embryonic muscle formation in a malacostracan that does not pass Methods through larval stages but develops directly. Development seems to be highly conserved among crayfish taxa (Vilpoux Embryo collection et al. 2006). The marbled crayfish has received a great deal of attention in recent years due to its strictly parthenoge- We used the staging system established by Alwes and Scholtz netic mode of reproduction, a feature never previously (2006) to identify embryonic stages during the collection of observed in or described for decapods. The ease with which series. Embryos were collected from the pleopods of gravid this species can be reared and egg material obtained has led females twice a day for 10 days, starting on day 5 after egg many authors to propose the establishment of this as deposition (embryonic stage 3, which corresponds to the model system for developmental studies (Alwes and egg-nauplius). The chorion and the vitelline membrane were Scholtz 2006; Scholtz et al. 2003; Seitz et al. 2005; Vogt removed by hand dissection. Fixation was carried out in 4% et al. 2004). paraformaldehyde (Electron Microscopy Sciences)/PBS

Kreissl et al. (2008) introduced an antibody with a (1.86 mM NaH2PO4,8.41mMNa2HPO4, 175 mM NaCl, particularly high sensitivity to the heavy chain of the pH 7.4). For fluorescent staining and CLSM or regular myosin protein which permits the early visualization of transmission microscopy, the soft yolk mass was dissected muscle pioneer cells in Isopoda. We apply the same method from the embryos. Additionally, eggs were fixed by boiling to the marbled crayfish and combine it with phallotoxin in PBS for 10 min at 85°C followed by incubation in staining of F-actin and fluorescent detection systems to paraformaldehyde. This procedure preserves the entire yolk, document the process of muscular development. which hardens at high temperatures, and was only used for Moreover, the study of insect myogenesis has led to the the combination of staining and signal detection using the discovery of single muscle founder cells (Steffens et al. alkaline phosphatase system. 1995, Xie et al. 1994) and to the establishment of the “founder cell model” of myogenesis, which has been most Generation of monoclonal antibodies and characterization thoroughly tested on Drosophila melanogaster. According of the antibody 017C5 to this model, mesodermal muscle progenitor cells are specified that undergo division to form muscle pioneer cells A library of monoclonal antibodies against proteins of (termed “muscle founder cells” in the fly). Muscle pioneer meso- and metathoracic locust muscles (flight muscles and Dev Genes Evol (2010) 220:89–105 91 intrinsic muscles) was generated and screened as loaded with 1–2 μg protein, and gels were run according to described earlier (Kreissl et al. 2008). In brief, a crude standard techniques. The proteins were transferred to myosin extract (Dalbis et al. 1979) of locust muscles was cellulose nitrate membranes (Protran, Schleicher & Schuell used as the antigen to immunize mice. Hybridoma clones GmbH, Dassel, Germany) and selective binding of cell were screened and subsequently selected by enzyme-linked culture supernatant of the clone 017C5 at a dilution of 1:10 immunosorbent assay, immunostaining on cryosections of was detected with the IgG-ABC-ELITE-POD kit (Vector native and paraformaldehyde-fixed locust muscles, as well Labs) using ECL (Pierce) as a substrate. Molecular weight as western blot analyses. The antibodies generated against markers were biotinylated, allowing direct detection by the meso- and metathoracic locust muscle proteins were also ABC reagent in the western blots. screened for their binding affinity to muscle tissue or myosin of rabbits. The supernatant of the clone 016C6 was Specificity of antibodies 016C6 and 017C5 also screened for binding to proteins of the isopod crustacean Idotea emarginata (Kreissl et al., 2008). For 016C6 antibodies have previously been shown to be the identification of the antigen of monoclonal antibodies specific for myosin-HCs of Locusta migratoria, I. emargi- produced by clone 017C5 in western blots, the crude nata (Isopoda), Eriphia spinifrons (Decapoda), and to the myosin extracts of locust flight and muscles from the slow myosin-HC-I isoform of rabbit muscles (Kreissl et al. meso- and metathorax and myosin from rabbit muscle 2008). Screening the supernatant of monoclonal hybridoma (SIGMA, Deisenhofen, Germany, cat. nr. M1636) were cell line 017C5 for binding affinity to muscle proteins in separated by sodium dodecyl sulfate-polyacrylamide gel western blots revealed strong labeling of locust muscle electrophoresis (Fig. 1a). The lanes of 10% linear gels were proteins with an apparent molecular weight of about 200 kDa and myosin heavy chains of rabbit muscle (Fig. 1b). These findings indicate that the monoclonal antibody 017C5 exhibited prominent affinity for myosin- HC of and arthropods. In addition, 017C5 strongly binds to high molecular weight compounds of the locust muscle extract which most likely represent non- dissociated dimers or multimeres of myosin-HC (Fig. 1b). We used Mab 017C5 as a general marker for differentiating muscle cells already containing myosin heavy chains. It was particularly useful for staining of the cardiac and gut muscle cells.

Antibody staining

Embryos were washed prior to staining in PBT (PBS pH 7.4; 0.3% bovine serum albumin (MERCK Darmstadt), 0.3% Triton X-100; 1.5% dimethylsulfoxide) for 3 h and incubated for 1 h in PBT and 3% normal goat serum (Dako) to block unspecific binding sites. Incubation was carried out overnight at 4°C in a 1:10 dilution of supernatant in PBT. Signal detection was performed using mouse antibodies (Cy3- labeled goat-AffiniPure anti-mouse IgG H + L, Jackson Immunoresearch or alkaline phosphatase conjugated Affini- Fig. 1 017C5 specifically binds to myosin heavy chains. a Separation of muscle proteins on 10% SDS-PAGE. Lanes were loaded as follows: Pure goat anti-mouse IgG H + L, Jackson Immunoresearch). molecular weight markers (MW), rabbit myosin, no loading to prevent Embryos were washed in PBT for 3 h to remove unbound spillover of rabbit and locust proteins, muscle homogenates of L. antibody and incubated in blocking solution for 1 h, as migratoria flight muscle, and muscle homogenates of L. migratoria described above. Secondary antibodies were diluted 1:200 walking muscles. Proteins were silver stained. Numbers at the left indicate molecular weight in kilodalton and the arrow indicates start and applied as described for the primary antibody incubation. of the separation gel. Myosin heavy chains are prominent at MW of Washing in PBT for 4 h followed to remove unbound about 200 kDa. b Identification of the 017C5 antigen by western blot antibody. Embryos were then counterstained with Hoechst analysis of a gel run in parallel with that shown at the left reveals (Bisbenzimide, Biochemica) 1 μg/ml in PBS and Alexa myosin heavy chain of rabbit muscle and locust muscle. Note that the antibody also binds to non-dissociated myosin heavy chain multimeres A488-conjugated phalloidin (Invitrogen Molecular Probes) to in the loading gel which did not enter the separation gel visualize nuclei and F-actin. All washing steps were carried 92 Dev Genes Evol (2010) 220:89–105 out on a horizontal shaker (neoLab1). Objects were mounted signal visualization in anti-myosin-HC stains only produced on microscopy slides or cover slips in glycerol containing poor results in embryos younger than stage 5, but it was 2.5 mg/ml of the anti-bleaching agent Dabco (ROTH). possible in these specimens to detect a signal using the alkaline phosphatase system. We have adapted the terminology of the “founder cell Microscopy model” of insect myogenesis for description of the cell types observed in crayfish muscle development. Single Light microscopy was carried out using an Axio-Imager cells positively stained for myosin-HC which lay down a M1 compound microscope and a Discovery V12 stereo scaffold for muscle formation early in development are microscope, both equipped with an Axio-Cam ICc3 camera termed “muscle pioneer cells”. The multicellular or syncy- (Zeiss, Jena). Image stacks were taken using a Leica DM tial primordia arising from these pioneers are referred to as IRE2 confocal laser scanning microscope equipped with a muscle precursors. Unfortunately, we cannot say if fusion Leica TCS SP2 AOBS laser scanning unit. Step sizes has taken place in the muscle precursors. The undifferen- ranged from 0.35 to 1.0 μm. Late-stage embryos could not tiated mesodermal cells that together with muscle pioneer be scanned at z-depths of more than 50 μm so they were cells, form the precursors, are termed “myoblasts”. We want mounted on 60 mm cover slips and scanned from both to emphasize, however, that the common use of insect sides. The image stacks were then inverted and aligned terms for crustaceans is used to maintain the simplicity of manually using “Autoaligner” (Bitplane AG, Switzerland). the descriptions but must not be interpreted as a statement Subsequent analyses were performed with “IMARIS 6.4.0” of between insect and crustacean muscle- (Bitplane AG, Switzerland). All figures were compiled forming cells. using “Corel Graphic Suite XIII”. The description of myogenesis is based on the staging system established by Development of cephalic muscles Alwes and Scholtz (2006). Muscle precursors of the head segments are first detected by immunostaining and after stage 4 by a combination of Results immunostaining and phalloidin. The role of the precur- sors in the juvenile is not always clear. To describe In crayfish development, the tissues of the prospective precursors observed here, we therefore use a code of trunk and are first laid down on the surface of lower case letters and numbers which specifies the body the large yolk mass. The caudal papilla, the posterior part of segment in which the primordium is seen (designated by the embryo containing the ectoteloblast and mesoteloblast its characteristic appendage, e.g., 1), the orien- cells that proliferate the cell material of new body seg- tation of the forming muscle primordium in relation to ments, is flexed anteriorly after stage 3. A specimen the appendage bud (lateral or medial), and a number that providing a ventral view of the segments anterior to the further specifies the primordium if more than one is fold therefore displays the segments posterior to the fold present in one segment. a2-l2, for example, represents the from a dorsal perspective (for orientation see Fig. 2a–c). second laterally orientated muscle precursor in the antenna A series of embryos stained with phalloidin is shown in 2segment. Fig. 2d–h. Anti-myosin-HC 016C6 has proven to effective- Initiation of muscle differentiation appears late in ly stain muscle-forming cells before myofibrils have stage 3 in groups of mesodermal cells which form formed within the cytoplasm. This is possibly due to the muscle precursors in the head segments of antenna 1 considerable affinity between these antibodies and mono- (a1-m1), antenna 2 (a2-l1, a2-l2), the mandible (md-m1, meric forms of the protein. Mononucleate muscle pioneers md-l1),andmaxilla1(mx1-l1)(Fig.3a). These precursors can therefore be identified in the earliest stages of the display significant myosin-HC immunoreactivity. At stage differentiation process by the increase in monomeric 4, the precursors also show a phalloidin signal. a1-m1 myosin in the cytoplasm. As differentiation continues, reaches anterolaterally from the ventral periphery of the fibrous patterns are also visible, caused by the emerging stomodaeum to a region anterior to the bud of antenna 1 filamentous actin (F-actin) signal. As a result, it was (Fig. 3b). It contains just two positively stained cells possible to identify both striated and non-striated muscles whose nuclei are positioned in the middle of the muscle in various embryonic tissues. Since phalloidin binds precursor (Fig. 3c). The nuclei of a2-l1, a2-l2,andmd-l1 specifically to F-actin, its use in combination with the are more evenly distributed within these precursors myosin antibodies makes it possible to identify muscle (Fig. 3d). At stage 5, e-m1, a muscle precursor reaching pioneer cells and precursors that differ in their grade of from the anterior of the stomodaeum to the medial region differentiation. The fluorescent detection system used for of the optic lobe, arises (Fig. 3e). The posterolaterally Dev Genes Evol (2010) 220:89–105 93

Fig. 2 Overview of Marmorkrebs' myogenesis. a Schematic drawing of have expanded anteriorly and posteriorly. g Musculature of the an egg in lateral view. b Drawing of an embryo (without yolky mass) in anlage has formed a longitudinal band on each side of the embryo. ventral view. c Stereomicroscopic image of embryo as in b, ventral view Intrinsic muscles of the postnaupliar appendages (asterisks) are visible. of living egg with the right half of the embryo outlined in white. d– h Dorsal view of same embryo. Longitudinal pleon musculature is h Overviews of phalloidin signal in embryos of stages 5 to 9. d First divided into multiple subunits. Letters in the upper left corners identify cephalic muscles (arrowheads) around the stomodaeum (St). Strong the developmental stages; Ca carapace anlage, cam carapace anlagen signal is seen in the early ventral nerve chord (Vnc). e Progenitors of muscles, Ch chorion, Cp caudal papilla, Ha heart anlage, mca myocard carapace muscles (cam), myocard (mca), and first longitudinal muscle anlage, slp subunits of longitudinal pleon musculature, St stomodeum, Y fibers (double arrows) are visible. f Carapace anlagen muscles (cam) yolk. Scale bars,200μmin all panels protruding precursor, mx1-l1, can be seen anteriorly of the bipartite structure. This precursor most likely represents anlagen of maxilla 1. At stage 7, a cephalic muscle a future adductor and possible rotator muscle of the precursor, mx2-l1, is added in the maxilla 2 segment mandible (Fig. 3j). The subsequent appendage anlagen (Fig. 3f). a1-m1 now contains multiple fibrous elements also reveal intrinsic muscle precursors which, however, (Fig. 3g). The median endings of a2-l1 and a2-l2 clearly are not the subject of this study. show an insertion into the bud of antenna 2 (Fig. 3h). md- m1 is partly positioned within the mandibular bud, Development of longitudinal muscles completely filling out this appendage anlage. At this stage, it contains a comparatively large number of cells, The longitudinal muscles of the trunk, which are especially going by the number of nuclei within the primordium prominent in the complex muscle apparatus of the adult (Fig. 3i). At stage 9, the median ends of e-m1 and a1-m1 pleon, are first laid down as a pair of undivided muscle are seen in close proximity to the stomodaeum and strands in the embryo. Their development involves a adjacent pharynx (Fig. 3j). We assume that they form transition from segmental single cell pioneers to multicel- pharyngeal dilator muscles, as seen in myosin heavy chain lular precursors that form multiple single muscles. staining in dissected juveniles (data not shown). All In stage 3 embryos, a row of spindle-shaped pioneer muscle precursors display a significant portion of con- cells exhibiting myosin immunoreactivity is found (lmp in densed fibrous elements, although striation is still difficult Fig. 3a). The anteriormost pioneer is located medially to make out in most areas. a2-l1 and a2-l2 take a more between the buds of antenna 2 and the mandible. A lateral position. The same applies to md-l1, which is now posterior pioneer between the limb buds of the mandible subdivided into two distinct units. md-m1 exhibits a and maxilla 1 follows. At stage 4, a continuous myosin-HC 94 Dev Genes Evol (2010) 220:89–105

Fig. 3 Embryonic muscles in the left half of the head anlage. Signal between a2-l1 (arrowhead) and a2-l2 (double arrowhead) and the detection of anti-myosin-HC was performed by alkaline phosphatase base of antenna 2 are seen more clearly here than in stage 5. i reaction (dark blue). Other samples are labeled with anti-myosin-HC Myocytes of the prospective mandible adductor muscle md-m1. The (red) and phalloidin (green). The Hoechst signal (light blue) is added muscle primordium contains a comparatively large number of nuclei. j in g, h, and i. a Anterior half of stage 3 embryo. Muscle precursors Muscular elements around the pharynx (Px) and stomodaeum (St) a1-m1, a2-l1, a2-l2, md-m1, md-l1, and mx1-l1 are seen, as well as the become visible. The progenitor em1 is now connected to this complex. unicellular precursors of the longitudinal muscle strand (lmp). b Progenitors of further extrinsic appendage muscles (asterisks) are seen Overview of anterior head segments at stage 4. Muscle progenitors a1- along the anlagen of maxilla 2 and the maxillipeds. Letters in the m1, a2-l1, a2-l2, and mx1-l1 are seen. c Detailed view of a1-m1. upper left corners identify the developmental stages; A1 antenna 1, A2 Asterisks mark the positions of nuclei. d Close-up of a2-l1, a2-l2 and antenna 2, Cp caudal papilla, hp prospective heart precursor, lm md-l1 (the latter is not seen in b). e Muscle progenitor e-m1 is now longitudinal muscle strand, lmp longitudinal muscle strand precursor, also visible. f Overview of anterior head segments. An additional Ol optic lobes, Md mandible, Mx1 maxilla 1, Mx2 maxilla 2, Mp1 muscle progenitor mx2-l1 is seen anteriorly of the second maxilla. maxilliped 1, Mp2 maxilliped 2, Mp3 maxilliped 3. Scale bars, a–d 20 mx1-l1 and mx2-l1 both intersect with the longitudinal muscle strand μm, e–i 50 μm, j 100 μm (lm). g Close-up of the median ending of a1-m1. h Connections Dev Genes Evol (2010) 220:89–105 95 and F-actin positive longitudinal muscle strand can be seen The same applies to the patch-forming cells in the in the cephalic and thoracic segments. The precursors are pereomeres. No segmental regularity or repetition of distributed metamerically along the AP axis and are each myocyte patterns is evident (Fig. 4n, o). Furthermore, the made up of two or three cells (Fig. 4b). At stage 5, the cell position in which the segmental arrangement of nuclei ends number continues to increase as mesodermal cells showing can vary between stages and individuals. no myogenic signal become associated with the precursors of the segments of maxilla 1 and maxilla 2 (Fig. 4c, d). At Posterior longitudinal muscle origin stage 6, the number of cells increases as development progresses and a greater number of longitudinal F-actin One particularly surprising feature of crayfish myogenesis fibers is seen (Fig. 4e). At stage 7, the first striated fibers relates to the myocytes which form the posterior end of the can be seen within the precursor. The precursors in the ventral longitudinal muscle strands. The strands elongate anterior segments are separated by segmentally reiterated posteriorly as new segments bud off from the caudal subdivisions of the fiber bundles. This feature is strongest papilla. The posteriormost myogenic cells of the ventral in the segment of maxilla 2 and decreases posteriorly longitudinal muscle strand, the longitudinal muscle origin, throughout the developing thorax (data not shown). The lie slightly posterior to the ring of mesoteloblast cells. Even borders between two adjacent clusters are marked by an those myocytes closest to the teloblastic proliferation zone area of particularly strong F-actin signal (Fig. 4f). At late show no sign of segmental arrangement. This is visible in stage 4, a pair of parallel longitudinal muscle strands has high magnification confocal images of Engrailed- and F- appeared in the posterior part of the caudal papilla that actin-labeled embryos at stages 4 to 6 (Fig. 5a–h). The protrudes anteriorly into the segment of maxilla 1. In the mesoteloblast cells themselves can be identified with the segments of the prospective pereon, a continuous fiber help of the parallel rows that they form with their early strand is seen, containing only a few nuclei (Fig. 4g). At progeny as well as their position. The mesoteloblasts are stage 6, myocyte clusters are seen inside the strand located directly beneath the ectoteloblasts and possibly positioned below the appendage bud. This is shown for shifted to the anterior (see Fig. 5c, e, g–i). Ectoteloblasts pereomere 4 in Fig. 4h. At stage 7, the first signs of again are readily visible as the posteriormost row in the striation can be seen (Fig. 4i). Surprisingly, the posterior- chess-board-like pattern seen in the posterior germ band most part of the longitudinal muscle strand already shows a during elongation (shown in Alwes and Scholtz 2006). strong F-actin signal at stages 4 and 5. In comparison, the Early mesoteloblast progeny show a segmental arrange- myogenic signal is dominated by myosin-HC reactivity in ment which is quickly lost in the more anterior segments. the more anterior segments at these stages of development. Three-dimensional reconstruction helped to visualize these The strand in pleomeres 2 to 4 at stage 5 (Fig. 4j) shares the cells and the relative position of the longitudinal muscle features described for the pereomeres at stage 4 (Fig. 4g). origin in embryos stained for Engrailed and F-actin At stage 6, the strand is made up of multiple cells for each (Fig. 5a, b). Since the newly formed mesoblasts proceed pleomere. F-actin fibers are continuous, and no subdivision with their own cell divisions, only the mesoteloblasts of the strand is seen. However, the beginning of lateral themselves and their first three generations of descendents protrusions from the longitudinal strands can be observed can be distinguished from the posterior mesoderm. The (Fig. 4k). Also, additional cells have appeared along the clearly visible segmentation in the mesoderm ends poste- strand that show only myosin-HC immunoreactivity but no riorly to the first visible Engrailed stripe (shown yellow in F-actin signal. Fig. 5). The posteriormost myogenic signal is seen close to In contrast to the cell clusters in the anterior parts of the the mesoteloblasts rMT1 and rMT2 on the right side of the strand, the nuclei of the earliest visible muscle-forming caudal papilla of a stage 5 embryo. cells in the posterior segments do not show a clear The cells of the longitudinal muscle origin themselves segmental arrangement along the AP axis until stage 7. show a loose cluster of nuclei that are significantly smaller This is evidenced by confocal laser scanning micrographs than those of the surrounding mesoteloblasts and their of Engrailed/F-actin double staining of stage 5 and 6 segmental progeny. At late stage 4, approximately four embryos. At these stages, only a few nuclei in the nuclei are found, surrounded by F-actin fibers (Fig. 5c, d). pleomeres can be clearly assigned to longitudinal muscle These myocytes exhibit long protrusions that extend strand-forming precursors on the basis of their position anteriorly and converge into condensed bundles. At stage within the F-actin positive fiber bundle (Fig. 4l, m). 5, nuclei in the longitudinal muscle origin cell cluster are However, these nuclei cannot be clearly assigned to a more tightly packed and elongated (Fig. 5e, f). This specific position within the prospective somite (visualized suggests that myoblast fusion is taking place. The same is here by the yellow Engrailed signal which marks the true of stage 6, where the F-actin signal in the posteriormost cells in the ectoderm of each body segment). longitudinal muscle origin is strongest (Fig. 5g, h). In 96 Dev Genes Evol (2010) 220:89–105 addition, single nuclei are seen in the fiber strand slightly Fig. 4 Development of the longitudinal muscle strands from stage 4 b to 7: a Schematic drawings of crayfish embryos of stages 4 to 7 with anterior to the posterior patch. The position of the the caudal papilla and pleon anlage flexed posteriorly. The relative longitudinal muscle origin suggests a mode of formation positions of images (b–o) are indicated by colored frames. The frames independent of mesoteloblast proliferation. The first of images (b–o) have been colored accordingly. Longitudinal muscle detectable phalloidin signal in the caudal papilla is seen strands of the right body half, stained with Anti-Myosin-16C6/Cy3 (red), phalloidin/Alexa488 (green), and Hoechst (light blue). Dotted at stage 4. It is not restricted to muscle precursors, but also lines mark the anlagen of the appendages (in b and i) or the segment seen in the posterior pioneer neurons. However, this borders (in l–o). Arrowheads mark the position of myocyte nuclei. feature is not observed in later stages. Throughout all the The Hoechst signal is not shown in b and e. b Longitudinal muscle stages, Hoechst-positive granules are seen in the caudal progenitors forming a strand in the segments posterior to the mandible. Progenitors in the segment of maxilla 1 and 2 are composed papilla medially of the longitudinal muscle strands of two myocytes (arrowheads). c–d Close-up of progenitors in the (Fig. 5i). This suggests that a considerable level of segments of maxilla 1 and maxilla 2 at stage 5. Mesodermal cells apoptosis takes place in these tissues as part of the showing no myogenic signal lie close to the progenitors. e The developmental process. progenitor of the longitudinal muscle strand in the segment of maxilla 1 is made up of several myocytes. f Myocytes of the longitudinal muscle strand show striated fibers (double arrowhead). Borders Differentiation of the pleon muscles between the myocyte clusters of the segments show particularly strong F-actin signal (asterisks). g Early longitudinal muscle strand By stage 7, the formation of new segments from the caudal myocytes in pereomeres 3 to 5 (P3–5). h Myocyte clusters forming below the appendage bud in pereomere 4. Additional cells are added papilla has ceased. From stage 7 to hatching, the longitu- to the myocyte clusters in stage 7 as shown in i (ventral section). dinal muscle anlagen in the pleon undergo a transition from Striated fibers are also seen (double arrow). j Pleon segments at stage continuous fiber bundles to the complex muscle apparatus 5. The longitudinal muscle strands only contain a few nuclei of the adult pleon. During this developmental process, the (arrowheads) but exhibit continuous F-actin fibers. At stage 6, more F-actin fibers are present, but they remain continuous. The number of anterior/posterior gradient of differentiation is maintained. myocytes has slightly increased. In the more ventral portion of the An overview is given in Fig. 6a–e. In addition to the ventral strand, some fiber elements begin to take on a more lateral orientation pair of longitudinal muscle strands, a second pair appears at as seen in k (dotted line). Arrowheads mark cells of the strand this stage running parallel but dorsomedially to the previous showing myosin-HC but no F-Actin signal. l–o Caudal papilla and – pleon anlage labeled with anti-Engrailed 4D9 (yellow), phalloidin one (Fig. 6a e). At stage 7, it reaches as far posterior as the (green), and Hoechst (light blue). Nuclei of longitudinal muscle strand third pleon segment and consists of segmentally arranged myocytes are marked with arrowheads. l Longitudinal muscle strand cells and cell clusters within the dorsal mesoderm that are in pereomere 5 and pleomeres 1 to 4. m Longitudinal muscle strand in positively stained by anti-myosin-HC, but not by phalloidin pleomeres 1 to 4. n Strand containing cell patches in pereomeres 3 to 5. o Posterior longitudinal muscle origin and adjacent strand in at the posteriormost end (Fig. 6a, k). Unlike the posterior pleomeres 1 to 4. Letters in the upper left corners identify the part of the ventral longitudinal muscle strands, dorsal developmental stages. Mx1 maxilla 1, Mx2 maxilla 2, Mp1 maxilliped muscle precursors display no stretches of non-nucleated 1, Mp2 maxilliped 2, Mp3 maxilliped 3, P1–5 pereomeres 1–5, Pl 1–6 fiber bundles at any time in development. The segmental pleomeres 1–6, Cp caudal papilla, lmo longitudinal muscle origin. Scale bars,(b) 50 μm, all remaining panels 20 μm cell patches of the ventral strand in the future pleon extend into pleomeres 6 at stage 7, but no subdivision of fibers can be seen (Fig. 6 a, l). Many cells located closer to the body midline in the ventral strand show myosin-HC immunore- staining. The developing myocytes of the ventral strand activity but no F-actin signal. This suggests that they are continue to diversify in terms of fiber orientation. The not as differentiated as their more lateral counterparts. The central muscles become distinguishable in the dorsal part dorsal muscle strands at early stage 8 (Fig. 6b, m) reach of the strand as cell groups directing their fibers from a into pleomeres 5. Their F-actin fibers already show clear lateral position slightly posterior to the segmental furrow subdivisions close to the posterior segment borders and to a median position in the next posterior segment are significantly condensed into bundles in the medial of (Fig. 6p). Anterior to the fourth pleomere, cells form a the strand units. The lateral portions of the dorsal myocyte medioventral ending approximately one third of a segment groups also lack phalloidin signal and are therefore length anterior to the posterior somite border (Fig. 6q). interpreted as younger myocytes. In the ventral strands, Multiple cells that have maintained their antero-posterior the segmented clusters have also increased in size and cell orientation are also seen. F-actin fibers within muscular number. Cells within them now show diversification in cell groups anterior to pleomere 5 reveal units that start at their previously parallel fiber orientation. Lateral endings a ventromedial position at approximately half of the are formed near the segmental furrows (Fig. 6n). At late segment length and extend posteriorly. They insert stage 8, the forming myotubes in the dorsal strand show dorsolaterally, just anterior to the segmental furrow in striation (Fig. 6o). Striated fibers are also revealed the following segment (Fig. 6r) and represent the early throughout the ventral strand by F-actin and myosin-HC anterior oblique muscles. Dev Genes Evol (2010) 220:89–105 97 98 Dev Genes Evol (2010) 220:89–105

Fig. 5 Caudal papilla of embryos stained with phalloidin/Alexa488 the fiber strand anteriorly to the first Engrailed stripe (yellow). f (green) and Hoechst (light blue). In a–h, only the left half of the More dorsal section of the same object. The nuclei of the myocyte caudal papilla is shown. a–b Mesoteloblast nuclei and nuclei of patch are more elongated and tightly packed. This may be a sign of early mesoteloblast progeny have been reconstructed from the myocyte fusion. g Ventral section at stage 6. h More dorsal section Hoechst signal (white: mesoteloblasts and second generation of ofthesameobject.i The F-actin signal is also visible in the progeny, light blue: first and third generation). The phalloidin signal cytoskeleton of the cell cortex. Myocytes of the longitudinal muscle of the forming longitudinal muscle strand myocytes is shown in strands show phalloidin-reactive processes that protrude anteriorly green. The Engrailed signal is shown in yellow. The nuclei of the (arrowheads), also seen in the paired posterior pioneer neurons remaining tissues are not shown. a Dorsal view. b Ventral view. c– (asterisks). Granules of degrading DNA lie medially of the posterior h Extended optical sections through the caudal papilla (left half)in myocytes (dotted line). Letters in the upper left corners identify the ventral view. Arrowheads mark the position of nuclei, asterisks mark developmental stages. hg hind gut, lMT1–4 left mesoteloblast cells ectoteloblast cells. c Ventral section. d More dorsal section of the 1–4, rMT1–4 right mesoteloblast cells 1–4. Scale bars,20μminall same stage. e Ventral section. A single nucleus (triangle)isseenin panels

At late stage 9, the characteristic subunits of the pleonal with three pairs of ostia and is surrounded by the pericardial muscle apparatus are more differentiated. The number of sinus. In contrast to longitudinal muscle development, the visible nuclei within the muscles has decreased significant- formation of myocardial tissues in the Marmorkrebs takes ly. The pattern of nuclei around the fiber bundles suggests place without segmentally arranged muscle precursors. that they have been moved to the periphery of the syncytial The heart-forming cells can first be clearly distinguished muscle precursor (Fig. 6s, t). The dorsal strand muscle units at stage 5 in the dorsal periphery of the third maxilliped have broadened and formed parallel elements. Crayfish segment, posterior to a myogenic cell group that will later pleon muscles reflect the general pattern of adult pleon form a muscular band surrounding the carapace anlage musculature in decapods. The pleonic muscle complex (cam) (Fig. 2c–f). Fibrous elements of the heart-forming comprises the muscles responsible for the flexion of the cells stain positive for F-actin (Fig. 7b, c) and myosin-HC pleon, the anterior and posterior oblique pleonal muscles, 017C5 (Fig. 7f) at this stage, though the myosin-HC signal the central muscles and the transverse (stator) muscles as is less strong in the cellular processes. The heart primor- well as the dorsal pleonal muscles which function as dium is passively shifted posterior as segments from the opposing extensor muscles (Fig. 6u, v). caudal papilla move anteriorly around the fold. The majority of myocyte nuclei remain close to the periphery of the caudal fold (Fig. 7f). At stage 6, the heart Cardiogenesis primordium is a contractile membrane between the epider- mis and the caudal fold (Fig. 7d). At this stage, irregular The heart, which is responsible for the distribution of contractions of the membrane can be observed in the living hemolymph in the adult animal, represents a muscular bulb egg. The fine structure of the membrane reveals a network Dev Genes Evol (2010) 220:89–105 99 of F-actin positive cellular processes (Fig. 7g). By stage 7, pioneers). At late stage 4, these cells show significant the density of the network has increased, and the membrane myosin-HC 017C5 signal and form a layer surrounding the has expanded anteroposteriorly and dorsally on both sides proctodaeum (Fig. 8d). Since these myogenic cells are not (Fig. 7e, h). In subsequent stages, the network becomes yet clearly separated, it is impossible to determine if they make denser, and the fibers begin to show striation (Fig. 7i). up an early syncytium. We suggest, however, that they Interestingly, the phalloidin signal is stronger in fibers represent pioneers or precursors of visceral ring muscles facing the heart lumen (Fig. 7j). At stage 8, the dorsal that control the peristaltic movements of the intestine. The extensions of the contractile network have closed under- most posterior visible cells of this group are located neath the dorsal epidermis, forming a contractile tube ventrolaterally to the gut, but anteriorly to the MT1 cells (Fig. 7k). Newly formed fibers pass through the heart and the first 2–3 mesoteloblast pairs (Fig. 8e, f). This lumen. Anteriorly and posteriorly, the myocardium narrows suggests that gut muscles are generated in a non-teloblastic and is connected to the anterior and posterior aorta. way. The signal is not seen more anteriorly in the gut Myosin-HC immunostaining using the alkaline phosphatase muscle precursors which are, however, strongly stained by detection system on whole eggs in which only the chorion phalloidin. At stage 9, striation is seen in the circular gut and vitelline membrane had been removed, revealed a muscles (Fig. 8g). signal in cells of the anterior aorta (Fig. 7k, l). These cells form longitudinal rows that extend anteriorly from the heart. However, this feature is lost after stage 8. Between Discussion stage 8 and hatching, the myocardium grows more compact, and the ostia become visible (Fig. 7m). At stages By using the antibody clones myosin-HC 016C6 and 9 and 10, the progenitors of the alary muscles are visible 017C5 in combination with F-actin staining by phalloidin, under the ventral surface of the myocardium (Fig. 7n, o). we have shown that muscle cells with differing grades of Initially, two parallel muscles form on each side of the differentiation can be distinguished in one and the same ventral heart midline, extending laterally. The muscles will object. This greatly improves interpretation of the processes eventually split, giving rise to the four muscle pairs seen in involved in forming the complex muscular system in the adult (data not shown). analyses of series of developmental stages. Steffens et al. (1995) and Xie et al. (1994) have described a characteristic Visceral musculature pattern of early muscle generation in the ventral thoracic embryonic segments of the grasshopper Schistocerca In living crayfish eggs, we observed slow contractions of gregaria. These authors report single cell muscle pioneers the yolk mass after stage 8, marking its incorporation into that form cone-like processes towards the ectodermal the forming midgut gland. Using immunostaining for insertion sites. One pair of transverse and one or two pairs myosin-HC protein on whole dechorionated eggs, we found of longitudinal muscle pioneers are seen per segment. a group of star-shaped mononucleate cells positively These muscle pioneers serve as scaffold for the formation stained by myosin-HC antibodies of the hybridoma line of multicellular muscle precursors by fusion of surrounding 017C5. They are first seen in the periphery of the carapace myoblast cells with the muscle pioneers. The transverse anlage at stage 5 and then spread out over the surface of the pioneers, for example, form the musculature of the ventral yolk mass (Fig. 8a). By stage 7, the cells have formed a diaphragm by fusion with myoblasts. This pattern is also muscular network covering the entire yolk surface observed in crayfish in the segments of maxilla 1 and (Fig. 8b). The density of the network has increased by maxilla 2 during early stage 4, where one pair of stage 9 (Fig. 8c) and areas with a particularly high longitudinal and one pair of transverse muscle pioneers condensation of fibers have formed. Unfortunately, we can be found. Transverse muscle pioneers are also found in cannot say whether this involves myocyte fusion. Increas- the antennal segments. ing fiber density is seen for example, in the neighborhood Our data therefore indicates that a conserved pattern of of the lateral cephalic muscles of the maxilla 1 segment early muscle founders is at least partly shared between (mx1-l1). Furthermore, we found that myosin-HC anti- and crustaceans. The development of a muscle bodies of the 017C5 hybridoma line not only stain early precursor in the early longitudinal muscle strands of the somatic muscles such as the longitudinal fiber strands, but crayfish thorax begins with the elongate mononucleate also show a strong signal in a posterior group of myogenic pioneer cell. This cell shows the specific myosin-HC signal cells associated with the hindgut in the caudal papilla (the likely caused by an increase in monomeric myosin in the 016C6 clone used in all the myosin-HC staining experi- cytoplasm. Next, F-actin fibers become visible. At the same ments described above shows stronger affinity to longitu- time, further myosin-HC positive cells from the surround- dinal, cardial, and cephalic muscles than to gut muscle ing mesoderm become attached to the pioneer. In this way, 100 Dev Genes Evol (2010) 220:89–105 a multicellular muscle precursor is formed in which the Fig. 6 Development of the pleon musculature: pleon anlagen of b embryos stained with phalloidin-Alexa 488 (green) and anti-myosin- grade of differentiation of the individual cells can be seen HC-Cy3 (red). All images (only the left body half is shown) show the from the relationship between F-actin and myosin-HC ventral view with the telson facing to the bottom of the page. Dotted reactivity. Unfortunately, we cannot determine the point at lines indicate segmental furrows of the ectoderm. Muscle precursors which cells fuse to form syncytial myotubes. The emer- of the dorsal strand (arrowheads) and the ventral strand (arrow) are shown. a Fibers of the dorsal and ventral longitudinal muscle strand gence of the striated sarcomere pattern of F-actin and are continuous. b The ventral strands show variations in fiber myosin-HC positive fibers, which is typical of adult orientation. c In both the dorsal strand and an anterior portion of the somatic musculature in most , is the next step ventral strand, single muscle units become distinguishable. d Single observed in muscle precursor differentiation in crayfish. muscle units are visible throughout the pleon. e Further differentiation and increase in size is seen. f–j Virtual transverse sections through the These findings show that the founder cell model for pleon anlage. Yellow lines indicate the positions of the extended muscle patterning described in Drosophila (Baylies et al. section planes shown in k–t. k The cells of the early dorsal strand in 1998; Paululat et al. 1999; Campos-Ortega and Hartenstein pleomere 3 (Pl3) show only myosin signal. The dorsal myocyte 1997) may also apply to crayfish. However, we cannot patches are arranged metamerically along the AP axis. l Cells of the ventral strand in the same pleomere. F-actin signal is seen in the draw conclusions about the mechanisms specifying lateralmost fibers, whereas medial cells are only positive for myosin. muscle pioneers and fusion-competent myoblasts in this m Myoctes of the dorsal strand in pleomere 3 forming metameric fiber animal. bundles with strong F-actin signal (arrowheads). n F-actin positive Unlike in dendrobranchiate crustaceans, the primordia of fibers of the ventral strand display the first lateral endings in the previously continuous strand (asterisks). o F-actin fibers in the dorsal all the major adult muscle groups are laid down during strand show striation. The signal intensifies at the ends of the muscle embryogenesis in the crayfish. However, the temporal gap units (asterisks). p Dorsal section plane within the ventral strand. The between the development of cephalic and postnaupliar central muscles (arrowheads) are distinguishable by their anterolateral intrinsic appendage muscles may well represent a relic of endings (arrow) near the epidermal infold. q Section plane from the middle of the ventral strand. Posteriorly and ventromedially of the developmental features involved in locomotion of nauplius anterior central muscle endings shown in p, the posterior end of this larvae. This staggering of cephalic muscle development muscle is recognizable (arrow). Other fibers in the strand show compared to the remaining trunk, however, seems to be striation but maintain their AP orientation at this stage (asterisk). r absent in direct developing embryos of the amphipods P. Ventral section through the ventral strand. The anterior oblique muscles can be seen. They originate at the epidermal infolds (asterisk) hawaiensis (Price and Patel 2008)andO. cavimana and protrude anteromedially to the middle of the next segment (stars). (Hunnekuhl and Wolff in preparation). We suggest that s The dorsal strand muscle units have broadened. t Ventral muscle these characteristics were lost in the evolutionary lineage strand at same stage. The central muscles (triangle) and transverse leading to the Peracarida. muscles (asterisk) are shown. In u and v, the muscles of pleomere 3 have been three-dimensionally reconstructed in IMARIS from the We still do not have a clear picture of intrinsic phalloidin signal and assigned different colors. The gut and right half appendage development in the marbled crayfish. It has of the pleon muscle complex are masked and hidden. Blue: dorsal been shown for isopod muscles (Kreissl et al. 2008) that pleon muscles (dpm), turquoise: transverse muscles (tm), light green: intrinsic muscle precursors of the appendages are formed central muscles (cm), orange: anterior oblique muscles with external arms (aom), pink: posterior loop of anterior oblique muscles (pla). by single muscle founder cells in the appendages that can Muscles of pleomere 2 and 4 are shown in dark green. Scale bars (a– be detected by staining with the myosin-HC antibody e) 100 μm, (f–u)20μm 016C6. Our data confirm that this is the case for the development of longitudinal musculature of the trunk and in cardiac development. In the lobster Homarus ameri- addition to the temporal continuum, a spatial gradient in canus, intrinsic appendage muscles are laid down as differentiation can also be seen along the AP axis. This is syncytial precursors that stretch into the end of a multi- most evident in stages 5 and 6. However, clearly distin- articulated appendage anlage to subdivide later into the guishable muscle pioneers are not evident at these stages individual muscles (Harzsch in press). In dendrobranchiate posterior to the last thoracic segments. In fact, these embryos, undivided muscle primordia reach from the trunk segments display strands of continuous F-actin fibers that into the tip of the cephalic appendages and remain reach into the caudal papilla. Nuclei are found in some undivided in the free-living larva (Hertzler and Freas cases within the strand, but their positions in relation to the 2009). For the marbled crayfish, however, investigations segment borders, represented by ectodermal stripes of concentrating on later stages are necessary. Engrailed, are variable. A group of muscle cells is found The ventral longitudinal muscle strand of the marbled at the posteriormost end of the strand. The origin of this crayfish is laid down as segmentally repeated patches of myogenic cell group in the caudal papilla appears to be muscle precursors from a row of longitudinal muscle independent of mesoteloblast activity, since it is located pioneers that connect to each other via cellular extensions. posteriorly of undivided mesoteloblast progeny. Evidence The patches originate from single pioneer cells to which of posterior migration from anterior mesodermal regions mesodermal cells are added as development proceeds. In was not found. We assume that the posterior longitudinal Dev Genes Evol (2010) 220:89–105 101 102 Dev Genes Evol (2010) 220:89–105

Fig. 7 Heart development: a–j, n and o dissected embryos, k–m F-actin, which is stronger on the side of the network facing the cardial undissected eggs. a Schematic drawing of an embryo at stage 6 with lumen. k–m Stereo micrographs of embryos stained with myosin-HC the caudal papilla in its natural position. The heart precursors are antibody and subsequently subjected to an alkaline phosphatase indicated by red dotted lines. Orientation of the embryo is the same in reaction. Dorsal views with anterior facing the top of the page. k all the images of this table. b–e CLSM images of the embryonic heart Myocardium and posterior aorta (Pa). The anterior aorta (Aa) shows precursors stained with phalloidin (green). b The first F-actin fibers two parallel rows of cells displaying myosin-HC signal. l More are visible at early stage 5. c Increased number of fibers forming a anterior view of same object. m Myocardium, anterior, and posterior broad band. d The fibers have formed a membrane that is closing aorta. A pair of ostia (arrows) can be seen. n Heart stained with anti- medially (asterisk). The dorsal longitudinal muscle strand (dlm) and myosin-HC (red) and phalloidin (green), dorsal view. Embryo is the muscles of the carapace anlage (cam) are shown. e The membrane oriented as in a. The myocardium is a three-dimensional network of has broadened and formed dorsolateral extensions. f–j Myocardial multiple muscle fibers. Anteriorly, four alary muscle primordia can be anlage (mca) stained for myosin-HC and F-actin, counterstained with seen. o The heart and the adjacent muscle groups of a stage 10 embryo Hoechst (light blue). f The early myocytes of the forming heart have each been reconstructed and assigned different colors: red membrane are arranged in a lateral area of the caudal fold and extend myocardium, yellow alary muscles, green circular gut musculature, dorsomedially with their fiber forming processes (arrowheads). g, blue carapace anlagen musculature, pink longitudinal muscle appara- h The same group of cells increases and expands to the anterior and tus of the trunk). The myocardium at this stage forms a condensed posterior. Additional cellular processes are seen protruding in different network of fibers. Fibers also take up an extensive part of the heart directions. These form the myocardial network. Myosin-HC immuno- lumen. Orientation as in a but in ventral view. Letters in the upper left reactivity is observed closer to the nucleus of the cardial myocytes corners identify the developmental stages. cam carapace anlagen (arrowhead) than the F-actin signal. i Horizontal section of the dorsal muscles, dlm dorsal longitudinal muscles, G gut, mc myocardium, Pa myocardial layer. Striation (arrowhead) of the muscle fibers can be posterior aorta, vlm ventral longitudinal muscles. Scale bars (b–e) seen. j Saggital optical section shows the asymmetrical distribution of 100 μm, (f–j) 20 μm, (k–m) 100 μm, (n–o) 200 μm muscle origin is formed by cells of the telson mesoderm muscle cells in question form cellular processes that (Alwes and Scholtz 2006), which is laid down behind the comprise a considerable part of the longitudinal muscle teloblast cell rows during gastrulation (Weygoldt 1961) and strands in the posterior thoracic and the pleonal segments. later forms musculature in the telson. We propose that the These processes are then elongated as the muscle cells Dev Genes Evol (2010) 220:89–105 103

Fig. 8 Visceral muscle development: a–c dechorionated eggs stained images. d The anti-myosin-HC signal identifies a group of cells with anti-Myosin-HC 017C5 and subjected to secondary staining with surrounding the posterior part of the gut in the caudal papilla. F-actin the alkaline phosphatase system (dark blue). Embryos are shown in is mainly seen in the ventral longitudinal muscle fibers left and right. e their natural position with the caudal papilla flexed anteriorly (Fig. 6a). A horizontal section showing the posteriormost cells of the forming gut a Star-shaped cells showing strong myosin-HC signal are visible on the muscles (double arrows) and partly the left mesoteloblast cell lMT2. f yolk surface on both sides of the carapace anlage. The cells carry More ventral section of the same object. The ventral mesoteloblast pair filamentous projections which extend distally from the nucleus. b Fine lMT1 and rMT1 (arrowheads) and their progeny (asterisks) are shown. myocyte projections form a network that covers the yolk. c The fiber g Forming circular myocytes of the gut. Letters in the upper left corners network diversifies and forms an area of converging fibers near the identify the developmental stages. cam carapace anlagen muscles, cgm lateral cephalic muscle of the second maxilla (mx1-l1), which extends circular gut muscles, dlm dorsal longitudinal muscle, gmp gut muscle dorsally. d–g Dissected embryos stained with anti-myosin-HC-017C5 precursors, mca myocardial anlage, P proctodaeum, vlm ventral (red), phalloidin (green), and Hoechst (light blue,ine and f). The longitudinal muscles, ymc yolk muscle cells. Scale bars (a)100μm, posterior end of the caudal papilla faces the bottom of the page in these (b–c) 200 μm, (d–g) 20 μm

themselves move posteriorly as a result of the addition of ancestor of malacostracans but has not been found in mesodermal segment primordia from the mesoteloblasts. amphipods or isopods so far (Wolff, unpublished data). No data are yet available on early longitudinal muscle We suggest that the mechanism responsible for the formation in the postnaupliar region of dendrobranchiates formation of early longitudinal fibers from posterior (Hertzler, personal communication). Kiernan and Hertzler myocytes is similar to the mode of posterior connective (2006) describe a pair of phalloidin-positive strands that development. The simultaneous formation of nervous or extend anteriorly from the telson anlage which they interpret muscular tissue from the anterior and posterior makes as possible nerves of the furcal spines. In both branchiopods when we consider the necessity of posterior sensory input (Blanchard 1986) and malacostracans (stomatopods: Fischer in a free-swimming nauplius or a muscularized trunk for the and Scholtz 2010; decapods: Biffis, personal communica- tail-flip reflex of a dendrobranchiate protozoea. These tion), posterior pioneer neurons have been described as part features may well also be relics inherited from ancestors of the developing central nervous system, neural cells which with free-swimming larvae and lost within malacostracans lie at the posterior end of the caudal papilla in a pair-wise in the lineage leading to Amphipoda and Mancoida. The arrangement. These pioneer neurons form the posterior development of the dorsal longitudinal muscle strand is connectives of the ventral nerve cord by sending axons entirely gradual. Segmentally repeated cell clusters are seen anteriorly, while the anterior connectives are formed by at dorsal positions within the mesoteloblast progeny. anterior–posterior progression from the developing ganglia. Muscle pioneers then become visible within these clusters This mechanism was most likely present in the last common and the surrounding cells are recruited to the precursor. 104 Dev Genes Evol (2010) 220:89–105

Crayfish pleon muscles at stage 9 reflect the general Our survey also produced data on visceral musculature. pattern first described for larval stages of caridean shrimp Muscle development in higher metazoans is characterized and (Daniel 1930a–c; Young 1959)whichalso by a split into somatic and visceral myogenic cell applies to the adult pleon musculature of the dendrobran- populations (see for example Bodmer 1993). Visceral chiate Penaeus setiferus as well as to dendrobranchiate muscles serve as contractile components for the functioning larvae (Hertzler and Freas 2009). This pattern may well be of inner organs in many animals. They are responsible, for important for the flipping movement of the pleon, which is example, for the peristaltic movement of the intestinal tract. typical for many adult decapods or larvae. In this case, it The formation of a fibrillar network covering the yolk was might have been established in the lineage leading to an unexpected finding at this stage. However, it explains decapods. However, we have no knowledge of muscle the contractile activity of the yolk surface observed in patterning in other malacostracans. living eggs. These contractile cells may well lead to the Cardiac development in crayfish strongly resembles myoepithelial layer of the adult midgut gland and, at this cardiogenesis in P. varians (Weygoldt 1961). The cells of time in development, facilitate the transport of nutrients dorsal mesodermal regions move dorsally with muscular from the yolk to the developing tissues. Mesodermal cells processes and form a membrane below the dorsal epidermis that become associated with the yolk during the develop- which subsequently closes to form a contractile, myocardial ment of P. varians have been reported by Weygoldt (1961). sack. The exact spatial relationship between the myocardial The specificity of the 017C5 antibody to early gut muscle- pioneer cells and the thoracic somites is difficult to discern forming cells may be explained by its relatively strong affinity because they are passively shifted to the posterior as the to characteristic isoforms of the myosin-HC protein that is number of trunk segments anterior to the caudal fold expressed in the gut muscle pioneers and precursors at a increases. The heart-forming cells are positioned laterally certain time in development. As in the case of longitudinal away from the thoracic mesodermal somites when first muscle pioneers, the posteriormost detectable gut muscle detectable by antibody and phalloidin staining. It is reason- founders are located posteriorly of the stereotypically able, however, to assume that these cells represent mesotelo- arranged descendants of the ventralmost mesoteloblast MT1. blast derivatives, though this could not be shown in this study. It therefore seems unlikely that early visceral myocytes of the In all arthropods studied so far, the heart tissue is formed from gut are formed by this cell. Possibly, gut muscle precursors are a dorsal subset of the mesoderm (Hartenstein and Mandal derived from median cells of the telson mesoderm. 2006, Janssen and Damen 2008). In vivo-labeling experi- The development of muscular tissue in non-model ments on mesodermal cells of the amphipod O. cavimana arthropods is a very young field of study. Modern provide clues that the dorsal mesoteloblast pair MT4 gives approaches to developmental processes in new organism rise to the heart (Hunnekuhl and Wolff, in preparation). The systems normally rely on the analysis and comparison of early development of the heart is still of particular gene expression. However, morphological resolution is an interest and deserves to be dealt with in further important prerequisite for the interpretation of these data. investigations. Crayfish and other malacostracans includ- We believe that our investigations provide an informative ing all the remaining decapods and euphausiaceans share picture of myogenesis in the crayfish which will form a the feature of a bulbous heart in the adult, in contrast to useful basis for molecular developmental studies. the tubular heart of stomatopods, leptostracans, or peracarids (Wirkner and Richter 2009a, b). The mecha- Acknowledgements The 4D9 anti-EN/INVECTED monoclonal an- nisms involved in the formation of the myocardium in tibody developed by Corey Goodman (University of , Berkeley) was obtained from the Developmental Studies Hybridoma these groups remain to be investigated. Interestingly, Bank, which was developed under the auspices of the NICHD and is prospective cells of the anterior aorta of the crayfish heart maintained by The University of Iowa, Iowa City, IA 52242. We also show a myosin-HC signal at stage 8 that is lost as express our gratitude to Frederike Alwes for instructions on crayfish development proceeds. Similar findings have been care, egg handling, dissection, and staining. Thanks also to Lucy Cathrow for improving the English of the manuscript. This study was reported for lobster arteries (Wilkens et al. 1997; Wilkens supported by the DFG grant Ri837/8-1 to SR, Wo1461/1-1 to CW. 1999). These cells may be part of a general mechanism of cell recruitment for the formation of a tubular heart but may later be lost when pumping is taken over by the References bulbous myocardium. In other regions of the crayfish dorsal vessel, such as the posterior aorta, a temporary Abzhanov A, Kaufman TC (2000) Evolution of distinct expression increase in myosin-HC protein as seen here was not patterns for engrailed paralogues in higher crustaceans (Mala- observed. costraca). Dev Genes Evol 210:493–506 Dev Genes Evol (2010) 220:89–105 105

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1 Jirikowski GJ, Richter S, Wolff C. 2013. Myogenesis of Malacostraca – the “egg- nauplius” concept revisited. Frontiers in Zoology. 10:76-103.

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Jirikowski et al. Frontiers in Zoology 2013, 10:76 http://www.frontiersinzoology.com/content/10/1/76

RESEARCH Open Access Myogenesis of Malacostraca – the “egg-nauplius” concept revisited Günther Joseph Jirikowski1*, Stefan Richter1 and Carsten Wolff2

Abstract Background: Malacostracan evolutionary history has seen multiple transformations of ontogenetic mode. For example direct development in connection with extensive brood care and development involving planktotrophic nauplius larvae, as well as intermediate forms are found throughout this taxon. This makes the Malacostraca a promising group for study of evolutionary morphological diversification and the role of heterochrony therein. One candidate heterochronic phenomenon is represented by the concept of the ‘egg-nauplius’, in which the nauplius larva, considered plesiomorphic to all Crustacea, is recapitulated as an embryonic stage. Results: Here we present a comparative investigation of embryonic muscle differentiation in four representatives of Malacostraca: Gonodactylaceus falcatus (Stomatopoda), Neocaridina heteropoda (Decapoda), Neomysis integer (Mysida) and Parhyale hawaiensis (Amphipoda). We describe the patterns of muscle precursors in different embryonic stages to reconstruct the sequence of muscle development, until hatching of the larva or juvenile. Comparison of the developmental sequences between species reveals extensive heterochronic and heteromorphic variation. Clear anticipation of muscle differentiation in the nauplius segments, but also early formation of longitudinal trunk musculature independently of the teloblastic proliferation zone, are found to be characteristic to stomatopods and decapods, all of which share an egg-nauplius stage. Conclusions: Our study provides a strong indication that the concept of nauplius recapitulation in Malacostraca is incomplete, because sequences of muscle tissue differentiation deviate from the chronological patterns observed in the ectoderm, on which the egg-nauplius is based. However, comparison of myogenic sequences between taxa supports the hypothesis of a zoea-like larva that was present in the last common ancestor of Eumalacostraca (Malacostraca without Leptostraca). We argue that much of the developmental sequences of larva muscle patterning were retained in the eumalacostracan lineage despite the reduction of free swimming nauplius larvae, but was severely reduced in the peracaridean clade.

Introduction in morphology and life style from the adult and juvenile. Malacostraca comprises approximately 30.000 species Throughout this paper we will address individuals which with a broad range of morphological and ecological di- have hatched from the egg shell but do not show the versity. Throughout the malacostracan clade an enor- complete number of segments or differ strongly from mous variety of reproductive strategies can be found. juveniles in appendage morphology, as larvae [1]. The Malacostracan ontogeny encompasses an embryonic larval period in these species is followed by a juvenile phase, which is restricted to the egg. In many taxa it is (or ‘postlarva’) phase in which the adult morphology in followed by a postembryonic phase, in which a larva respect of body segments, appendage number and hatches and passes through a series of larval phases, sep- morphology, is apparent but may differ from the sexually arated by molts. This developmental mode is referred to mature adult in size and proportion. The remaining as ‘indirect development’. The larva differs significantly malacostracan taxa possess an ontogenetic mode in which all pre-juvenile development takes place within the egg shell and an individual with adult-like morph- * Correspondence: [email protected] 1Universität Rostock, Allgemeine & Spezielle Zoologie, Institut für ology hatches from the egg. This is commonly referred Biowissenschaften, Universitaetsplatz 2, Rostock 18055, Germany to as ‘direct development’. Full list of author information is available at the end of the article

© 2013 Jirikowski et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 2 of 27 http://www.frontiersinzoology.com/content/10/1/76

The nauplius is a planktonic larva with three pairs of the so called ‘egg-nauplius stage’ is traversed. It shows functional appendages (mandibles, first and second prominent appendage anlagen in the naupliar segments antennae) that are together used for feeding and (first antennal-, second antennal and mandible buds). locomotion and is often considered being part of the This is the result of a (more or less) synchronous forma- ground pattern for Crustacea [2-5] or Tetraconata, tion of the naupliar appendage buds with a significant assuming paraphyletic crustaceans [6]. Within malacos- temporal advance compared to appendage anlagen of tracans, however, only dendrobranchiate decapods and the following posterior segments (Figure 1a and -b). Euphausiacea possess a pelagic nauplius larva [7]. In Whether the Mysidacea show temporal advance in these groups several nauplius stages are passed through, development of the nauplius appendages is a matter of sequentially adding segments to the trunk. They are definition, since here the advance is seen only in the first followed by stages with thoracic exopods functioning in and second antenna. We will consider it as advance in propulsion, but without functional pleopods (dendro- development of nauplius segments here. The so-called branchiate zoea, euphausiid calyptopis) and stages with ‘nauplioid-larva’ of Mysidacea is sketched in Figure 1e natatory pleopods (dendrobranchiate postlarva, euphaui- and -f. The remaining Peracarida lack the clear temporal siid cyrtopia)[1,8-14].Themajorityofdecapodspossess advance in nauplius appendage development. Instead an intermediate form of ontogeny in which more ad- they show only a slight difference in size between nau- vanced pelagic, mostly planktotrophic, larval forms pliar and postnaupliar appendage anlagen. Apart from hatch and show correspondence to the dendrobranchiate that, segment differentiation along the anterior-posterior zoea [15,16]. These larvae, which we will refer to as axis is continuous and follows a comparatively weak ‘zoea-like’ larvae, generally carry the complete- or nearly gradient (Figure 1g and -h) as demonstrated for amphi- complete set of body segments, though the number and pods and isopods [20,30,31]. In all representatives post- morphology of appendages differs strongly from the naupliar segment anlagen are formed subsequently as adult situation, as well as a paddle-shaped telsonic plate. cells are proliferated in the teloblastic growth zone and There is considerable variation in number and morphology can be distinguished by intersegmental furrows or ap- of larval stages in the Decapoda (see [4] for review). pendage buds in the followingembryonicstages.This Stomatopods (with the exception of Lysiosquillidae) have growth zone is located in the preanal region of the so called Pseudozoea-larvae. In these pelagic larvae the head germ band in all malacostracan representatives. In as well as the first and second thoracic segments bear Leptostraca, Stomatopoda, Anaspidacea, Thermosbae- appendages, while the pleomeres carry one pair of natatory nacea and Pleocyemata the preanal region is repre- appendages each [17,18]. sented by a yolk-free posterior structure called the Direct development is found in a large number of caudal papilla (Figure 1c and -d), which is flexed ante- malacostracan lineages such as Leptostraca, Astacidea roventrally [32-41]. Here mesodermal units are prolif- (Decapoda), Anaspidacea, Thermosbaenacea [19]. In erated from the mesoteloblast cells, and ectodermal these cases all of development takes place inside the units from the ectoteloblast cells, respectively. egg shell (i.e. is embryonic) and ends with the hatching Fritz Müller [42] was probably the first one who em- of the juvenile. Peracarida are generally regarded as phasized the evolutionary importance of the nauplius direct developers [20-24]. This group has evolved a larva. Based on the discovery of the dendrobranchiate specialized mode of brood care, where eggs and larvae nauplius larva he suggested that all crustaceans evolved are carried in a ventral broodpouch(marsupium)until from a nauplius larva like ancestor, which implies that they are released. We classify the Mysidacea as larval- the nauplius as a larval stage represents a recapitulation developers because hatching occurs early in development, of an adult stage. Jägersten [43] also stressed the import- and the inert larva, called ‘nauplioid’ with an incomplete ance of the nauplius larva. He suggested the nauplius set of segments but prominent first and second antennae larvae as the ‘primary larvae’ of all arthropods directly [25,26], remains in the marsupium. This particular form derived from a trochophora and that characters of the of larval development is herein referred to as ‘pseudodir- adults were transferred to the larva by a process he ect development’, a term which has been recently called “adultation” (which is quite the contrary from introduced for development of Cladocera [27]. In certain Müller’sideas).Anderson[44]formulatedamodelofan- other Peracarida, ie. in certain amphipods and isopods cestral crustacean development. According to him devel- the free hatchlings also show aberration from adult opment in the crustacean ground pattern comprised the morphology, e.g. the pantochelis and protopleon –stages formation of a nauplius larva bearing three functional of Hyperiida, and the manca of isopod [28,29]. pairs of appendages and four undifferentiated postnau- In a large number of malacostracan representatives pliar segment anlagen. Advanced larval forms or direct with either direct development or an indirect develop- development are interpreted as derived in his view. The mental mode where the hatchlings are zoea-like larvae, potentially primitive nature of a nauplius larva was Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 3 of 27 http://www.frontiersinzoology.com/content/10/1/76

Ol Ol Ol Ol A1 A1 A1 A2 A1 A2 Md Md A2 A2 Mx1 Md Mx2 Md Cp Cp Mx1

a c e g

Ol Ol Ol Ol A1 A1 A1 A2 A2 A1 Md A2 Cp Mx1 Md A2 Mx1 Md Mx2 Cp

b d f h Figure 1 Schematic overview of malacostracan germ band morphology in embryonic- and pseudodirect development. a, c, e, and g represent ventral views of the germ band, b, d, f and h represent lateral views. a-d Simplified drawings of crayfish embryo modified from [68]. The general pattern applies to all Malacostraca with a yolk-free caudal papilla. a Ventral view of germ band at the egg-nauplius stage. Anlagen of the optic lobes, nauplius appendages and the caudal papilla are present. b Lateral view of same embryo. c Embryo at advanced, but still incom- plete germ band stage. Optic lobe- and nauplius appendage rudiments are larger than posterior appendage anlagen. The caudal papilla is flexed anteriorly. d Lateral view of same embryo. e Early mysidacean nauplioid larva. An egg-membrane is missing. First and second antennae show advanced external morphology. Posterior appendage anlagen follow a gradual decrease in differentiation. f Lateral view of nauplioid larva. g Embryo representative of Peracarida except Mysidacea. The gap between naupliar- and postnaupliar appendage development is less distinct. The postnaupliar germ band displays gradual a/p -differentiation. The gradient is exaggerated in this drawing. h Lateral view of same embryo. Areas containing yolk are shaded grey in all drawings. Abbreviations: Ol optic lobes, Cp caudal papilla, A1, A2, Md, Mx1, Mx2, appendage anlagen. Yolk-rich areas are shown in light grey. further suggested by interpreting the situation in em- necessarily one of an adult stage) that has been con- bryogenesis with advanced and synchronous develop- served and which is expressed during development” [7], ment of the naupliar appendages, as described above, as 182p. In this view the developmental program respon- a transient nauplius-phase in embryonic development – sible for constructing a nauplius larva is still active in the so-called egg nauplius (Figure 1a and -b). The nau- embryogenesis of species which have lost the larval plius either as free-swimming larva or as egg-nauplius stage. Furthermore he argues that the egg-nauplius stage has been interpreted as representing a crustacean phylo- is plesiomorphic for Malacostraca and that the free liv- typic stage [45-47], meaning that development is con- ing nauplius larvae of dendrobranchiates and euphausia- strained to form a nauplius morphology in the early ceans have evolved independently to non-malacostracan germ band as a prerequisite to subsequent morpho- nauplius larvae, as phylogenetic hypotheses of Malacostraca genetic processes in all members of the clade. The always place these groups in a nested position within validity of such a concept must be questioned [48-50], if the tree [5,19,51]. Scholtz [7] also suggests that the only because a nauplius/egg-nauplius stage is missing in egg-nauplius facilitated secondary evolution of the free some Malacostraca. Scholtz [7] argued that, based on swimming nauplius larva of dendrobranchiates and correspondences between the nauplius larva and the euphausiaceans. He argues that from the starting point egg-nauplius, the latter can be viewed as a developmen- of an egg-nauplius stage only few evolutionary changes tal stage which is recapitulated: “The egg-nauplius are necessary to generate a free swimming nauplius represents clearly a Müllerian (Haeckelian) recapitula- larva, compared to a starting point in which no egg- tion in the modern sense of an ancestral information (not nauplius is present. The caseoftheegg-naupliusraises Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 4 of 27 http://www.frontiersinzoology.com/content/10/1/76

the question to what extent development must be Development of muscle patterns using genetic tools has modified during evolution to transform larval ontogeny only been studied on the amphipod Parhyale hawaiensis into embryogenesis and vice versa. If the malacostracan [53] and using histochemistry- and immunohistochemistry- egg-nauplius truly represents a transient larval stage in techniques on dendrobranchiates [54,55], the American arecapitulatorysense(i.e.alarvalstagewhichisnow lobster Homarus americanus [56], the marbled crayfish embryonized), then the developmental advances should Procambarus fallax f. virginalis [52], the amphipod comprise more than just external morphology. Hereafter crustacean Orchestia cavimana [57] and two isopod species we will use egg-nauplius as a descriptive term for embry- Porcellio scaber and Idotea baltica [58]. The species investi- onic semaphoronts externally characterized by the ad- gated here comprise the stomatopod Gonodactylaceus vanced three first appendage buds in the epidermis, but falcatus (FORSKÅL, 1775) (Figure 2a) the decapod without implying recapitulation of a nauplius larva. Neocaridina heteropoda (KEMP, 1918) (Figure 3a) and two The problem inherent to the recapitulatory egg- peracarids: the mysidacean Neomysis integer (LEACH, nauplius concept is the lack of detailed morphological 1814) (Figure 4a) and the amphipod Parhyale hawaiensis data that can be drawn from the observation of such (DANA, 1853) (Figure 5a). Gross development of sto- early embryonic stages, as knowledge of tissue differenti- matopods is known from few studies using histology ation at a cellular level, especially within the mesoderm, and external morphology [17,18,59,60]. N. heteropoda is still scarce. Also, if heterochrony is a possible evolu- is a Southeast-Asian freshwater shrimp and a popular tionary mechanism involved in transformation of a nau- pet for aquarists. Postembryonic ontogeny has been plius larva or egg-nauplius, the complete developmental described for related species [61,62]. Neomysis integer trajectories in naupliar- and postnaupliar tissues must has been subject to previous studies of germ band be taken into account when comparison between species development [26] and other aspects of ontogeny is performed. We have chosen to put muscle tissue to [25,63]. Parhyale hawaiensis has recently become a the focus of our ontogenetic study. Muscles form func- popular model organism for developmental biology tional units together with the more readily observable and is cultured in several laboratories around the epidermis/cuticle. Muscle patterns can therefore be as- world [64]. sumed to evolve together with the structural capacity to perform certain body movements and behavioral fea- tures. That is why muscle development is particularly Methods well suited to address questions of larval recapitulation Egg material of Gonodactylaceus falcatus was collected and the applicability of the egg-nauplius concept. Here from females caught in the wild on Coconut Island we present the first attempt to study evolution of mala- (Hawai‘i Institute of Marine Biology) in September 2008. costracan larval developmental features using muscle Animals were kept in a large tank under a continuous morphology. flow of fresh sea water. Eggs were collected from gravid Our previous work on muscle development of the females in periods of approximately 12 h and preserved crayfish Procambarus fallax f. virginalis [52] has re- for further investigation. Egg material of Neocaridina vealed abundant data that can now be utilized to com- heteropoda was collected from the pleopods of gravid fe- pare muscle development between taxa and reconstruct males of an established lab culture in the facilities of the the evolutionary history of myogenesis. Here we apply a zoology department at Rostock University. Collection comparative ontogenetic approach to five malacostracan was performed at time periods of approximately 24 h. species, extending the methodology used on the crayfish, Embryos were freshly dissected from the egg shells in to obtain the temporal sequence of myogenic events. PBS (1.86 mM NaH2PO4, 8.41 mM Na2 HPO4, 175 mM Our aim is (i) to describe developmental patterns of NaCl, pH 7.4). Gravid females of Neomysis integer were myogenesis for all five taxa, (ii) to test the consistency of caught from the south pier of the Rostock harbor be- muscle developmental patterns across taxa and (iii) by tween April and August of 2011. Eggs or nauplioid comparison draw first conclusions on the evolutionary larvae were removed from the marsupium and kept in history of myogenic sequences and their relation to the artificial sea water (15PSU) for further processing. concept of a cryptic nauplius stage (a recapitulatory Animals could not be reared successfully but material egg-nauplius). We believe that a detailed spatiotempo- covering all developmental stages could be collected ral study of muscle tissue patterning and differenti- from a larger batch of females. Egg material of Parhyale ation during embryogenesis can reveal the extent to hawaiensis was collected from the marsupium of female which the egg-nauplius is anchored to the develop- animals kept in a permanent lab culture at Rostock mental system and possibly uncover further hetero- University (original stock from Anastasios Pavlopoulos, chronic developmental traits that may be related to MPI CBG Dresden). Embryos were freshly dissected as larval development. described above for Neocaridina heteropoda. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 5 of 27 http://www.frontiersinzoology.com/content/10/1/76

Figure 2 Muscle ontogeny of Gonodactylaceus falcatus. a Macroscopic image of adult female. b-g Maximum intensity projections of confocal image stacks taken from whole mount fluorescent staining. Nuclear stain (SYTOX) is shown in blue; muscle stain (Myo16-C6) is shown in red (c-g). b blend projection of reconstructed nuclei with overlaying transparent image of ectodermal nuclei (grey). Dorsal view of Gf I caudal papilla. Mesoteloblasts (labeled for the right hemisegment: rMT1-rMT4) and two rows of mesodermal cells are reconstructed and highlighted in artificial colors alternating yellow and red. Mesoderm anlagen of more anterior segments showadvancedproliferationwhichmakesdelineation of segments difficult. They are not highlighted. Mesoteloblasts form a ring in the caudal papilla.Thedottedlinemarkstheborderbetweenmesoteloblastsandmesodermalcells for the right hemisegment. c-g Ventral views of semaphoronts Gf I - Gf V.Ine-g only the anterior left hemisegments are shown. cGfIthe external egg-nauplius morphology is apparent but the first maxilla bud is visible and the caudal papilla is elongate and flexed anteriorly. Intrinsic muscle precursor in antenna 1 is marked with an asterisk. dGfII. st, st-2, a2-l1, a2-l2, md-l2, md-l3 and lmp are visible (a clear distinction between lmp-t1 and lmp-post cannot be made). Intrinsic muscle precursors of the first antenna appear (asterisk). eGfIII. a1-m1, mx2-l1, lmp-md and lmp-mx2 appear. Intrinsic muscle precursors of the second antenna appear (asterisk). fGfIV. st-1, lmp-mx1, a1-m2, a2-m1, a2-m2 and mx2-l2 are shown. lmp-md is no longer visible. gGfV.Muscle precursors are slightly enlarged and md-l1 has appeared. Abbreviations: Ol Optic lobes, Cp Caudal papilla, A1, A2, Md, Mx1, Mx2, T1 appendage anlagen. Scalebars are 1 cm in a,50μminb,100μminc-g.

Staining and microscopy needles to acquire full exposure of tissues to the fixative Fixation of dissected embryonic material was carried out and staining solutions. Immunohistochemical staining was by incubation in 4% paraformaldehyde (Electron Micros- applied following standard protocols as described previ- copy Sciences)/PBT (PBS with 0.3% triton X-100 as ously [52]. Monoclonal antibody 16C6 which is reactive to detergent). Incubation times varied between 30′ and 60′ myosin-heavy chain [56,58] was used to label early muscle and were adapted for the individual developmental stage. progenitors at 10× dilution. 10% ROTI-Block (Carl Roth Larvae of N. integer were opened dorsally with tungsten GmbH, Karlsruhe) was used as blocking agent in the Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 6 of 27 http://www.frontiersinzoology.com/content/10/1/76

Figure 3 (See legend on next page.) Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 7 of 27 http://www.frontiersinzoology.com/content/10/1/76

(See figure on previous page.) Figure 3 Muscle ontogeny of Neocaridina heteropoda.aMacroscopic image of adult. b and d blend-projection-, c-g maximum intensity projections of confocal image stacks taken from whole mount fluorescent staining. Nuclear stain (TOPRO-3): cyan (b), grey (d), blue (e-f); muscle stain (Myo16C6-Cy3): red (c, e and f); muscle stain (phalloidin-ALEXA564 ): multiple artificial colors (g). b Egg-nauplius stage. Arrowheads mark the ectoteloblast row in the caudal papilla. c-g Ventral views of semaphoronts Nh I - Nh IV.Inc and e-g only left body half is shown. cNhI.Postnaupliar appendage anlagen are not seen but the caudal papilla is elongated and flexed anteriorly. Muscle precursors st, st-2 and a2-m1, md-l1 and md-m are present. d Dorsal view of Nh I caudal papilla (as in Figure 2b). e Nh II. st-1. a1-m, a2-l1, a2-l2, mdl2 are present, as well as anterior longitudinal muscle precursors lmp-mx2 and lmp-d . Intrinsic muscles appear (asterisks). f Nh III. Novel lateral extrinsic precursors: a2-l3, mx1-l1, mx1-l2, mx1-l3, mx2-l1, mx2-l2, t1-l1 and t1-l2.Novelmedialextrinsicprecursors:mx1-m, mx2-m and t1-m.Novellongitudinalmuscleprecursor:lmp-mx1. gNhIV.Individual precursors or groups of precursors arereconstructedandassignedartificialcolorsforbetter orientation. The color code is also applied to the labels. a1-m is no longer seen.lmp-t1is not shown. Medial precursors: a2-m2, mx1-m1, mx1-m2, mx2-m1, mx2-m2, t1-m1 and t1-m2.Abbreviations:Olopticlobes, Cp caudal papilla, A1, A2, Md, Mx1, Mx2, T1 appendage anlagen. Scalebars are 5 mm in a,50μminb–e, 100 μminf and g.

rinsing- and staining solutions. For late developmental According to the model these cells serve as scaffold for stages 1.5% DMSO (AppliChem, Darmstadt) was added to muscle formation. Fusion of surrounding mesodermal all solutions to increase tissue permeability, along with 0.3% cells (myoblasts) leads to multinucleate units, also de- BSA (Merck, Darmstadt). Incubation times were adapted tectable with myosin and phalloidin. These units are individually for large specimens. Goat-AffiniPure anti- called muscle primordia.Thetermmuscle precursor mouse IgG H + L labeled with Cy3 (Jackson Immunore- refers to muscle pioneer cells and muscle primordia search) was applied for antibody detection. likewise. The scaffolding role of the muscle precursor F-Actin was labeled histochemically with ALEXA-488- or pattern implies that parts of the initial pattern can be ALEXA-561 conjugated phalloidin (Invitrogen Molecular lost during development as observed in grasshoppers Probes) following the manufacturers protocols. Nuclear [65]. Therefore it is not possible to assign a certain muscle staining was performed using TOPRO-3 (Invitrogen pioneer or precursor to a specific adult muscle or function Molecular Probes) or SYTOX (Invitrogen Molecular in every case. We use a modified version of the termin- Probes). CELL MASK (Invitrogen Molecular Probes) ology presented in [56] to specify individual precursors. was used as unspecific tissue stain on Gonodactylaceus The terms relate only to the position and orientation of falcatus. All samples were mounted in Vectashield muscle precursors within the embryo and do not immedi- (Vector Laboratories, Burlingame CA) for microscopy. ately imply adult function or even homology between spe- Confocal image stacks were recorded with a Leica DMI cies. A list of all precursors is given in Table 1. 6000 CFS confocal laser scanning microscope, equipped We define the following muscle precursor groups with a conventional scanning system Leica TCS SP5 II. Step following [67]: sizes between successive scanning planes ranged from 0.4 to 1.0 μm. Volume Data were calculated from confocal ‘stomodeal muscle precursors’ stacks and edited using IMARIS 7.0 (Bitplane, Switzerland). Editing included manual reconstruction of volume parti- Precursors of ring- and dilatator muscles associated tions (e.g. mesoteloblast cells or muscles at advanced devel- with the stomodeum. opmental stages) and assignment of individual colors to improve clarity for complex structures. Image tables were ‘intrinsic appendage muscle precursors’ created in COREL DRAW X3. Micrographs of adult speci- mens of N. integer and P. hawaiensis were recorded using Muscle precursors located within the appendage aDISCOVERYV12stereomicroscopeequippedwithan anlage and extending distally from the coxa. They AxioCam ICc 3 – Camera (Carl Zeiss, Jena). Images were function in moving podomeres of the appendage in edited in COREL PHOTOPAINT X3. respect to each other. Laboratory work on any of the crustacean species which are part of our study does not raise ethical issues. Therefore ‘extrinsic appendage muscle precursors’ approval from a research ethics committee is not required. Extrinsic muscles serve to move the coxa of the Myogenesis terminology appendage in respect to the trunk. We apply a terminology adapted from the founder cell model of myogenesis proposed for insect development ‘medial extrinsic appendage muscle precursors’ [65,66]. Spindle shaped mononucleate cells which stain positive for muscle specific proteins (in our case Muscle precursors extending medially into the trunk myosin-heavy chain) are termed muscle pioneer cells. from the proximal region of the appendage anlage. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 8 of 27 http://www.frontiersinzoology.com/content/10/1/76

Figure 4 Muscle ontogeny of Neomysis integer.aMacroscopic image of adult. b Blend-projection, c-g maximum intensity projections of confocal image stacks taken from whole mount fluorescent staining. Nuclear stain (TOPRO-3): cyan (b), blue (d-h); muscle stain (anti- Myo16C6-Cy3): red (d-h); Muscle stain (phalloidin-ALEXA488): green (f, g), multiple artificial colors (h, i). b Early nauplioid larva. The telson anlage is marked with brackets. c Ventral view of the growth zone (as in Figure 2b and 3d). Mesoteloblasts and mesodermal cells form transverse rows instead of rings. dNiI. Anterior portion of lmp (lmp-mx2) and lmp-d is visible. e Overview of same specimen. White rectangle marks the field shown in d. f and g show anterior left half of the larva. f Ni II. st, md-m and intrinsic musculature (asterisk) have appeared. g Ni III. st-1, st-2, md-l2, lmp-mx1, mx1-m1, mx2-m1 and mx2-m2 are seen. h Ni IV. Lateral view; lateral and medial precursors are reconstructed and assigned artificial colors white and yellow respectively, the remaining muscle signal is shown in red. Novel lateral precursors: a2-l1/2, a2-l3, md-l1, mx1-l1, mx1-l2, mx2-l and t1-l. Novel medial precursors: a2-m1, a2-m2, mx1-m2 and t1-m. iNiV. Reconstructed precursors and precursor groups, as in Figure 3g. Novel lateral precursors: a2-l1, a2-l2, a2-l3 (derived from a2-l1/2). md-l2 is lost. Abbreviations: Ol optic lobes, A1, A2, Md, Mx1, Mx2, T1, appendage anlagen, T telson anlage. Scalebars are 2 mm in a, 100 μm in b, e, h and I, 50 μm in c, f and g, 25 μmind.

‘lateral extrinsic appendage muscle precursors’ Muscle precursors are given in italics throughout this paper. Muscle precursors extending laterally into the trunk from the proximal region of the appendage anlage. Semaphoront specification and ontogenetic sequences ‘longitudinal muscle precursors’ We avoid established staging nomenclature (as given for example for P. fallax f. virginalis in [68]). Instead Muscle precursors which extend in anterior-posterior we will apply a numerical code using roman num- direction within the trunk. bers to specify an individual at the respective time Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 9 of 27 http://www.frontiersinzoology.com/content/10/1/76

Figure 5 (See legend on next page.) Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 10 of 27 http://www.frontiersinzoology.com/content/10/1/76

(See figure on previous page.) Figure 5 Muscle ontogeny of Parhyale hawaiensis.aMacroscopic image of adult. c-g Maximum intensity projections of confocal image stacks taken from whole mount specimens. h and i blend projections. Nuclear stain (TOPRO-3): blue in b-g. Muscle staining (anti- Myo16C6-Cy3): red in b-g, multiple artificial colors in h; muscle stain (phalloidin-ALEXA488): green in b-f, multiple artificial colors in i. b Extended optical section showing anterior portion of lmp (Overview shown in c). c Ph I. Dorsal view of embryo expressing phalloidin-signal in the CNS and first myogenic signals. Ventral portions of CNS are partly blocked by yolk. d Magnification of cephalic region in Ph I, ventral view. The CNS is shown as well as muscle pioneers of st. e Magnification of c, only left body half, dorsal view, showing lmp and lmp-d. Thoracic segments are marked with dotted lines. f Ph II. st2, ppg, lmp-t1 and lmp-d are present. Midgut caeca muscle anlagen (mgc) are shown. g-i: ventral views of the anterior left embryonic region. g Ph III. Novel precursors: lb (labral muscle precursors), st-1, st-3, a2-l1, mx2/t1-l, a2-m1, md-m, mx1-m1, mx1-m2, mx2-m3 and t1-m. h and i Individual precursors or groups of precursors are reconstructed and assigned artificial colors as in Figure 4i. hPhIV.Novelprecursors:a2-l2, md-l1, md-l3/mx1-l, mx2-l, t1-l1 and t1-l2, and a2-m2. iPhV.Additionalprecursors:md-l2, mx2-l1, mx2-l2, mx2-m1, mx2-m2 and mx2-m3. Abbreviations: A1, A2, Md, Mx1, Mx2, T1 appendage anlagen, T2, T3, T4, T5, T6, T8 thoracic tergite anlagen. Scalebars are 2 mm in a,25μmin b,100μminc-i.

of its life, hereafter referred to as ‘semaphoront’ [69]. progeny. (Gf II) The muscle progenitor complex of The code does not imply any correspondence be- semaphoront Gf II is characterized by two lateral extrin- tween taxa and only refers to the semaphoronts in sic muscle primordia (a2-l1, a2-l2) associated with the relation to each other. Emergence of novel muscle second antenna and two lateral extrinsic precursors pioneer cells or muscle precursors represent the cri- (md-l2, md-l3) associated with the mandible anlage teria by which these semaphoronts are specified. The (Figure 2d). Around the developing stomodeum a ring- development of embryonic musculature in the cray- like assembly of muscle forming cells (st) has formed. From fish Procambarus fallax f. virginalis was described the posterolateral margin of st another precursor (st-2) earlier [56]. Developmental stages St3, St5, St7 and extends anterolaterally and dorsally. Longitudinal muscle St9 described therein are utilized for comparison primordia (lmp)formaventralstrandwhichextends here and specified as semaphoronts Pf I, Pf II, Pf from the first thoracic segment into the caudal papilla. III and Pf IV respectively. The posterior end of the longitudinal precursor strand (lmp-post) is located just medially of the lateral-most Results mesoteloblast MT2 (Figure 6a), which can be specified In the following section semaphoronts are listed and de- by the characteristic cell shapes and -arrangements in scribed (roman numbers). All description refers to the the growth zone mesoderm. Slightly anterior to the hemisegments of the left body half in ventral view. Our mesoteloblast the longitudinal strand contains a cluster investigation focuses on two body regions which we find of nuclei which show no trace of segmental order. (Gf III) to be most enlightening in respect of myogenic evolu- As development proceeds, an extrinsic muscle prim- tion: the anterior six segments of the trunk and the ordium of the first antenna (a1-m1) arises and extends telson anlage. Therein we concentrate on the muscle medially from the base of the appendage rudiment and precursor groups listed above. This excludes visceral- posteroventrally towards the stomodeal muscle ring (st) and heart-musculature. Intrinsic muscles of the append- (Figure 2e). A muscle primordium (md-m) has arisen in ages are also largely excluded from our discussion for the mandible segment and a longitudinal muscle primor- reasons of clarity, but are considered in early develop- dium (lmp-md) is attached to the posterolateral margin mental stages. In the caudal papilla (if present), the of st. The anterior end of the ventral longitudinal muscle posterior pleon segments and the telson anlage we strand can now be found in the second maxilla segment focus on longitudinal muscle precursors (Table 1). The where it touches a novel lateral muscle primordium position of mesoteloblast cells and early mesodermal (mx2-l1). The posterior end of the longitudinal muscle segmental units are recorded as well. strand has enlarged slightly and shows an increased Gonodactylaceus falcatus (Figure 2a): number of nuclei. The cluster of nuclei within the strand (Gf I) The first muscle signals could be detected in in- appears more condensed anterior to the teloblasts trinsic muscle precursors of the second antenna anlage (Figure 6b). (Gf IV) Yet later in development a novel after the egg-nauplius stage. At this developmental stage muscle primordium (st-1) extends from the anterior mar- unsegmented rudiments of the first and second antenna, gin of st anterodorsally (Figure 2f). An additional medial the mandible and the first maxilla are visible while the extrinsic muscle primordium (a1-m2) of the first antenna second maxilla bud at this stage is not yet visible is now present and additional medial extrinsic primordia (Figure 2c). In the caudal papilla the undifferentiated an- (a2-m1, a2-m2) have appeared in the second antenna lagen of the thoracic segments are present. Figure 2b segment. In the mandible segment the medial muscle shows the mesoteloblasts and two rows of mesoteloblast primordium (md-m) is now composed of several units Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 11 of 27 http://www.frontiersinzoology.com/content/10/1/76

Table 1 The table gives a list of all muscle precursors Table 1 The table gives a list of all muscle precursors described and discussed throughout the paper described and discussed throughout the paper Body region Muscle Appendage Muscle (Continued) precursor anlage/segment precursor Mx2 lmp-mx2 group Stomodeal region Stomodeal - st T1 lmp-t1 precursor group - st-1 All segments lmp-d (dorsal region) - st-2 Growth zone/ lmp-post - st-3 telson - ppg Muscle precursor terms represent a code of letters and numbers, given in italics, which relate to the body region (e.g. stomodeum st) appendage anlage Appendage anlagen Intrinsic - Intr or body segment (e.g. first antenna a1). It also refers to the anteroposterior (distal region) precursors arrangement of precursors from anterior to posterior (numbers). Muscle Appendage anlagen Medial extrinsic A1 a1-m precursors are sorted into groups by appendage/segment affiliation (A1, A2, (proximal region) precursors etc.), muscle precursor group (stomodeal precursor group, intrinsic appendage a1-m1 muscle precursors, medial extrinsic appendage muscle precursors, lateral extrinsic appendage muscle precursors, longitudinal trunk muscle precursors), a1-m2 and body region (stomodeal region, distal region of appendage anlage, A2 a2-m proximal region of appendage anlage, trunk region). Certain precursors (a1m, mx1-m, mx2-m and t1-m) give rise to multiple precursors found at later stages a2-m1 (e.g. a1-m gives rise to a1-m1 and a1-m2) but the way this is achieved is uncertain (there are three possibilities: a precursors splits into two precursors, a2-m2 an additional precursor arises at a more anterior position, or an additional Md md-m precursor arises at a more posterior position). Three precursors (a2-l1/2, md-l3/ mx1-l1, mx2-l/t1-l) give rise to two different muscle units each (a2-l1, a2-l2; Mx1 mx1-m mx2-l, t1-l and md-l3, mx1-l1, respectively). mx1-m1 mx1-m2 Mx2 mx2-m with transverse orientation. Interestingly the longitudinal mx2-m1 muscle primordium of the mandible segment is no mx2-m2 longer observable. However, a prominent longitudinal primordium (lmp-mx1) now extends throughout the first mx2-m3 maxilla segment from the posterior of the medial man- T1 t1-m dible muscles. An additional lateral muscle primordium t1-m1 of the second maxilla segment (mx2-l2) can be seen. t1-m2 The posterior longitudinal muscle strand has increased Lateral extrinsic A2 a2-l1/2 in width and now shows striation. The mesoteloblasts precursors are no longer visible as all body segments have been a2-l1 formed and cell division has proceeded within all meso- a2-l2 dermal units at this time of development (Figure 6c). Md md-l1 Segmental furrows have formed throughout the entire md-l2 trunk (Additional file 1: Figure S1a). (Gf V) In the final md-l3 stage of muscle development presented here an add- md-l3/ itional lateral extrinsic precursor has formed in the man- mx1-l1 dible segment (md-l1) and a medial muscle precursor of Mx1 mx1-l1 the second maxilla anlage is becoming visible (mx1-m) mx1-l2 (Figure 2g). In the caudal papilla differentiation of the telson flexor muscles has begun, which insert dorsally mx1-l3 and ventrally (not shown) at the anterior margin of the Mx2 mx2-l/t1-l telson (Figure 6d). mx2-l1 Neocaridina heteropoda (Figure 3a): mx2-l2 No muscle primordia can be detected at the egg- mx2-l3 nauplius stage (Figure 3b). (Nh I) The earliest detectable T1 t1-l1 muscular pattern is found after the egg-nauplius stage, when the caudal papilla has elongated (Figure 3c). Ap- t1-l2 pendage rudiments of the head segments up to the man- Trunk region Longitudinal Md lmp-md dible are distinguishable, while the segments of the first precursors (lmp) Mx1 lmp-mx1 and second maxillae are not yet fully differentiated. The Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 12 of 27 http://www.frontiersinzoology.com/content/10/1/76

caudal papilla, containing the mesoteloblasts and at this progeny have proliferated into mesodermal cells of the stage the early mesodermal units of the thoracic seg- mesodermal units (Figure 6g). The intersegmental furrows ments is folded ventrally and oriented anteriorly. The of the posterior pleon segments and the telson can be dis- initial set of muscle precursors comprises the stomodeal tinguished (Additional file 1: Figure S1b). (Nh IV) Finally, muscle ring (st), a laterally extending precursor associ- as adult morphology becomes apparent we find a new ated with the stomodeum (st-2) and a medial extrinsic muscle primordium of the second antenna (a2-m2), which muscle precursor of the second antenna (a2-m1). The extends anteromedially from the posterolateral margin of latter is oriented from the second antennal base towards the appendage base (a2-m1 is no longer present outside of the stomodeum at this initial stage of morphogenesis. the appendage anlage). The first and second maxilla seg- The mandibular segment contains one muscle precursor ments, as well as the first thoracopod segment now each (md-m) extending medially and one (md-l1) extending contain two medial extrinsic precursors (mx1-m1, mx1- laterally from the limb bud. In the caudal papilla the m2, mx2-m1, mx2-m2, t1-m1, t1-m2). In the developing single mesoteloblasts can be identified (Figure 3d). To- telson the telson flexor muscles (tf) are present and ex- gether with undivided mesoteloblast progeny they form tend anteriorly into the sixth pleomere (Figure 6h). a stereotypic pattern of segmentally arranged cell rings. Neomysis integer (Figure 4a): (Nh II) One step later in muscle development of N. Mysid ontogeny differs from the species described heteropoda intrinsic muscle primordia of the first and above as a large part of development is confined to the second antenna are visible (Figure 3e). Also an extrinsic nauplioid cuticle [63]. No muscle precursors can be muscle primordium (a1-m1) running medially from the detected in the embryonic stages. A small inert larva first antenna bud towards the stomodeum is found. An called ‘nauplioid’ hatches but remains in the marsupium anterior extension of the stomodeal muscle group is (Figure 4b). The prominent uniramous first and second present (st-1), and the second antenna and mandible antennae are sheeted by cuticle and bear setae but segment now each possess two lateral extrinsic primor- posteriorly to them no appendage buds are visible. Yet dia (a2-l1, a2-l2, md-l1, md-l2). a2-m1 has moved to a the formation of segment anlagen in the nauplioid larva position within the elongate second antenna-anlage, is comparatively advanced as can be seen from the regu- where it will give rise to intrinsic musculature (marked lar arrangement of ectodermal and mesodermal cell ma- with an asterisk in Figure 3e). Finally longitudinal terial in the germ band. The mesoteloblast cells and muscle precursors become visible in the segment of the early trunk mesoderm anlagen are arranged in transverse second maxilla. The formed strand (lmp-mx2) extends rows (not in rings, as in G. falcatus and N. heteropoda) into the caudal papilla. At the posterior end (lmp-post)a (Figure 4c). (Ni I) The first myogenic signals in N. inte- small accumulation of nuclei is visible medially to the ger larvae are detected after all segments have been laid mesoteloblasts (Figure 6e and -f). An additional parallel down and appendage rudiments are present in the entire strand of longitudinal musculature (lmp-d) is being trunk (not shown). The ventral longitudinal muscle formed at a more dorsal position but it does not extend strand extending posteriorly from the second maxilla as far posteriorly as the ventral strand (Figure 3e). segment (lmp-mx2), is the first muscular primordium to (Nh III) In semaphoront III the medial mandible muscle become visible (Figure 4d and -e), together with the dor- precursor md-m has now formed an extension that crosses sal longitudinal muscle strand (lmp-d) (Figure 4d, only the median region of the germ band (Figure 3f). Medial anterior segments shown). The ventral longitudinal extrinsic precursors are now present associated with muscle strand extends from the second maxilla segment both maxillae- and the first thoracopod anlagen (mx1-m, (lmp-mx2) to the anterior pleon segments, though the mx2-m, t1-m) and also lateral extrinsic precursors have exact position of the posterior end is difficult to specify emerged in the same segments (mx1-l1, mx1-l2, mx1-l3, due to insufficient staining intensity at this early stage. mx2-l1, mx2-l2, t1-l1, t1-l2). A longitudinal muscle The initial strands are continuous and are not separated primordium (lmp-mx1) is visible for the first time in the into distinct segmental units. Yet internal segmentation first maxilla segment. A third lateral extrinsic precursor can be seen later on and we will treat the longitudinal of the second antenna has also become visible (a2-l3), muscle precursors of the second maxilla- and first which extends posterolaterally and crosses md-l1 and thoracopod-segments as discrete units for comparison in md-l2. The posterior end of the dorsal longitudinal our discussion (see below). Also the posterior end of the strand (lmp-d) has extended posteriorly into pleomere 4 germ band is free of muscular tissue at this stage (not (Figure 6g). The posterior end of the ventral longitudinal shown) and remains free also in the second semaphoront. muscle strands (lmp-post)hasenlargedandalsothe (Ni II) In semaphoront II the stomodeal muscle ring (st), more anterior portions have obtained additional nuclei. the intrinsic muscles of the first antennae and the median The mesoteloblasts can no longer be seen, indicating that muscle primordium of the mandible (md-m) have been segment formation has stopped and the mesoteloblast added to the nauplioid muscle pattern (Figure 4f). Both Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 13 of 27 http://www.frontiersinzoology.com/content/10/1/76

Figure 6 (See legend on next page.) Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 14 of 27 http://www.frontiersinzoology.com/content/10/1/76

(See figure on previous page.) Figure 6 Muscle ontogeny in the caudal papilla, posterior pleon- and telson anlage of all four species. a-f extended optical sections, g-q maximum intensity projections. a-h, n-q dorsal view, i ventral view, j-l lateral view, m dorsolateral view. Anterior is oriented to the top in all images, a-c, e-g, n and o show only the right body half. a Gf II. lmp-post, mesoteloblast rMT2 and reconstructed mesodermal cells are shown. b Gf III. lmp-post is enlarged, mesoteloblasts and mesodermal cells are visible. c Gf IV. Broad shape of lmp-post. Mesoteloblasts and mesodermal cells are no longer visible. d Gf V. Telson is delineated (bracket). tf inserts in the anterior dorsal region of telson. e Nh II. lmp-post, mesoteloblasts and mesodermal cells are visible. Telson is delineated. f Nh II slightly later in development (specimen not shown in Figure 3). lmp-post, has enlarged, only one mesodermal cell row can be identified. Telson is delineated. g Nh III. Broad shape of lmp-post, striation is visible. Mesoteloblasts and mesodermal cells are no longer visible. lmp-d is visible in P4. Telson is delineated. h Nh IV. tf show the adult tripartite pattern and insert anterodorsally in the telson. i Ni II. Telson and uropods are delineated. Mesoteloblasts and mesodermal cells are no longer visible. lmp shows a-p gradient of differentiation and reaches P6. j Ni III Posterior part of lmp (tf) extends into telson. k Ni IV. tf is enlarged and separated from lmp. k Ni IV. Slightly later in development (specimen not shown in Figure 4). l Ni IV. Yet later in development: tf show tripartite structure. m Ni V. tf show adult morphology and insert anterodorsally in the Telson. n Ph III. Telson and uropods are delineated. Mesoteloblasts and mesodermal cells are no longer visible. lmp shows a-p gradient of differentiation and reaches P1. lmp-d reaches P3. o Ph IV. lmp and lmp-d are enlarged and show metameric subdivision. Both reach P6. p Ph IV. Slightly later in development (specimen not shown in Figure 5). Individual segmental muscle units are enlarged and gut muscle becomes apparent. q Ph V. lmp and lmp-d are differentiated in P6, but telson remains free of musculature. The muscle precursors marked upm lie within the uropods, ventrally of the telson. Abbreviations: P proctodeum, rMT2 second mesoteloblast cell of left body half, tf telson flexor muscles, T telson anlage, UP uropod anlagen, upm uropod muscle precursors, P1-P6 pleon segments 1–6, gm gut muscle primordia. Scalebars are 50 μm in all panels.

longitudinal strands have increased in width. A gradual of the second antenna a2-l1/2 has become clearly sepa- decrease in differentiation from anterior to posterior is ap- rated into two distinct units (a2-l1, a2-l2 ). Also an add- parent and the ventral strand reaches the sixth pleomere itional lateral extrinsic muscle precursor has emerged (only the ventral strand lmp is shown in Figure 6i). A pos- (mx2-l2) and one of the lateral extrinsic precursors as- terior muscle precursor (lmp-post) associated with the sociated with the mandible (md-l2) is no longer visible. growth zone, such as in G. falcatus and N. heteropoda is The telson and uropods are clearly differentiated and not found. Segmental furrows have formed throughout the telson flexor muscles display the adult arrangement the entire trunk (Additional file 1: Figure S1c). (Ni III) (Figure 6m). The next observed myogenic events are the origination of Parhyale hawaiensis (Figure 5a) the anterior and lateral stomodeal muscle precursors (st-1, Unlike in G. falcatus and N. heteropoda, muscle st-2), together with a median muscle precursor in the first development in P. hawaiensis initiates at a relatively and second maxilla segment (mx1-m1, mx2-m1, mx2-m2) late developmental stage and is completed rapidly. (Figure 4g). A lateral extrinsic muscle precursor appears Semaphoronts (Ph I), (Ph II) and (Ph III) correspond to in the mandible segment (md-l2). Also an additional lon- stage S22 following Browne et al. (2005), (Ph IV) corre- gitudinal muscle precursor (lmp-mx1) has formed in the sponds to S24 and (Ph V) to S28 respectively. (Ph I) At segment of the first maxilla. The longitudinal muscle the onset of myogenesis the germ band contains the strands have now reached the last pleon segment and ex- complete set of segments, each with elongated append- tend even into the telson rudiment (Figure 6j). (Ni IV) age anlagen. The antennae and thoracic limbs are fully The following semaphoront possesses two lateral extrinsic subdivided into final podomeres. Figure 5c shows an muscle primordia of the second antenna (a2-l1/2, a2-l3), overview of an embryo at the earliest stage where muscle the mandible (md-l1, md-l2), the first maxilla (mx1-l1, formation is detectable. F-actin which is marked by the mx1-l2), as well as a single lateral extrinsic muscle primor- green phalloidin signal in these early muscle primordia dium of the second maxilla (mx2-l) and the first thoraco- is restricted to the cell cortex and is not co-localized pod (t1-l) (Figure 4h). The second antenna has obtained with myosin-signal in the initial muscle precursors medial extrinsic muscle primordia (a2-m1, a2-m2). The (Figure 5b). F-actin staining also revealed that the first maxilla- anlage now shows two medial muscle pre- central nervous system is developed to a large extent; cursors (mx1-m1, mx1-m2) and a medial muscle primor- showing paired ganglion anlagen (Figure 5d). The initial dium is now present in the first thoracic segment (t1-m). set of head muscles includes a pioneer cell located close The posterior portion of the ventral longitudinal muscle to – and dorsally of the developing tritocerebral hemi- strand (lmp) has differentiated into an elongate muscle ganglion, on each side of the stomodeum. This muscle precursor giving rise to the telson flexor muscles in the pioneer cell represents the anlage of the stomodeal anteroventral region of the premature telson (Figure 6k muscle ring (st) and exhibits cytoplasmic protrusions and -l). (Ni V) After the larva has molted the nauplioid which extend anteriorly. At the same time pioneer cells cuticle the first antenna still lacks extrinsic musculature forming the dorsal and ventral longitudinal muscle (Figure 4i). The first lateral extrinsic muscle primordium strands (lmp, lmp-d) can be found in the first thoracic Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 15 of 27 http://www.frontiersinzoology.com/content/10/1/76

segment extending posteriorly (Figure 5e). The ventral md-l2, md-l3/mx1-l1). The second maxilla shows three longitudinal strands terminate in the 7th-, the dorsal medial extrinsic muscles (mx2-m1, mx2-m2, mx2-m3) longitudinal strands in the 6th thoracic segment. No and two lateral extrinsic muscles (mx2-l1, mx2-l2). The metameric pattern can be recognized within the strands telson is still devoid of musculature (Figure 6q). at this stage. (Ph II) The stomodeal muscle anlage (st) now encloses the stomodeum anteriorly (Figure 5f). A Semaphoront specification novel muscle pioneer st-2 is connected to st at the pos- Semaphoronts described in the Results-section are terolateral margin and extends laterally. Slightly poster- shown in a schematic overview in Figure 7 and Figure 8. ior to the stomodeum a pair of pioneer cells forms yet 45 muscle precursors are specified (Table 1) and color another novel muscular unit which protrudes ventrally coded. One color is used for lmp-md, lmp-mx1, lmp- and inserts medially of the paragnaths. This muscular mx2 and lmp-t1. The lateral extrinsic muscle precursors unit, which is not observed in any of the other investi- of one segment are shown separately but also only gated malacostracan species in this study, is termed assigned one color. The same is true for the medial ‘pharyngo-paragnathal muscle’ (ppg). The ventral longi- extrinsic precursors of one segment. Five different tudinal muscle strand primordium lmp is detectable semaphoronts could be identified for G. falcatus, N. from the first thoracic- to the second pleon segment integer and P. hawaiensis, while four of them could be (not shown). (Ph III) The following stage is character- distinguished in N. heteropoda and P. fallax f. virginalis. ized by the appearance of further muscle pioneer cells in Schematic drawings of myogenesis in the posterior the head region (Figure 5g). Intrinsic muscle pioneers embryonic region (Figure 8) refer to the same semaphor- are now present in both proximal podomeres of the sec- onts also shown in Figure 7. ond antenna. Also the anlage of the labrum now con- Since Malacostraca exhibit a conserved number of tains paired muscle primordia. A novel precursor (st-3) body segments (5 head segments, 8 thorax segments, 6 associated with the stomodeal muscles is observed in a pleon segments) segment position is used as overall ref- transverse position posterolaterally of the stomodeum, erence for comparison of development. Four additional which displays a thin cytoplasmic extension across the morphological features were used to align the temporal medial region of the germ band. Two medial and lateral sequences of muscle development (Figure 9, Additional extrinsic muscle primordia (a2-m1, a2-l1) associated file 2: Figure S2): N, the ‘egg-nauplius’-stage, showing with the second antenna can be seen. The medial prominent appendage buds of the first antenna, second mandible muscle anlage (md-m) is present, as well as antenna and mandible; PN, presence of postnaupliar additional medial extrinsic muscle primordia (mx1-m1, appendage buds (at least one appendage bud of first mx1-m2, mx2-m3, t1-m) associated with the respective maxilla to sixth pleopod); FS, the emergence of the full appendage anlagen. One lateral extrinsic precursor set of segments, meaning that the ectodermal and meso- (mx2/t1-l) is found which gives rise to extrinsic muscu- dermal cell material responsible for generating all post- lature of second-maxilla and first thoracopod. The naupliar segments of the adult has been proliferated posterior ends of lmp and lmp-d are detectable only an- from the ectoteloblasts and mesoteloblasts respectively terior to the third pleon segment and first pleon seg- (the mesoteloblasts, can no longer be detected once FS ment respectively (Figure 6n). Segmental furrows have is acquired because their directional proliferation has formed throughout the entire trunk (Additional file 1: ceased and cell division has continued within the meso- Figure S1d). (Ph IV) Formation of additional lateral ex- dermal units); IF, presence of intersegmental furrows trinsic muscle primordia in the remaining appendage an- (Here the embryo is developed to a degree where seg- lagen from the mandible to the first thoracopod has mentation is visible externally over the entire length of occurred (a2-l2, md-l1, md-l3/mx1-l, mx2-l, t1-l1, t1-l2) the trunk and the telson is clearly delineated from the (Figure 5h), but extrinsic primordia can be observed also last pleomeres, as shown in Additional file 2: Figure S2). in the following segments down to the sixth pleopod For N. integer and P. hawaiensis N and PN can be taken (not shown). An additional medial extrinsic muscle pre- as one event (NPN) (Figure 9d and -e) due to the lack of cursors associated with the second antenna has formed significant temporal difference between naupliar and (a2-m2). The longitudinal muscle strands have diversi- postnaupliar appendage formation in these species. FS fied into multiple muscular elements and display a meta- and IF coincide in all species investigated at the given meric pattern. lmp and lmp-d terminate in the sixth temporal resolution, except for N. integer. pleon segment, while the telson remains free of muscu- lature (Figure 6o, -p). (Ph V) Close to hatching, embryos Discussion of P. hawaiensis display additional diversification within Cephalic muscle development of Malacostraca the existing muscle pattern (Figure 6i). The mandible is Comparison between investigated species reveals that equipped with three lateral extrinsic muscles (md-l1, stable chronological order is not prevalent in muscle Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 16 of 27 http://www.frontiersinzoology.com/content/10/1/76

a b c d e

Gonodactylaceus falcatus FS FS

Ol Ol Ol Ol Ol A1 A1 A1 A1 A1 A2 * A2 A2 A2 A2 Md Md Md Md Md Mx1 Mx1 Mx1 Mx1 Mx1 Mx2 Mx2 Mx2 Mx2 Mx2 T1 T1 T1 T1 T1

FS

Ol Ol Ol Ol Ol A1 A1 A1 A1 A1 A2 * A2 * A2 A2 * A2 Md Md * Md * Md Md Mx1 Mx1 Mx1 Mx1 Mx1 Mx2 Mx2 Mx2 Mx2 Mx2 T1 T1 T1 T1 T1

FS

Ol Ol Ol Ol Ol A1 A1 A1 A1 A1 A2 * A2 * A2 A2 * A2 * Md * Md * Md * Md * Md * Mx1 Mx1 Mx1 Mx1 Mx1 Mx2 Mx2 Mx2 Mx2 Mx2 T1 T1 T1 T1 T1

FS

Ol Ol Ol Ol Ol A1 * A1 * A1 * A1 * A1 * A2 A2 A2 * A2 A2 Md * Md * Md Md * Md * Mx1 Mx1 Mx1 Mx1 Mx1 Mx2 Mx2 Mx2 Mx2 Mx2 T1 T1 T1 T1 T1

Ol Ol Ol A1 * A1 * * A1 * A2 A2 * A2 * Md * Md Md Mx1 Mx1 Mx1 Mx2 Mx2 Mx2 T1 T1 T1

st st-1 st-2 st-3 ppg a1-m1 a1-m2 a2-m1 a2-m2 md-m mx1-m1 mx1-m2 mx2-m1 mx2-m2 mx2-m3 t1-m1 t1-m2 a2-l1 a2-l2 a2-l3 md-l1 md-l2 md-l3 mx1-l1 mx1-l2 mx1-l3 mx2-l1 mx2-l2 t1-l1 t1-l2 * intr lmp Figure 7 (See legend on next page.) Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 17 of 27 http://www.frontiersinzoology.com/content/10/1/76

(See figure on previous page.) Figure 7 Schematic overview of head muscle development and summary of results presented in Figures 2-5. a Gonodactylaceus falcatus, b Neocaridina heteropoda, c Procambarus fallax f. virginalis, d Neomysis integer, e Parhyale hawaiensis. The specified semaphoronts (roman numbers) are shown as simplified drawings containing the optic lobes and anterior six hemisegments of the left body half. Muscle precursors are color coded. The color code is shown at the bottom of the figure. Lateral or medial extrinsic precursors of one segment are numbered in the order they are positioned from anterior to posterior. Abbreviations: Gf (I-IV), Nh (I-V), Pf (I-IV), Ni (I-V), Ph (I-V) species- and semaphoront affiliation; Ol optic lobes, A1, A2, Md, Mx1, Mx2, T1 appendage anlagen, FS developmental event: full set of segment anlagen present, intrinsic appendage muscle precursors are marked by asterisks.

development. For example the set of muscle units that in G. falcatus and P. hawaiensis (lmp-t1). Previous stud- appear first in development differs greatly. For G. ies of malacostracan development gave strong indica- falcatus it consists of just the intrinsic muscles of the tions that the first maxilla segment has a developmental first antenna (Figure 7a), in N. heteropoda we find origin that differs from that of the more posterior seg- stomodeal muscles (st, st-2), medial extrinsic primordia ments, which are formed by teloblastic proliferation of the second antenna and mandible (a2-m1, md-m) and [31,41,57,68,70]. It has been observed in several species lateral extrinsic precursors of the mandible (md-l1) that differentiation of the first maxilla appendage anlage (Figure 7b). In P. fallax f. virginalis we find the lateral is delayed, compared to the more posterior appendages. muscle precursor of the stomodeum (st-2) along with Interestingly, formation of the longitudinal muscle pre- lateral extrinsic muscles of the second antenna, man- cursor in this segment (lmp-mx1) is very dynamic in our dible and first maxilla (a2-l1, a2-l2, md-1 l, mx1-l1), species. It is formed only after lmp-md, lmp-mx2 and medial extrinsic precursor of the mandible (md-m) and lmp-t1 in G. falcatus and only after lmp-mx2 in N. het- longitudinal muscle precursors in the mandible- and eropoda and N. Integer. In P. fallax f. virginalis it is first maxilla segment (lmp-md, lmp-mx1) (Figure 7c). In present already at the earliest myogenic stage together N. integer myogenesis begins with the longitudinal with lmp-md, while it is never formed in P. hawaiensis. muscle precursors which form a strand, beginning in the G. falcatus also lacks lateral extrinsic muscles in the second maxilla segment (Figure 7d). In P. hawaiensis the second maxilla segment throughout all semaphoronts longitudinal muscle strands beginning in the first - investigated here. We conclude that the delay of muscle acic segment (lmp-t1) are the major components of the differentiation in the first maxilla segment compared to initial pattern, together with muscle pioneer cells of the the posterior segments belongs already to the eumala- stomodeal muscle ring (st) (Figure 7e). Also subsequent costracan ground pattern. patterns of cephalic muscle precursors in the ontogen- Despite the variation in temporal and spatial patterns etic series vary greatly. of muscle development in the species investigated, G. All investigated species exhibit ventral longitudinal falcatus, N. heteropoda and P. fallax f. virginalis show muscle strands (shown in pink in Figure 7) at some onset of myogenesis before the offset of segment prolif- point in development. Metameric organization of longi- eration (FS) from the mesoteloblasts and the delineation tudinal muscle precursors in the anterior six segments is of all segment primordia by formation of intersegmental problematic to see in some cases, especially in early furrows (IF) (Figure 7a, -b and -c, Figure 9a, -b and -c, stages (Figure 5e). However, metameric organization of Additional file 2: Figure S2a, -b and -c). In both N. inte- longitudinal musculature becomes evident eventually ger and P. hawaiensis, myogenesis is delayed compared and therefore precursors are shown as individual units to overall body patterning. This is reflected by the fact (lmp-md, lmp-mx1, lmp-mx2 and lmp-t1) for the re- that germ band elongation is completed (FS) before the spective segments. Also a dorsal longitudinal strand onset of myogenesis in these species (Figure 7d and -e, (lmp-d) is formed in the cephalic region of all species in- Figure 9d and -e, Additional file 2: Figure S2d and -e). vestigated. However the position of the anterior end of In P. hawaiensis even full external segmentation is vis- the dorsal strand is not clear in every case and its large ible (IF) and the podomeres of the head- and thoracic distance to the appendage buds makes it difficult to as- appendages are distinguishable before muscle precursors sign segment positions to lmp-d primordia. They are appear. In G. falcatus, N. heteropoda and N. integer early therefore excluded from further discussion. In P. fallax f. onset is observed in the formation of extrinsic append- virginalis the first ventral longitudinal precursors to age muscles of the nauplius segments (Gf II in Figure 7a, appear are found in the segment of the mandible and Figure 9a, Additional file 2: Figure S2a, Nh I and Nh II first maxilla (lmp-md, lmp-mx1), in N. integer and N. in Figure 7b, Figure 9b, Additional file 2: Figure S2b, Ni II heteropoda they are located in the segment of the sec- in Figure 7d, Figure 9d, Additional file 2: Figure S2d), ond maxilla (lmp-mx2), but in the first thoracic segment though in N. integer this includes only md-m.InP. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 18 of 27 http://www.frontiersinzoology.com/content/10/1/76

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Gonodactylaceus Neocaridina falcatus heteropoda FS FS

m m m m m

FS

m m m T T

FS

m T T T T

FS

T T T T T

T T T lmp lmp-d

? lmp-post

tf T T m Mesoteloblast

Figure 8 (See legend on next page.) Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 19 of 27 http://www.frontiersinzoology.com/content/10/1/76

(See figure on previous page.) Figure 8 Schematic overview of muscle development in the posterior germ band, posterior pleon- and telson anlagen. Summary of results presented in Figure 6. a Gonodactylaceus falcatus, b Neocaridina heteropoda, c Procambarus fallax f. virginalis, d Neomysis integer, e Parhyale hawaiensis. For G. falcatus the late embryonic stages are unknown. The specified semaphoronts (roman numbers) are presented as simplified drawings. Only hemisegments of the right body half are shown. Anterior is oriented to the top in all panels. lmp and lmp-d are shown as continuous lines and color coded (pink and purple respectively). The posterior pioneer muscle strand lmp-post is treated as a single unit though it cannot be precisely delineated from lmp. The mesoteloblasts and mesodermal cells are shown as circles with colors alternating yellow and white in a-p direction. Only one of four mesoteloblast cells is shown (yellow). Units of the trunk mesoderm, in which cell proliferation has progressed beyond the stereotypic pattern of mesodermal cell rows, are shown in the same color code. Abbreviations: Gf (I-IV), Nh (I-V), Pf (I-IV), Ni (I-V), Ph (I-V) (species- and semaphoront affiliation); FS (developmental event: full set of segment anlagen present), T telson anlage, m mesoteloblast.

fallax f. virginalis extrinsic muscle primordia of the first a posterior pioneer muscle primordium is a continuous maxilla, together with muscle primordia in the naupliar muscle strand connecting the telson to more anterior seg- segments appear in advance compared to the more ments before the germ band is fully segmented (FS, IF) posterior ones (Pf I in Figure 7c, Figure 9c, Additional (Gf II in Figure 8a, Nh II in Figure 8b, Pf II in Figure 8c). file 2: Figure S2c). In P. hawaiensis extrinsic muscle The fate of lmp-post cannot be determined with certainty, precursors of naupliar and postnaupliar segments appear but in G. falcatus, N. heteropoda and P. fallax f. virginalis synchronously with no temporal gap in the onset of we find strong support that it eventually forms the flexor development (Ph II in Figure 7e, Figure 9e, Additional muscles of the telson, because the position of lmp-post file 2: Figure S2e). and tf show strong correspondence and no additional muscle precursors are observed in the telson at any time. Also the dorsal strand (lmp-d) appears to remain anterior Posterior longitudinal muscle development to the telson, a feature also observed for N. integer and P. in Malacostraca hawaiensis, and therefore is unlikely to contribute to the The different ontogenetic events of posterior longitu- tf-muscles. Although we cannot exclude the possibility dinal muscle development are summarized schematically that mesodermal cells from anterior segments contribute in Figure 8. Following the previous findings of extensor- to these muscles and that lmp-post merely serves as a scaf- and flexor muscle development in the pleon of P. fallax fold for muscle morphogenesis which progresses by pos- f. virginalis [52] we find it reasonable to assume an terior migration, we find the existence of an independent equivalent scaffolding role of these strands for the adult posterior longitudinal muscle origin (lmp-post) to be longitudinal muscle pattern of G. falcatus and N. hetero- much more consistent with our observations. Both pera- poda. The first muscle primordium detectable in the carids, N. integer and P. hawaiensis, do not exhibit a posterior region of the ventral longitudinal strand (lmp- posterior longitudinal muscle origin (lmp-post)asob- post) shows strong correspondences in G. falcatus, N. served in G. falcatus, N. heteropoda and P. fallax f. - heteropoda and P. fallax f. virginalis. This primordium ginalis (Figure 8d and -e, Figure 9d and -e, Additional was described as ‘posterior longitudinal muscle origin’ in file 2: Figure S2d and -e). Rather the longitudinal muscle P. fallax f. virginalis [52] and is formed before the full strands (lmp) follow a clear anterior-posterior gradient set of segments is present as mesodermal anlagen (FS) of differentiation. The posterior-most pleon segments or visible in external morphology (IF) (Figure 8a, -b, therefore are the last ones in which longitudinal muscle and -c). It is located very close to the mesoteloblasts in primordia are formed. In N. integer, which possesses a the caudal papilla and also the newly formed mesodermal decapod-like tail fan, flexor muscles are formed in the tel- cells, which have been formed by them, representing the son nevertheless. Our observations suggest that the longi- mesodermal units of the adult body segments. Also no tudinal strand eventually extends into the telson and gives metameric pattern, not even metameric arrangement of rise to them, but without any posterior advance in differ- nuclei within the primordium is seen. These features entiation (Figure 8d). Typically for an amphipod crust- justify the interpretation of lmp-post as an independent acean [71] and unlike N. integer, P. hawaiensis lacks a tail posterior longitudinal muscle primordium or ‘pioneer fan and possesses a comparatively small telson in the ju- muscle strand’, which is not formed from mesodermal venile stage. Telson flexor muscles are lacking completely somites and hence from the mesoteloblast cells, but in P. hawaiensis (Figure 8e). more likely from a separate mesodermal origin, most likely coming from the telson [68]. This interpretation is in accordance to Weygoldt [39] who observed anterior The validity of the egg-nauplius concept migration of telson mesoderm in embryos of the decapod Correspondences between functional larvae, such as the Palaemonetes varians. The developmental consequence of anostracan and dendrobranchiate nauplius, and embryonic Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 20 of 27 http://www.frontiersinzoology.com/content/10/1/76

Gf II Gf IV Ni II Ni IV Ni V

N PN FS/IF NPN FS IF

A1 A1 A2 A2 Md Md Mx1 Mx1 Mx2 Mx2 T1 T1 lmp-post lmp-post ad

Nh II Nh III Nh IV Ph II Ph IV Ph V

N PN FS/IF NPN FS/IF

A1 A1 A2 A2 Md Md Mx1 Mx1 Mx2 Mx2 T1 T1 lmp-post lmp-post b e

Pf II Pf IV 13h 15h N1 N4 N5 Z1 Z2-Pl

N PN FS/IF hatch N hatch PN FS/IF

A1 A1 A2 A2 Md Md Mx1 Mx1 Mx2 Mx2 T1 T1 lmp-post lmp-post c f

Figure 9 Simplified timeline representation of developmental events. a Gonodactylaceus falcatus, b Neocaridina heteropoda, c Procambarus fallax f. virginalis, d Neomysis integer, e Parhyale hawaiensis, f Sicyonia ingentis. Ontogenetic data on extrinsic appendage muscle development of the naupliar-, first- and second maxilla-, and first thoracopod segments, are combined to four general categories and compared between species. A more detailed comparison of myogenic sequences between species is given in Additional file 2: Figure S2. Extrinsic muscle precursors and posterior longitudinal muscle precursors in the respective semaphoronts are mapped in the sequence they first occur in each species. Categories of extrinsic muscle precursors, namely of the nauplius segments, the first maxilla segment, the second maxilla segment and the first thoracic segment, are shown in specific shades of grey. The posterior pioneer muscle strand (lmp-post) is also mapped (pink). General developmental events (N, PN, FS, IF) are added in the sequence they occur relative to the muscle precursors. FS is marked by a bold vertical dotted line. Regular vertical dotted lines mark the chronological boundaries between all semaphoronts. Abbreviations: Gf (I-IV), Nh (I-V), Pf (I-IV), Ni (I-V), Ph (I-V) species- and semaphoront affiliation, N appendage anlagen in nauplius segments present, PN appendage anlagen in postnaupliar segments present, FS full set of segment anlagen present, IF intersegmental furrows present in entire trunk. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 21 of 27 http://www.frontiersinzoology.com/content/10/1/76

stages, such as the malacostracan egg-nauplius, are prob- respective medial extrinsic precursors (corresponding lematic, because fully differentiated, functional tissues are to a1-l, md-l, a1-m, a2-m, md-m), prior to hatching compared with undifferentiated, developing tissues. The (Additional file 2: Figure S2f). At nauplius stage 4 longi- free swimming nauplius larva itself is generated from spe- tudinal muscle strands become visible, which extend cific embryonic stages, (which also correspond to the egg- from the second maxilla segment into the telson anlage, nauplius) and those should also be compared. In the taxa corresponding to lmp-mx1, lmp-mx2, lmp-t1 and lmp- which have no nauplius larva the egg-nauplius is followed post. At nauplius stage 5 the first extrinsic muscle by an advanced embryonic stage comprising additional primordia of the first and second maxilla and the segment anlagen and first muscle precursors. For this maxillipeds (mx1-m, mx1-l, mx2-m, mx2-l, t1-m, t1-l) reason, when development between malacostracan spe- appear. Kiernan and Hertzler [54] also described muscle cies is compared, it is better to consider developmental morphology in nauplius stages of the branchiopod trajectories, more specifically the chronological sequence , although not in the preceding embry- of developmental events necessary to form a specific onic stages. Yet, as with S. ingentis,alsoA. salina must semaphoront, rather than just a single semaphoront of the possess an embryonic phase in which larval tissues are developmental sequence. The developmental trajectory of formed through a series of developmental events. An nauplius-larva formation should include the events, from egg-nauplius stage should therefore be present also in early embryonic anlagen to hatching, which are necessary this species and must contain developing musculature. to establish the tissue structure of the functional larva. If we compare the egg-nauplius of G. falcatus, N. hetero- Furthermore the developmental trajectories of nauplius- poda and P. fallax f. virginalis with that in S. ingentis larva formation should affect all tissues of the nauplius (and the expected egg-nauplius in A. salina)weseethat segments which must be functional at hatching (the epi- epidermal- and muscle development are uncoupled in dermis and developing exoskeleton, connective tissue, ner- our species and that the egg-nauplii are not directly vous system, digestive system, vascular system and of comparable. Nevertheless a distinct gap remains be- course musculature). The egg-nauplius concept as formu- tween naupliar and postnaupliar muscle formation. This lated by Scholtz [7] implies that the developmental system finding suggests that part of an active ancestral develop- involved in formation of a free swimming nauplius larva is mental trajectory within muscle development related to active in species which exhibit an egg-nauplius stage but nauplius larva formation is still present and can be assigned lack a free-swimming nauplius larva. If, as suggested by to the ground pattern of Eumalacostraca or perhaps further Williams and Dahms [46,47] the egg nauplius was part of down the tree (as no data is available for Leptostraca). The a crustacean phylotypic stage, or as argued by Scholtz [7], egg-nauplius concept therefore describes a phenomenon the nauplius larva “[…] was conserved in the egg nauplius, of heterochrony in parts of the developmental system (i.e. which is still characterized by advanced development in in the epidermis), but not the whole. Our results are in ac- the naupliar region […],”. correspondences in the develop- cordance with Alberch et al. [72], who argues that the con- mental trajectory should be found in more than just the cept of heterochrony targets only specific traits of the epidermal tissue. In this light an egg-nauplius would be organism, in this case of the embryo or larva. A theory of expected to include anlagen of musculature, which, how- recapitulation (in an inclusive interpretation) of an ances- ever, is not the case in any of the species studied herein. tral nauplius larva must be rejected. The egg-nauplius stage (N) of G. falcatus, N. heteropoda, P. fallax f. virginalis and N. integer, is free of detectable An approach to the evolution of malacostracan muscle precursors. Only after postnaupliar segment for- myogenesis mation becomes apparent in external morphology (PN) Recent attempts to infer phylogenetic relationships from they are formed. To date the only published documenta- different sources of molecular data yield contradictory re- tion comparable to our approach, which describes muscle sults [73-75]. We follow the phylogeny of Malacostraca development in a species with a free swimming nau- proposed by Richter & Scholtz [19] which is based on plius larva, has been performed on a malacostracan, the morphological data and shown in a simplified form in dendrobranchiate decapod Sicyonia ingentis (Figure 9f, Figure 10 and Figure 11. Within Eumalacostraca the Additional file 2: Figure S2f) [54]. In this species nauplius stomatopods, represented by G. falcatus are the sister muscle precursors are formed in an egg-nauplius stage, an group to the remaining taxa. N. heteropoda, P. fallax embryo with distinct appendage buds in the nauplius f. virginalis and S. ingentis form the clade Decapoda. segments. Muscle precursors are by no means formed Decapoda and Peracarida appear as sister groups in the synchronously in this species. Rather myogenesis begins simplified tree because Euphausiacea and Anaspidacea with lateral extrinsic precursors of the second antenna are omitted. (corresponding to a2-l), followed by the lateral extrinsic The phylogenetic relationships of Malacostraca proposed precursors of the mandible, the first antenna and the by Richter & Scholtz [19] imply that Dendrobranchiata Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 22 of 27 http://www.frontiersinzoology.com/content/10/1/76

and Euphausiacea evolved from ancestors which lacked a observed for G. falcatus, N. heteropoda and P. fallax f. nauplius larva. In this light the entomostracan and the virginalis. In this case loss of a nauplius larva and of malacostracan nauplius larva are not homologous. Further- muscle precursors in the preceding egg-nauplius stage more Scholtz [7] listed several properties of the dendro- would have occurred in the malacostracan stem lineage. branchiate (and euphausiacean) nauplius larvae which are The re-acquisition of a nauplius larva in Dendrobranchiata not shared with nauplius larvae of non-malacostracans, but (and Euphausiacea, not shown in Figure 10 and Figure 11) with malacostracan egg-nauplii: lack of a labrum, lack of would have included an acceleration of muscle develop- articulations in the appendages, relatively low cell number, ment in the nauplius segments, resulting in an egg- an undifferentiated growth zone, rudimentary stomodeum nauplius stage with muscle precursors. This transformation and proctodeum, sparse setation, undeveloped midgut would have been accompanied by an accelerated differenti- and lack of mandibular gnathobase and masticatory spines ation also of the non-muscular tissues of the nauplius seg- of second antenna. According to Scholtz [7] these fea- ments, an earlier hatching event and a delay in embryonic tures can be taken as additional support for the hypothesis development and differentiation of postnaupliar segments. of independent origin of the nauplius larva in dendro- The latter scenario (Figure 11) is clearly more parsimoni- branchiates (and euphausiaceans). Kiernan & Hertzler [54] ous, as it requires a single loss of egg-nauplius musculature describe differences in the functional muscular pattern of and single reacquisition in Dendrobranchiata (as well as nauplius larvae of S. ingentis and A. salina, which are another reacquisition in Euphausiacea, not shown). On the consistent with the secondary-evolution hypothesis of the contrary multiple loss of the nauplius larva (Figure 10) im- dendrobranchiate nauplius. Our observations of muscle plies three evolutionary losses of egg-nauplius musculature development in five malacostracan species, however, show (not counting Leptostraca, Anaspidacea Syncarida and that corresponding muscle precursors can give rise to di- Thermosbaenacea, which would require four more in- verse patterns of juvenile musculature. We think that stances of reduction), which is clearly less parsimonious. homology of embryonic musculature of Branchiopoda The early onset of muscle development in the naupliar and Malacostraca should not be excluded based on segments described for G. falcatus and N. heteropoda these findings alone. If the embryonic muscle precursors (Figure 9a and -b) refers only to the extrinsic appendage of A. salina and S. ingentis are homologous, an egg- muscles. In either of the solutions presented above the nauplius stage with extrinsic appendage muscle precursors pattern of myogenesis was altered in P. fallax f. virgina- can be postulated for the malacostracan ground pattern. lis so that the initial set of extrinsic appendage muscle Certainly independent evolution of the dendrobranchiate precursors includes the naupliar segments and also the and euphausiacean nauplius larvae cannot be concluded first maxilla segment (Figure 9c). The nauplioid larva of from observation of potential symplesiomorphies of egg- mysids, more precisely the advanced formation of first- nauplii and nauplius larvae alone. A phylogenetic test of and second antenna and the early timing of hatching are these developmental features based on the phylogeny of likely to be relics of a developmental trajectory including Richter & Scholtz [19], however, can be used to argue in a free swimming larva. However, the early onset of ap- favor of secondary nauplius larva evolution (Figure 10 and pendage development is in no way reflected by the de- Figure 11). velopmental pattern of extrinsic musculature. Only the If, hypothetically, a free-swimming nauplius larva was medial extrinsic appendage muscle precursor (md-m) present in the malacostracan (and eumalacostracan) last arises before the remaining extrinsic appendage muscle common ancestor (Figure 10), this ground pattern also precursors (Additional file 2: Figure S2d). Since md-m in included an egg-nauplius stage with muscle precursors, the mysid juvenile represents a comparatively large as observed in S. ingentis. This pattern would then have muscle the early onset of its development can also be re- been transformed by a heterochronic delay in embryonic lated to the fact that a relatively large amount of muscle nauplius muscle formation three times independently in tissue has to be generated from this precursor, and not the lineages leading to the Stomatopoda, Pleocyemata that early emergence of md-m is caused by cryptic larva and Peracarida, which resulted in an egg-nauplius stage development. Yet, given the considerably nauplius- lacking muscle precursors. These transformations were related characteristics of the ‘nauplioid’ larva we favor accompanied by the loss of the nauplius larva as a an interpretation in which mysids have retained part of hatching stage and with an advanced onset of postnau- the ancestral myogenic program. In this light early onset pliar segment formation and -differentiation in embryo- of naupliar myogenesis, as proposed for the eumalacos- genesis. If, however, an egg-nauplius (but no succeeding tracan last common ancestor, would have been present nauplius larva) was part of the eumalacostracan ground also in the ground pattern of Peracarida. P. hawaiensis pattern (Figure 11), as part of direct development or de- shows the most derived characteristics of muscle devel- velopment with an advanced larva, this egg-nauplius opment among the investigated species. Myogenesis is stage would also have lacked muscle precursors, as completed rapidly and certain muscle primordia observed Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 23 of 27 http://www.frontiersinzoology.com/content/10/1/76

Branchiopoda Eumalacostraca

Caridoida

Decapoda

Pleocyemata Peracarida

A. salina G. falcatus S. ingentis N. heteropoda P. fallax f. virg. N. integer P. hawaiensis

Nauplius egg nauplius larva

Advanced embryonic stage lmp-post

Onset of myogenesis: No lmp-post

Early onset in segments Zoea-like (A1, A2, Md) larva

Early onset segments (A1, A2, Md) + Mx1

No early onset

Figure 10 (See legend on next page.) Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 24 of 27 http://www.frontiersinzoology.com/content/10/1/76

(See figure on previous page.) Figure 10 Phylogram of hypothesized evolutionary history of myogenesis. Phylogenetic relationships refer to [19,51]. Anostraca are used as an outgroup without implying that they are sister group to Eumalacostraca or Malacostraca. Dendrobranchiata are included, represented by Sicyonia ingentis,[54].Evolutionarychangesofmyogenesisareshownasinferredfromcomparisonofthesimplified myogenic sequences shown in Figure 9. Only extrinsic muscle precursors and the posterior pioneer muscle strand are considered and the three nauplius segments are combined to one category. The features plotted on the tree are: egg-nauplius, advanced embryonic stage, nauplius larva, zoea-like larva, early onset of myogenesis in embryonicnaupliarsegments,earlyonsetofmyogenesisinembryonicnaupliar segments and the first maxilla segment, lack of advanced myogenesis in nauplius segments relative to postnaupliar segments. The features are shown as small icons. Loss of either larval form is indicated by a ‘ghost’-icon. Presence of a posterior pioneer longitudinal muscle strand (lmp-post)iscodedtothebranches(pink).Absenceoflmp-post is given in black. A free swimming nauplius larva is part of the ground pattern and has been lost three times independently in the Stomatopoda, Pleocyemata and Peracarida. In clades which possess lmp-post,zoea-likelarvaearecommonlyfound,indicatingthatbothfeatures are dependent upon each other. Loss of a zoea-like larva is clearly derived in N. heteropoda and P. fallax f. virginalis.Thereforeazoea-likelarvaisshownforthelastcommonancestor. Abbreviations: A1, A2, Md, Mx1 Body segments bearing respective appendages.

here are not found in the other species (for example ppg, with other embryonic relics of larva myogenesis. These md-l3/mx1-l, mx2-l/t1-l). In this species the early onset of reductions are most likely connected to the evolutionary extrinsic appendage muscle development in the naupliar acquisition of advanced brood care using a marsupium segments was lost (Figure 9e). in Peracarida. Posterior longitudinal muscle development involving lmp-post is interpreted as part of a developmental trajec- Conclusions tory of myogenesis in zoea-like larval forms, because it The observations on muscle development in five repre- allows the posterior pleon segments to function in sentatives of Malacostraca contribute to our understand- movement of the trunk before differentiation of these ing of the changes in the developmental system that may segments is complete. In combination with a broad, have caused evolutionary transitions between larval- and paddle-shaped telson, a rapid escape movement (similar embryonic phases. Also we conclude from our study that to the tail flip reflex of adult decapods [19]) could be concepts of recapitulation of a larva stage, such as the performed by these larvae. lmp-post is not found in A. egg-nauplius [46,47] are incomplete. First of all func- salina [54] which lacks a zoea-like larva. We conclude tional larval- or adult organs and tissues, must always that lmp-post is an autapomorphy of Eumalacostraca (or develop from specific organ- or tissue- anlagen which, in Malacostraca, the situation in Leptostraca is unknown). the case of the nauplius larva, are formed already in Within the Malacostraca lmp-post is commonly a feature embryogenesis. This implies that a nauplius larva and an of embryogenesis, but is also observed in the larval egg-nauplius do not represent alternative situations. Rather phase of the dendrobranchiate S. ingentis. Nauplius stage the nauplius larva must be viewed as a hatching stage 4 of S. ingentis is reported to exhibit developing muscle which directly follows an egg-nauplius stage. The corre- strands that extend from the second maxilla segment spondences possibly representing recapitulated ancestral into the furcal processes [54]. These strands precede the features do not reside in overall morphology of embryos longitudinal trunk musculature of the protozoea larva, but in the ontogenetic trajectories of developing tissues or which extend through the trunk into the telson before tissue parts. Our data shows that the sequence of emer- the full set of pleon segments can be distinguished exter- ging muscle precursors related to formation of a nauplius nally [55]. The common occurrence of zoea-like larval larva are partly retained in embryogenesis of species with forms in ontogeny of stomatopods and marine decapods advanced larval stages or direct development, but do not leads us to conclude that lmp-post is a crucial element correspond to morphogenesis of the ectoderm. Our ob- of development and – together with a zoea-like larval servations demonstrate that heterochronic events which form- part of the eumalacostracan ground pattern can be discussed in the light of larva recapitulation can (Figure 10 and Figure 11). It has been retained in these take place at different rates in different tissues, even the lineages, even if the zoea-like larva was lost with the particular organ anlagen therein. Retention of larva mor- adaptation to a freshwater environment (as in N. hetero- phology must therefore always be discussed across all poda and P. fallax f. virginalis). This conclusion is fur- stages of pre-adult ontogeny. However, in-depth investi- ther supported by findings which suggest that zoea-like gation of malacostracan development in further tissues larvae can be lost and reacquired multiple times, as in using cladistic methods for heterochrony analysis is be- the genus [76]. In the peracarid lineage yond the scope of the present study and will be dealt lmp-post was lost (Figure 10 and Figure 11) together with in the future. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 25 of 27 http://www.frontiersinzoology.com/content/10/1/76

Branchiopoda Eumalacostraca

Caridoida

Decapoda

Pleocyemata Peracarida

G. falcatus N. heteropoda N. integer A. salina S. ingentis P. fallax f. virg. P. hawaiensis

egg nauplius Nauplius larva

Advanced embryonic stage lmp-post

Onset of myogenesis: No lmp-post

Early onset in segments Zoea-like (A1, A2, Md) larva

Early onset segments (+A1, A2, Md) Mx1

No early onset

Figure 11 Phylogram of hypothesized evolutionary history of myogenesis. Phylogenetic relationships, taxa and compared developmental features as in Figure 10. An egg-nauplius stage is part of the ground pattern and the free swimming nauplius larva evolved independently in dendrobranchiates (and euphausiaceans, not shown). Abbreviations: A1, A2, Md, Mx1 Body segments bearing respective appendages. Jirikowski et al. Frontiers in Zoology 2013, 10:76 Page 26 of 27 http://www.frontiersinzoology.com/content/10/1/76

Additional files 6. Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin JW, Cunningham CW: Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 2010, 463:1079–1083. Additional file 1: Figure S1. External morphology of ventral posterior 7. Scholtz G: Evolution of the nauplius stage in malacostracan crustaceans. germ band region shown by nuclear staining with TOPRO-3 (cyan). The J Zool Syst Evol Res 2000, 38:175–187. first stage that reveals intersegmental furrows throughout the entire trunk 8. Knight M: Larval development of Euphausia fallax Hansen (Crustacea: is shown for embryos of G. falcatus, N. heteropoda, P. hawaiensis and a Euphausiacea) with a comparison of larval morphology within the nauplioid larva of N. integer. Anlagen of pleomeres 4 to 6 are demarcated E. gibboides species group. Bull Mar Sci 1978, 28:255–281. by dotted lines. Brackets point out the anteroposterior expansion of the telson anlage in a, b and c. In N. integer and P. hawaiensis uropod 9. Knight MD: Larval development of Euphausia sanzoi Torelli (Crustacea: – anlagen are visible at this stage. a Gf IV. b Nh III. c Nh II. d Ph III. Euphausiacea). Bull Mar Sci 1976, 26:538 557. Abbreviations: UP uropod anlagen, T Telson anlage, P4-P6 10. Zimmer C: Euphausiacea. In Klassen und Ordnungen des Tierreichs. Edited by – Pleon segments 4–6. Scalebars are 100 μm in all panels. Gruner HE. Leipzig: Akademische Verlagsgesellschaft Geest; 1956:1 286. 11. Mauchline J, Fisher LR: The biology of euphausiids. Mar Biol 1969, 7:1–454. Additional file 2: Figure S2. Detailed timeline representation of 12. Anderson WW, King JE, Lindner MJ: Early stages in the life history of the developmental events. a Gonodactylaceus falcatus, b Neocaridina common marine shrimp, Penaeus setiferus (Linnaeus). Mar Biol 1949, heteropoda, c Procambarus fallax f. virginalis, d Neomysis integer, 96:168–172. e Parhyale hawaiensis, f Sicyonia ingentis. All Muscle precursors shown in 13. Dobkin S: The larval development of Palaemonetes paludosus (Gibbes, Figure 7 are considered and mapped in the sequence they occur, as well 1950) (Decapoda, Palaemonidae), reared in the laboratory. Crustaceana as lmp-post, but not lmp-d. The color code from Figure 7 is used. For 1963, 61:41–61. lateral and medial extrinsic appendage muscle precursors the color 14. Dobkin S: Early developmental stages of the pink shrimp Penaeus specifies segment affiliation. Gross morphological features (N, PN, FS, IF) duorarum. Fish. bull 1961, 61:321–348. are added in the sequence they occur relative to the muscle precursors. 15. Gurney R: Larvae of decapod Crustacea. Discovery Reports 1936, 12:377–440. FS is marked by a bold vertical dotted line. Abbreviations: Gf (I-IV), Nh 16. Heegaard P: Larvae of decapod Crustacea - the Amphionidae. In The (I-V), Pf (I-IV), Ni (I-V), Ph (I-V) species- and semaphoront affiliation, N Carlsberg Foundations Oceanographical Expedition Round the World 1928–1930. appendage anlagen in nauplius segments present, PN appendage Edited by Heegard P. Copenhagen: Anrd. Fred. Høst & Søn; 1969:5–81. anlagen in postnaupliar segments present, FS full set of segment anlagen 17. Manning RB, Provenzano AJJ: Studies on development of stomatopod present, IF intersegmental furrows present in entire trunk, Crustacea 1. Early larval stages of Gonodactylus oerstedii Hansen. Intr intrinsic muscle precursors. Bull Mar Sci 1963, 13:468–487. 18. Provenzano J, Manning RB: Studies on development of stomatopod Crustacea 2. The later larval stages of Gonodactylus oerstedii Hansen Competing interests reared in the laboratory. Bull Mar Sci 1978, 28:297–315. The authors hereby declare that they have no competing interests. 19. Richter S, Scholtz G: Phylogenetic analysis of the Malacostraca (Crustacea). J Zool Syst Evol Res 2001, 39:113–136. Authors’ contributions 20. Weygoldt P: Die Embryonalentwicklung des Amphipoden Gammarus All three authors conceived the project and approved the final version of the pulex pulex (L). Zool Jahrb Abt Anat 1958, 77:51–110. manuscript. GJ carried out the immunohistochemistry, microscopy, 21. Scholl G: Embryologische Untersuchungen an Tanaidaceen (Heterotanais computational data processing, and drafted the manuscript. SR und CW oerstedi KroÈ yer). Zool Jahrb Abt Anat 1963, 80:500–554. critically revised the manuscript and collected material of G. falcatus. 22. Dohle W: Über die Bildung und Differenzierung des postnauplialen All authors read and approved the final manuscript. Keimstreifs von Leptochelia spec. (Crustacea, Tanaidacea). Zool Jahrb Abt Anat 1972, 89:505–566. Acknowledgements 23. Dohle W: Die Bildung und Differenzierung des postnauplialen Keimstreifs Work on P. fallax f. virginalis and collection of G. falcatus- material was von Diastylis rathkei (Crustacea): I. Die Bildung der Teloblasten und ihrer funded by the German science foundation (DFG grant Ri837/8-1 to SR, Derivate. Z. Morphol. Tiere 1970, 67:307–392. Wo1461/1-1 to CW). Individuals of P. hawaiensis were kindly provided by 24. Dohle W: Die Bildung und Differenzierung des postnauplialen Anastasios Pavlopoulos, MPI CBG Dresden. Furthermore we thank Jo Wolfe Keimstreifs von Diastylis rathkei (Crustacea, Cumacea): II. Die for giving helpful comments on the manuscript and improving the English. Differenzierung und Musterbildung des Ektoderms. Zoomorphologie Two anonymous reviewers are thanked for their fruitful comments on the 1976, 84:235–277. manuscript. 25. Manton SM: On the Embryology of a Mysid Crustacean, Hemimysis. Philos Trans R Soc Lond B Biol Sci 1928, 216:363–463. Author details 26. 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7 Jirikowski GJ, Wolff C, Richter S. 2015. Evolution of Eumalacostracan development – new insights into loss and reacquisition of larval stages revealed by heterochrony analysis. Evo Devo. 6:4.

97 98 Jirikowski et al. EvoDevo 2015, 6:4 http://www.evodevojournal.com/content/6/1/4

RESEARCH Open Access Evolution of eumalacostracan development —new insights into loss and reacquisition of larval stages revealed by heterochrony analysis Günther Joseph Jirikowski1*, Carsten Wolff2 and Stefan Richter1

Abstract Background: Within Malacostraca (Crustacea), direct development and development through diverse forms of larvae are found. Recent investigations suggest that larva-related developmental features have undergone heterochronic evolution in Malacostraca. In the light of current phylogenetic hypotheses, the free-swimming nauplius larva was lost in the lineage leading to Malacostraca and evolved convergently in the malacostracan groups Dendrobranchiata and Euphausiacea. Here we reconstruct the evolutionary history of eumalacostracan (Malacostraca without Phyllocarida) development with regard to early appendage morphogenesis, muscle and central nervous system development, and determine the heterochronic transformations involved in changes of ontogenetic mode. Results: Timing of 33 developmental events from the different tissues was analyzed for six eumalacostracan species (material for Euphausiacea was not available) and one outgroup, using a modified version of Parsimov-based genetic inference (PGi). Our results confirm previous suggestions that the event sequence of nauplius larva development is partly retained in embryogenesis of those species which do not develop such a larva. The ontogenetic mode involving a nauplius larva was likely replaced by direct development in the malacostracan stem lineage. Secondary evolution of the nauplius larva of Dendrobranchiata from this ancestral condition, involved only a very small number of heterochronies, despite the drastic change of life history. In the lineage leading to Peracarida, timing patterns of nauplius-related development were lost. Throughout eumalacostracan evolution, events related to epidermal and neural tissue development were clearly less affected by heterochrony than events related to muscle development. Conclusions: Weak integration between mesodermal and ectodermal development may have allowed timing in muscle formation to be altered independently of ectodermal development. We conclude that heterochrony in muscle development played a crucial role in evolutionary loss and secondary evolution of a nauplius larva in Malacostraca. Keywords: Malacostraca, Muscle development, Larval development, Nauplius Larva, Egg Nauplius, Heterochrony, Phylogeny

Background However, comparatively few investigations focus on het- Heterochrony—evolutionary change in timing of develop- erochrony in invertebrate evolution [12-16]. , mental events—is a central concept in understanding the such as the crustaceans (the potential paraphyly of crusta- diversity of animal form [1-3]. Investigations of sequence ceans has no impact to our study), display an enormous heterochrony have provided support for the important disparity in development, exemplified by the multitude of role of this mechanism in morphological evolution, e.g., in larval forms and life histories found throughout this the case of ossification timing in amphibians [4-6], snakes group. Here we set out to explore the impact of sequence [7], [8], mammals [9], internal organ development of heterochrony on life history evolution of the crustacean amniotes [10], or limb development of [11]. group Malacostraca (‘higher crustaceans’). Malacostraca represents a large and morphologically * Correspondence: [email protected] highly disparate taxon within crustaceans. Although 1Universität Rostock, Institut für Biowissenschaften, Allgemeine und Spezielle Zoologie, Universitätsplatz 2, 18055 Rostock, Germany malacostracans also have a rich fossil record, including Full list of author information is available at the end of the article larvae, we refer throughout our study to recent taxa only

© 2015 Jirikowski et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jirikowski et al. EvoDevo 2015, 6:4 Page 2 of 30 http://www.evodevojournal.com/content/6/1/4

because musculature and nervous system is hardly part of a recapitulated developmental program originally known for fossil larvae or embryos. The plesiomorphic involved in formation of a free-swimming nauplius larva. developmental mode generally accepted for crown- This egg nauplius concept has drawn our attention to the group Crustacea (or crown-group Tetraconata if crusta- question how exactly transitions between larval and em- ceans are paraphyletic in relation to Hexapoda) bryonic development are achieved in evolution. The comprises hatching of a free-swimming, planktonic larva presence of a larval developmental program in the mala- with conserved morphology, called nauplius [17-19]. costracan ground pattern can help to explain the secondary The nauplius larva (i.e., orthonauplius) bears three pairs (and potentially independent) origin of the dendrobranchi- of appendages (first antenna, second antenna, and man- ate and euphausiacean nauplius larva [59]. Though this egg dible), which are used for feeding and locomotion. In nauplius concept is of great value for understanding mala- Malacostraca, such a larva is found only in two groups costracan evolution, it does not sufficiently consider devel- (Figure 1): Dendrobranchiata and Euphausiacea [20-23]. opmental timing. For example, it treats the egg nauplius Moreover, despite controversies concerning the phylo- and the free nauplius larva as two alternative situations. genetic relationships within Malacostraca, Dendrobran- Yet nauplius larvae are themselves preceded by embryonic chiata and Euphausiacea are always placed at nested stages which show three pairs of appendage buds (Figure 2). positions within the tree [24-32]. In this light, the They differ from the egg nauplius stages of direct devel- nauplius larva in Malacostraca has evolved secondarily opers or species with zoea-like larvae, only by the lower from ancestors, which either showed direct development amount of yolk and the more lateral position of the limb or hatched as a more advanced larval stage with a higher buds [67]. We prefer a more inclusive definition of the number of segments (Figure 1). term ‘egg nauplius’ which applies also to all crustacean rep- A variety of malacostracan taxa show yet another larval resentatives with nauplius larvae. In our view, the egg nau- form that we call zoea-like larva (Figure 1). Contrary to plius represents a part of an ancestral developmental the nauplius larva, the zoea-like larva is characterized by a program which is shared between species with and without complete or nearly complete number of body segments, a a free-swimming nauplius larva, before two different paths motile trunk including a paddle-shaped telson or tail fan. can be taken in development: (i) development of postnau- Zoea-like larvae are found in stomatopods (pseudozoea, pliar tissues, leading to a larger number of functional seg- antizoea) [20,33-36], decapods (mysis, zoea)[37-43],bath- mentsathatching(Figure2a)or(ii)differentiationand ynellaceans (parazoea) [44-46], and euphausiaceans early functionality of the naupliar segments and hatching (calyptopis) [47-50]. Stomatopod larvae of the pseudozoea- of a nauplius larva (Figure 2b). type have functional pleopods that are used for swimming. The evolutionary scenario of recapitulated nauplius The antizoea larva of Lysiosquillidae (Stomatopoda) larva development in the egg nauplius [68] is largely based differs from the pseudozoea in that it swims using the on gross external morphology of epidermal limb buds. De- throracic appendages and lacks pleopods. Following the velopment of the nervous system or mesodermal tissue, phylogeny proposed by [51], the pseudozoea larva can be such as musculature, has not played an important role, considered the ancestral condition for Stomatopoda. and only one publication discusses neurogenesis of a The zoea-like larvae of Decapoda, Euphausiacea, and malacostracan species in an egg nauplius context [69]. Re- Bathynellacea bear at least one pair of functional thoraco- cently, we have found that anlagen of musculature develop pods, while the pleonal appendages are lacking. in the naupliar segments only after the egg nauplius stage Leptostraca, Anaspidacea (Syncarida), Thermosbaenacea, in several malacostracan representatives [67] (Figure 2a). and Peracarida show different kinds of direct (or pseudodir- The dissociated timing of mesodermal and ectodermal de- ect [12]) development and lack planktonic larvae (Figure 1) velopment suggests that retention of the ancestral larval [52-58]. Direct development also evolved several times developmental program does not occur in all tissues like- within the Decapoda, like the lineage leading to Astacidea wise and underwent heterochronic change in evolution. which changed to a freshwater environment [28]. Here we will apply a developmental sequence approach to Leptostraca, Stomatopoda, Caridea (Decapoda), Reptan- malacostracan development considering different tissue tia (Decapoda), Anaspidacea, Bathynellacea, and Thermo- types (epidermis, nervous tissue,andmuscle tissue)togain sbaenacea lack a nauplius larva but pass through a a more fundamental understanding of the evolutionary characteristic embryonic stage known as egg nauplius [59]. changes to developmental timing which caused loss and In the egg nauplius, the first antennal, second antennal, reacquisition of larvae in malacostracan evolution. and mandibular buds (naupliar appendage buds) appear Following Alberch [68], heterochrony affects only par- prior to the posterior (postnaupliar) appendage anlagen ticular features of the organism, never the whole. In the [44,52,53,60-66]. Timing of naupliar and postnaupliar ap- case of the egg nauplius, we want to determine the evolu- pendage bud formation is separated by a distinct gap. tionary changes of developmental timing in different body Scholtz [59] suggested that the egg nauplius is formed as regions and tissue types. Thus the relation between the Jirikowski et al. EvoDevo 2015, 6:4 Page 3 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 1 Overview of malacostracan phylogeny. Simplified representation of malacostracan phylogeny, following [28]. The major malacostracan monophyla suggested by these authors (Eumalacostraca, Caridoida, Decapoda, Pleocyemata, Xenommacarida, Peracarida) are marked with horizontal brackets. The taxa Anaspidacea and Bathynellacea are shown together as Syncarida. The peracaridan subtaxa , Spelaeogriphacea, Mictacea, Tanaidacea, and Cumacea are excluded. Therefore, Amphipoda and Isopoda appear as sister groups. The developmental mode of the taxa is indicated by symbolic drawings at the bottom. The developmental mode is color coded to the branches and the most parsimonious character states of the ancestral lineages are shown. Outgroups are not depicted. Color coding: Direct/pseudodirect development (black); nauplius larva as hatching stage (blue); zoea-like larva as hatching stage (green). modular organization of the developmental program and developmental sequence, in which only developmental heterochronic evolution in Malacostraca is at the heart of events of the naupliar segments occur (but none of the our study. A conservative view on malacostracan develop- postnaupliar segments), regardless of the respective tissue mental evolution would assume that all embryonic nau- type, as ‘egg nauplius phase.’ Transition of developmental pliar tissues develop in species without a nauplius larva by events from the egg nauplius phase to later positions in de- the same timing pattern as they would in nauplius larva- velopment, would prevent formation of a viable nauplius bearing species. In this case, in the malacostracan last com- larva. Such changes would have had to be reversed during mon ancestor, a developmental path would be taken that secondary evolution of the nauplius larva in Dendrobran- accelerates tissue development in the postnaupliar seg- chiata and Euphausiacea. ments relative to the naupliar segments after the egg nau- The morphological features to be investigated here in plius stage. We will refer to the initial part of the terms of developmental timing were chosen in a manner Jirikowski et al. EvoDevo 2015, 6:4 Page 4 of 30 http://www.evodevojournal.com/content/6/1/4

segments and the first trunk segment, the last trunk seg- ment, and the telson (Figure 3). This allows us to record heterochronic changes in patterning of the naupliar and postnaupliar segments and to draw conclusions on their relation to loss or gain of a nauplius larva. Also features without obvious segment affiliation but with relevance to the evolution of nauplius larva development are included, such as the anlage of the nauplius eye, muscle anlagen of the stomodeum, and hatching from the egg envelope. Other features are included which are potentially rele- vant for formation of a zoea-like larva and will serve to de- termine heterochronic changes that relate to this larval form: Formation of appendage buds, ganglion anlagen, and muscle precursors in the sixth pleonal segment, for- mation of a posterior longitudinal muscle precursor in the telson, and offset of segment formation. Offset of segment formation describes the point in development at which generation of body segments from the posterior growth zone terminates, and the full set of trunk segments is present as anlagen [67]. In Malacostraca, mesoderm and ectoderm of the trunk segments are formed by repeated asymmetric cell divisions of stem-like cells, the mesotelo- blasts and ectoteloblasts, which are located in the growth zone in the posterior part of the embryo and can be recog- nized by the specific arrangement of stained nuclei. We use the mesodermal segment anlagen as reference for over all body segmentation here. Timing of the first appearance (onset) of the specified features is recorded for six eumalacostracan representa- tives and one outgroup: Gonodactylaceus falcatus (FORSKÅL, 1775) (Stomatopoda), Sicyonia ingentis (BURKENROAD, 1938) (Dendrobranchiata), Neocaridina heteropoda (KEMP, 1918) (Caridea), Procambarus fallax forma virginalis (Astacidea), Neomysis integer (LEACH, 1814) (Mysidacea), Parhyale hawaiensis (DANA, 1853) (Amphipoda), and Artemia franciscana (KELLOGG, 1906) (Anostraca). G. falcatus hatches as a zoea-like larva (pseu- dozoea), S. ingentis hatches as nauplius larva, while the remaining species develop directly. A decapod representa- Figure 2 Simple examples of malacostracan life histories. tive that hatches as a zoea-like larva was not available. Schematic representation of two Malacostracan life histories both involving a zoea-like larva. (a) Left: The larva is formed in embryogenesis, However, the late embryonic stages of N. heteropoda differ an egg nauplius stage is succeeded by an advanced embryonic stage only little in morphology from other caridean zoea larvae. with postnaupliar segments developed. (b) Right: The zoea-like larva is Thus, in terms of developmental timing, N. heteropoda formed after preceding larval stages. An egg nauplius stage is traversed can be considered a legitimate representative of zoea-bear- in both cases (a and b). Naupliar musculature develops in the egg ing decapods. A representative of Euphausiacea could not nauplius if a nauplius larva follows. Appendage buds are labeled only in the upper left egg nauplius drawing. be sampled for this study, because our methods demand fresh or appropriately fixed material and these animals are difficult to obtain. Thus we focus on the lineage leading to that allows comparison between tissue types, as well as be- Dendrobranchiata to infer heterochrony related to evolu- tween the germ layers ectoderm and mesoderm. Also, we tion of a nauplius larva. N. integer differs from most other rely on a large number of segmentally repeated features, Peracarida in that it shows pseudodirect development. In namely appendage buds, ganglion anlagen, and muscle this species, hatching occurs early in development, but the precursors to allow detection of timing differences also be- inert larva (nauplioid) remains in the brood pouch until tween segments. Such features are recorded for the head juvenile morphology is established. Jirikowski et al. EvoDevo 2015, 6:4 Page 5 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 3 Overview of investigated morphological features. Schematic overview of malacostracan embryo or larva. The left hemisegments are shown in ventral view and hemisegments from the second thoracomere to the fifth pleomeres are excluded. A vertical dotted line at the left marks the longitudinal body axis. Anlagen of appendages, muscle precursor groups, and primordial elements of the nervous system are outlined and color-coded. The naupliar segments (bearing first antenna, second antenna, and mandible) and postnaupliar segments (all segments posterior to the mandible-bearing segment) are indicated by brackets on the right. Abbreviations: Ol optic lobe; pc protocerebral ganglion anlage; dc deutocerebral ganglion anlage; tc tritocerebral ganglion anlage; NGA naupliar ganglion anlagen; NEA nauplius eye anlage; PPN posterior pioneer neurons; A1-P6 appendage anlagen of the first antennal to the sixth pleonal hemisegments; T2-P5 excluded trunk segments; st stomodeal muscle precursor group, m medial extrinsic appendage muscle precursor groups; l lateral extrinsic appendage muscle precursor groups; lmp-post posterior longitudinal muscle precursor strand.

Based on the timing data, we apply a dynamic program- ming approach using the software Parsimov-based genetic inference (PGi) to trace evolution of the developmental sequences. The method used was first introduced by Harrisson & Larsson [70] and applied successfully in ana- lyses of heterochrony since [4,8,71]. We have chosen the malacostracan phylogeny proposed by [28] and [72] as framework for the reconstruction of developmental evolu- tion. It is in our view still the best supported one. We are aware that other suggested phylogenies [31] would give different results. The reconstruction of the ancestral devel- opmental sequences of Eumalacostraca, Caridoida, Deca- poda, Pleocyemata, and Peracarida, and inference of the heterochronic events that occurred along the different branches, will allow us to shed light on the evolutionary transformations of the segment and tissue-specific devel- opmental processes that were involved in alteration of the developmental mode. The following questions will be addressed (all taxon names refer to the respective crown-groups):

 How did the last common ancestor (LCA) of Eumalacostraca develop and to what degree did it show larval developmental patterns?  Which heterochronies were involved in evolution of the nauplius larva of Dendrobranchiata?  Which changes of the developmental sequence caused the emergence of zoea-like larval forms?  In which way were developmental sequences altered in the lineage leading to Peracarida?

Methods Specimen preparation, staining, and imaging procedure Collection of embryo and larva material, fixation, fluores- cent staining, and confocal microscopy followed by 3D image processing was performed in a previous investiga- tion [67]. The respective methodology applied to P. fallax Jirikowski et al. EvoDevo 2015, 6:4 Page 6 of 30 http://www.evodevojournal.com/content/6/1/4

forma virginalis is described in [73]. Visualization of cases where data could not be provided by our own investi- muscle tissue was performed on larval stages L4, L6, and gations, events were coded using the literature. An over- L9 [74] of A. franciscana. The fixation protocol was previ- view of these events and a list of the publications used are ously described in [75]. Larvae of A. franciscana were in- given in Table 2. A detailed overview of semaphoronts and cubated with Phalloidin-ALEXA 561 overnight to visualize events is given in Additional file 1. Early appendage mor- muscle tissue by f-actin labeling. Imaris software Version phogenesis of Astacidea and Amphipoda was coded from 6.1 (Bitplane AG) was used to adjust image quality in pro- [80,81], early neurogenic events for Stomatopoda, Astaci- jections of the volume data and to reconstruct and high- dea, Amphipoda, and Artemia sp. were coded from light single muscle precursors. Confocal image stacks of [82-84], and early myogenic events for Dendrobranchiata S. ingentis embryos and nauplius larvae labeled with and Artemia sp. were coded from [76,84]. Features which BODIPY-FL-phallacidin were kindly provided by Phillip were not recorded to be formed in development but are re- Hertzler for detection of nervous system development ported to be present in adults were coded as events at the [76]. Embryos of 13h, 15h, 17h, and 20h after end of the sequence. This is an effort to avoid missing data fertilization were analyzed, as well as nauplius stages 1 where possible, even if this results in artificial simultaneity and 4. Immunohistochemical labeling of developing ner- between late events. This was the case for developmental vous tissue was performed on G. falcatus, N. heteropoda, sequences of all groups [85-91], except for Artemia sp. P. fallax forma virginalis,andN. integer by application of Following the position of the events in the sequence, ranks an antibody against anti-acetylated α-tubulin (clone 6–11 were assigned to every event (Table 3). An overview of the B-1, Sigma T6793) which labels neurites, even at early de- event series for all species, also showing stage specifications velopmental stages. For this the same preparation and from the literature, rank values, and literature sources used staining, protocols as for muscle precursor labeling were for specific events, is provided in Additional file 1. Ranks used. Also histochemical staining with phalloidin- ranged from 9 to 11, depending on the total number of ALEXA488 (Molecular Probes, A12379) was applied to semaphoronts described for each species. visualize developing ganglion anlagen of early embry- The table gives a list of all events coded for comparative onic stages. Graphics were drawn and image tables were analysis of malacostracan development. The events specify assembled using CorelDRAW Graphics Suite X3 (Corel the first appearance of a specific morphological structure Corporation, Ottawa). (e.g.) appendage primordium or property (e.g.) hatched larva. Muscle precursor terminology was adapted from Developmental sequence data [58] for myogenic events. Abbreviations of muscle precur- The ontogeny of an individual organism can be viewed as sor groups are given in italics. The morphological features an array of semaphoronts [77]. Hennig’s concept of the are sorted by the following categories: Epidermal append- semaphoront has recently been revived to improve age development, segmentation, myogenesis, neurogenesis, morphology-based phylogenetic inference on Pancrusta- and Hatching. Events are numbered from 1–33 and this cea/Tetraconata [78]. A semaphoront, in the sense of order is maintained for the analysis. Descriptions are given Hennig is ‘[…] the individual at a certain, theoretically in- for every event, as well as abbreviations. Abbreviations of finitely small, period of its life’ [77, p6]. Sequences of devel- developmental events are given in brackets and used con- opmental events always refer to series of semaphoronts. sistently throughout the paper. We will speak of semaphoronts instead of developmental stages throughout this paper, because (i) staging systems Heterochrony analysis are not established for all of the species we investigate and Ranks were coded as character states for the respective (ii) staging systems rely on specific criteria that limit their events in a matrix of 7 × 33 cells and exported as a resolution. For the present work however, we must allow NEXUS file (Additional file 2) together with the phylogeny timing to be recorded even within stages and based on from [28], using the open source software package new criteria, thus defining new operational stages that we Mesquite 2.75 [92]. The tree was simplified by excluding refer to as semaphoronts. Developmental event sequences all taxa that are not represented in our sampling. We use [79] were recorded for each species from the semaphoront A. franciscana as an outgroup. Since a free-swimming series. Every event refers to the first appearance of a nauplius larva is found throughout the ‘entomostracan’ morphological feature (listed in Table 1). We restrict the crustaceans, it must also have been present in the linage present investigation to such ‘onset events’ to limit the size leading to the crown-group Malacostraca. We added the of the data set. The majority of events in our data set were developmental sequence of A. franciscana twice to the coded from our previously published comparative study of matrix to allow optimization of this condition in the ana- malacostracan muscle development [67]. A substantial part lysis. Analysis of heterochrony was performed using a of the event data was acquired from new observations pre- modified version of PGi [69], kindly provided by Luke sentedintheresultssection(Figures4,5,6,7,8,9).In Harrisson. The method uses a dynamic programming Jirikowski et al. EvoDevo 2015, 6:4 Page 7 of 30 http://www.evodevojournal.com/content/6/1/4

Table 1 Overview of events and event abbreviations Event group Event Information Abbreviation Epidermal appendage development 1 Anlage of the first and second antenna present. [A1/A2] 2 Anlage of the mandible present. [Md] 3 Anlage of the first maxilla present. [Mx1] 4 Anlage of second maxilla present. [Mx2] 5 Anlage of the first thoracopod present. [T1] 6 Anlage of the sixth pleopod present. [P6] Segmentation 7 All mesodermal segment anlagen present. [FS] Myogenesis 8 Stomodeal muscle precursor group present. [st] 9 Medial extrinsic appendage muscle precursor of first antenna present. [a1-m] 10 Lateral extrinsic appendage muscle precursor of second antenna present. [a1-l] 11 Medial extrinsic appendage muscle precursor of second antenna present. [a2-m] 12 Lateral extrinsic appendage muscle precursor of second antenna present. [a2-l] 13 Medial extrinsic appendage muscle precursor of mandible present. [md-m] 14 Lateral extrinsic appendage muscle precursor of mandible present. [md-l] 15 Medial extrinsic appendage muscle precursor of first maxilla present. [mx1-m] 16 Lateral extrinsic appendage muscle precursor of first maxilla present. [mx1-l] 17 Longitudinal muscle precursor in first maxilla segment present. [lmp-mx1] 18 Medial extrinsic appendage muscle precursor of second maxilla present. [mx2-m] 19 Lateral extrinsic appendage muscle precursor of second maxilla present. [mx2-l] 20 Longitudinal muscle precursor in second maxilla segment present. [lmp-mx2] 21 Lateral extrinsic appendage muscle precursor of first thoracopod present. [t1-m] 22 Medial extrinsic appendage muscle precursor of first thoracopod present. [t1-l] 23 Longitudinal muscle precursor in first thoracopod segment present. [lmp-t1] 24 Longitudinal muscle precursor in sixth pleopod segment present. [lmp-p6] 25 Posterior longitudinal muscle primordium present. [lmp-post] Neurogenesis 26 Anlagen of naupliar ganglia present. [NGA] 27 Anlagen of first maxilla ganglion present. [mx1-g] 28 Anlagen of second maxilla ganglion present. [mx2-g] 29 Anlagen of first thoracopod ganglion present. [t1-g] 30 Anlagen of sixth pleopod ganglion present. [p6-g] 31 Anlage of the nauplius eye present. [NEA] 32 Posterior pioneer neurons with anterior longitudinal neurite bundles present. [PPN] Hatching 33 Larva or Juvenile hatches from the egg membrane. End of embryogenesis. [HAT] approach which treats the event sequence as a single com- ancestral developmental sequences to be retained at each plex character. Therefore, it avoids the assumption of node. For each run, the most parsimonious solutions of event independence which is inherent to event pair-based equal cost are collected by the algorithm and used to cal- methodology of heterochrony analysis [93]. PGi uses a culate a pseudoconsensus tree. Heterochronies that occur simplified genetic algorithm-based heuristic on the event in the equally parsimonious solutions are included in the sequence, and Parsimov event pairing [94] is used as edit pseudoconsensus tree if they fulfill the 50% majority rule cost function. The program runs in the ape package [95] criterion and the percentage of each heterochrony is given and was carried out using the open source statistics envir- as bootstrap support [69]. The pseudoconsensus method onment ‘R’ (version 3.0.1) [96]. We performed three runs was set to ‘semi exhaustive’ and the limit of evaluated solu- with the following parameters for the PGi simplified gen- tions of equal score was set to 3,000. The pseudoconsen- etic algorithm: 100 cycles of selection per node, 200 se- sus trees of the three independent runs were combined to quences per cycle of selection, and a maximum of 100 a superconsensus tree (Additional file 3). The ancestral Jirikowski et al. EvoDevo 2015, 6:4 Page 8 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 4 Investigated morphological features of different semaphoronts of G. falcatus (Stomatopoda) and S. ingentis (Dendrobranchiata). External morphology and developing nervous system of G. falcatus (a–d) and developing nervous system/muscular tissue of S. ingentis (e–h). (a) Semaphoront Gf EN1, showing naupliar appendage buds. (b) Same semaphoront, showing acetylated alpha-tubulin signal. Ganglion anlagen are not yet visible. (c) Semaphoront Gf V, ventral view of complete embryo, showing anlagen of all appendages. (d) Same semaphoront, developing pleon and telson anlage. Ganglion anlagen are not developed posterior to the fourth pleomeres. Continuous longitudinal neurite bundles extend into the telson. (e) S. ingentis semaphoront Si En 17. Anlagen of naupliar ganglia are present in the embryo. (f) semaphoront Si N3. The larva shows four pairs of postnaupliar appendage buds. (g) Semaphoront Si N4. Ganglion anlagen are present in the first and second maxilla segment. (g) Semaphoront Si N5. Ganglion anlagen of the first and second thoracic segments are present. (a, c) Blend projection of nuclear signal (TOPRO-3) from confocal image stack; (b) maximum intensity projection of acetylated alpha-tubulin signal; (d) extended section of acetylated alpha-tubulin (red)- and TOPRO-3-signal (blue); (e–h) extended sections of phallacidin-BODIPY-labeled specimens, with permission of Phil Hertzler. Abbreviations: BA brain anlage, Ol optic lobes, A1–P6 appendage anlagen of respective segments, T telson anlage, pc protocerebrum, dc deutocerebrum, tc tritocerebrum, md-g–P6-g ganglion anlagen of respective segments, lonb longitudinal neurite bundles, md-m medial extrinsic mandible muscle precursor, lmp longitudinal muscle precursor, P proctodeum, CP caudal papilla. Scale bars 50 μmin(a, b, e–h); 200 μmin(c), 100 μmin(d). Jirikowski et al. EvoDevo 2015, 6:4 Page 9 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 5 (See legend on next page.) Jirikowski et al. EvoDevo 2015, 6:4 Page 10 of 30 http://www.evodevojournal.com/content/6/1/4

(See figure on previous page.) Figure 5 Investigated morphological features of different semaphoronts of N. heteropoda (Pleocyemata). External morphology and developing nervous system of N. heteropoda. (a) Semaphoront Nh EN, showing naupliar appendage buds, but no ganglion anlagen. (b) Semaphoront Nh I+ showing postnaupliar appendage anlagen Mx1–T1. (c) Same semaphoront, showing naupliar ganglia and longitudinal neurite bundles. (d) Higher magnification image of the specimen shown in (c). Anlagen of the protocerebrum, deutocerebrum, tritocerebrum, mandible ganglion and nauplius eye are visible. (e) Semaphoront Nh II showing postnaupliar appendage anlagen T3 and additional posterior segment anlagen. (f) Same semaphoront showing postnaupliar ganglion anlagen from the first maxillary to the second thoracic segment. (g) Semaphoront Nh III showing the full set of segment anlagen. Pereiopod anlagen are present. (h) Same semaphoront, higher magnification of posterior pleon and telson anlage. No ganglion anlagen are observed in the pleonal segments, but continuous longitudinal neurite bundles extend anteriorly from the posterior pioneer neurons. (i) Semaphoront Nh IV, posterior pleon and telson anlage. Ganglion anlagen with extending lateral nerves are observed in all pleomeres, as well as a seventh pleonal ganglion anlage. (a, c, d, f, h, i) Maximum intensity projections of (a, f) phalloidin-signal (green), and (c, d, h, i) acetylated alpha-tubulin signal (red). In (h) and (i), nuclei are shown in blue. Intersegmental furrows are demarcated by dotted lines. (b) Blend projection, (e) and (i) normal shading projection of nuclear signal (TOPRO-3) shown in cyan. Abbreviations: Ol optic lobes, A1–P6 appendage anlagen of respective segments, T telson anlage, pc protocerebrum, dc deutocerebrum, tc tritocerebrum, md-g–P6-g ganglion anlagen of respective segments, lonb longitudinal neurite bundles, NEA nauplius eye anlage, NEN nauplius eye nerve, lmp longitudinal muscle precursor, P proctodeum, CP caudal papilla. Scale bars 100 μmin(a–c, e, f, h, i);25μmin(d);500μmin(g). sequences are constructed by PGi from mean ranks that observations presented here. Table 2 gives an overview of are calculated from the multiple equally parsimonious the literature sources used and of the events coded from solutions of the pseudoconsensus trees [69]. Because of the them. The new observations are depicted in Figures 4, 5, high variation in the data set, the reconstructed ancestral 6, 7, 8, 9. Furthermore, specifications of literature sources sequences can show slight differences in event position and reference to the corresponding figures depicting the that are not given as heterochronies by the analysis. new observations in the present work are shown for each Calculations were carried out on a Dell Optiplex790—- event in Additional file 1. Information on development of computer with an i3-2100 [email protected] and 8GB RAM, other species is used in several cases to complete the running 64Bit Microsoft Windows 7. Graphic representa- semaphoront sequence. In Additional file 1, these species tions of the superconsensus tree and transformation of are also shown for the respective events. developmental sequences were edited using CorelDRAW Epidermal morphogenetic events (1–6): The events re- Graphic Suite X3. corded for epidermal morphogenesis represent the ap- For all events, heterochrony rates were calculated. The pearance of distinct appendage buds of the first antenna, heterochrony rate of an event in our case represents the second antenna, mandible, first maxilla, second maxilla, number of heterochronic changes recovered by PGi for that first thoracopod, and sixth pleopod. An event is scored event in the superconsensus tree, multiplied by its mean when the appendage anlage is recognizable as protuber- bootstrap value. Tissue-specific heterochrony rates were ance in the epidermal layer. Formation of first and second calculated which represent the mean heterochrony rate per antenna is scored as a single (event 1) [A1/A2] because event for all events specific to epidermis, neural tissue, or they are always observed to occur simultaneously. muscle tissue development. These values were also used to Myogenic events (8–25): First appearance of the muscle determine mean heterochrony rates for the germ layers precursors described in [67] is recorded here as myogenic ectoderm and mesoderm. Likewise heterochrony rates were events. For the present investigation, we reduced the total compared between segments and tabulated. For this, events number of muscle precursors by combining some precur- with problematic segment affiliation (FS, st,NEA,and sors to groups (Table 1): The stomodeal muscle precursors HAT) were excluded. Two events represent combinations are combined to one group (event 8). The same applies to of several segment-specific events ([A1/A2], [NGA]). In the medial extrinsic appendage muscles of the first and these cases, the heterochrony rate of the combined event is second antenna, mandible, first and second maxilla, and used for each of the single segments, because simultaneity first thoracopod, respectively (events 9, 11, 13, 15, 18, 21), is observed in each of the investigated species and can thus as well as the lateral extrinsic appendage muscles of the be assumed also for the ancestral sequences. Where mul- same body segments (events 10, 12, 14, 16, 19, 22). First tiple events are affiliated with the same segment (myogenic appearance of a group is registered as a developmental events), the mean heterochrony rate of these events is used. event, if any of the muscle precursors of one group is seen. Furthermore, the first appearance of longitudinal muscle Results precursors in the first maxilla, the second maxilla, and the Recorded events first thoracopod segment (events 17, 20, 23) are recorded, Event sequences were assembled for A. franciscana, but longitudinal muscle precursors of the mandible G. falcatus, S. ingentis, N. heteropoda, P. fallax forma segment are excluded, as they occur only transiently in virginalis, N. integer,andP. hawaiensis by combination of G. falcatus and P. fallax forma virginalis. Longitudinal our previous descriptions, literature data, and new muscle precursors of the sixth pleomeres (event 24) are Jirikowski et al. EvoDevo 2015, 6:4 Page 11 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 6 Investigated morphological features of different semaphoronts of P. fallax forma virginalis (Pleocyemata). Developing nervous system of P. fallax forma virginalis. (a) Semaphoront Pf I, ventral view of embryo. The naupliar ganglia are present, as well as posterior pioneer neurons [NGA], [PPN]. Appendage anlagen of the first antennal to the second thoracic segments are present. (b) Semaphoront Pf III, pleon and telson anlage. Ganglion anlagen are observed in none of the pleonal segments, but continuous longitudinal neurite bundles extend anteriorly from the posterior pioneer neurons. (c) Semaphoront Pf As8, posterior pleon and telson anlage. Developing ganglia are observed in all pleomeres, including the sixth pleomere [p6-g]. All panels show maximum intensity projections of acetylated alpha-tubulin signal (red) and nuclear signal (TOPRO-3), (blue), from confocal image stacks. In (a–c), appendage anlagen are demarcated with dotted lines. Abbreviations: Ol optic lobes, A1- P6 appendage anlagen of respective segments, T telson anlage, pc protocerebrum, dc deutocerebrum, tc tritocerebrum, md-g–p6-g ganglion anlagen of respective segments, lonb longitudinal neurite bundles, CP caudal papilla. Scale bars 100 μm in all panels. recorded. They are recognizable as metameric, non- Formation of the naupliar ganglia (protocerebrum, deuto- continuous muscle precursors in the sixth pleopod cerebrum, tritocerebrum, mandibular ganglion) are scored segment. The posterior longitudinal muscle primordium as a single event [NGA] because they are always observed (event 25) represents a portion of the longitudinal muscle to occur simultaneously. Developing ganglia are observed strand that extends posteriorly into the growth zone and also posterior to the sixth pleomeres, e.g., a seventh pleonal telson anlage. This event is lacking in Peracarida and is ganglion in N. heteropoda or N. integer. For our purpose, coded as absent for N. integer and P. hawaiensis. we will record only the emergence of the sixth pleonal neu- Neurogenetic events (26–32): Data that are lacking in romere. Furthermore, we record the presence of the anlage the published material for G. falcatus, P. fallax forma vir- of the nauplius eye, a feature commonly present in crust- ginalis, as well as data on neurogenesis of N. heteropoda, acean nauplius larvae (event 31), and of posterior pioneer N. integer,andP. hawaiensis were obtained by the meth- neurons (event 32). odology described above. We specified eight events for de- Overall development and segmentation (7, 33): We velopment of the nervous system (Table 1), six of which specified two features which are relevant for over all seg- relate to the first appearance of ganglion anlagen. ment formation and differentiation: Offset of segment for- Ganglion anlagen are defined here as metameric cellular mation (event 7), recognizable by the presence of arrangements in the neuroectoderm, with a developing mesodermal segment anlagen of all thoracic and pleonal central neuropile [97]. Developing nerve fibers of commis- segments, and hatching from the egg membrane, which is sures, connectives and lateral nerves can be present. Gan- at the same time the end of embryogenesis, is coded glion anlagen which are preceded by longitudinal neurite (event 33). bundles originating from the posterior pioneer neurons are recognizable as spindle-shaped regions formed by Description of developmental sequences these longitudinal fibers as shown by [82]. We specified For convenience, we use specific font style for event and the first appearance of ganglion anlagen in the naupliar, semaphoront abbreviations throughout this paper. Sema- the first maxillary, second maxillary, first thoracic segment phoront abbreviations are given in bold letters and contain (events 26–29), and in the sixth pleomere (event 30). a two-letter code for the species name, followed by a Jirikowski et al. EvoDevo 2015, 6:4 Page 12 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 7 Investigated morphological features of different semaphoronts of N. integer (Peracarida). External morphology and developing nervous system of N. integer. (a) Semaphoront Ni EN, showing buds of the first and second antenna [A1/A2], but not the mandible. (b) Semaphoront Ni Naup-d showing mandible buds and postnaupliar appendage anlagen from the first maxilla to the fifth thoracic segment [Md], [Mx1], [Mx2], [T1]. (c) Semaphoront Ni I. The full set of segment anlagen is present, including the sixth pleomere. (e) Magnified lateral view of same semaphoront showing the posterior trunk and telson anlage. Appendage buds are visible down to the second pleomere. (d) Same semaphoront, showing phalloidin signal in naupliar ganglia [NGA]. (f) Semaphoront Ni II. Faint ganglion anlagen are visible also in the first and second maxillary segment [mx1-g], [mx2-g ]. (g) Semaphoront Ni III, with ganglion anlagen observable also from the first thoracomere [t1-g] down to the seventh thoracomere. The more anterior neuromeres show extensive formation of nerve fibers. The phalloidin signal also shows developing musculature. (a, e) blend projection, (b, c) normal shading projection of nuclear signal (TOPRO-3) from confocal image stacks (cyan). (d, f) Maximum intensity projections of phalloidin-signal (green). (g) Maximum intensity projections of phalloidin-signal (green), acetylated alpha-tubulin signal (red), and nuclear signal (TOPRO-3), (blue). Abbreviations: Ol optic lobes, A1–P6 appendage anlagen of respective segments, T telson anlage, pc protocerebrum, dc deutocerebrum, tc tritocerebrum, md-g–p6-g ganglion anlagen of respective segments, lonb longitudinal neurite bundles, lmp longitudinal muscle precursor, CP caudal papilla. Scale bars 100 μm in all panels. roman number specifying the position of this semaphor- brackets. The abbreviations for ontogenetic events are ont in the sequence, or by an abbreviation of an given in square brackets throughout this paper. In the established stage name. The semaphoront abbreviations following section, the coded semaphoronts of the investi- are adapted from [67]. For hatching individuals, the abbre- gated species are described for G. falcatus, S. ingentis, viation ‘HAT’ is used instead of the roman number. N. heteropoda, P. fallax forma virginalis, N. integer, Abbreviations that refer to specific developmental stages P. hawaiensis,andA. franciscana, together with the that were coded from the literature are given in italics in respective developmental events. Jirikowski et al. EvoDevo 2015, 6:4 Page 13 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 8 Investigated morphological features of different semaphoronts of N. integer and P. hawaiensis (Peracarida). Developing nervous system of N. integer and P. hawaiensis. (a) Semaphoront Ni IV. Ganglion anlagen are observable down to the first pleomeres. (b) Semaphoront Ni V. Ganglion anlagen and extending lateral nerves are observable in all pleomeres, including the sixth pleomeres [p6-g]. Also an additional seventh pleonal ganglion is seen. (c) Semaphoront Ph I. Overview of an entire embryo. Appendage buds are present in all segments. Ganglion anlagen are observable down to the third pleomere. (d) Semaphoront Ph II. Developing pleon. Faint ganglion anlagen are visible in the sixth pleomere [p6-g]. (e) Semaphoront Ph III. Ganglia of the pleonal segments are enlarged and show developing commissures. (a, b) N. integer, maximum intensity projections of acetylated alpha-tubulin signal (red) and nuclear signal (TOPRO-3), (blue). Dotted lines demarcate appendage anlagen. (c) P. hawaiensis, maximum intensity projection of phalloidin signal (green) and nuclear signal (TOPRO-3), (blue). (d, e) P. hawaiensis, extended confocal sections generated by the same staining procedure. Abbreviations: A1–P6 appendage anlagen of respective segments, T telson anlage, md-g–p6-g ganglion anlagen of respective segments, lmp longitudinal muscle precursor. Scale bars 100 in (a–c),50μmin(d) and (e).

Gonodactylaceus falcatus: The first semaphoront Gf bundles are not yet seen, we did not assign [PPN] to this EN1 (Figure 4a) shows small buds of the first antenna, stage. Gf EN2 possesses an enlarged caudal papilla, as well second antenna, and mandible [A1], [A2], [Md]. Acety- as anlagen of the protocerebral, deutocerebral, tritocereb- lated α-tubulin-immunohistochemical labeling shows a ral, and mandibular ganglia, anlagen of the nauplius eye, signal scattered throughout the embryo, but it cannot be and the posterior pioneer neurons [NGA], [NEA], [PPN] specifically assigned to ganglion anlagen (Figure 4b). The (Additional file 1). Gf I shows the appendage bud of the terminal cellular processes of the posterior pioneer neu- first maxilla [Mx1]. In Gf II, the second maxillary and first rons are detectable, but since the longitudinal neurite thoracic appendage buds appear [Mx2], [T1], as well as Jirikowski et al. EvoDevo 2015, 6:4 Page 14 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 9 Investigated morphological features of different semaphoronts of A. franciscana (Branchiopoda). External morphology and developing musculature of A. franciscana larvae. (a) Overview of semaphoront Af II. Postnaupliar muscle precursors are not present, except for visceral musculature of the gut and proctodeum. (b) Higher magnification image of the region demarcated by a white rectangle in (a). Appendage anlagen are seen in the first [T1] and second thoracic segment, but not in the first and second maxilla segment (demarcated with a bracket). (c) Semaphoront Af III. Appendage buds become visible in the first and second maxillary segment. Longitudinal muscle precursors extend from the first maxilla segment into the anterior thoracomeres [lmp-mx1], [lmp-mx2], [lmp-t1]. Medial and lateral extrinsic appendage muscle precursors appear in the first maxilla segment and the first thoracopod segment [mx1-m], [mx1-l], [t1-m], [t1-l]. (d) Semaphoront Af IV. A lateral extrinsic muscle precursor appears in the second maxillary segment [mx2-l]. (e) Semaphoront Af V. All muscle precursor groups show advanced differentiation and a medial extrinsic muscle precursor of the second maxilla has appeared [mx2-m]. (a,c,d)Maximum intensity projection of phalloidin-signal (red) and nuclear signal (TOPRO-3), (blue). In (a),also autofluorescence signal of the cuticle is shown (green). (b) Blend projection of phalloidin-signal (orange) and nuclear signal (TOPRO-3), (light blue) and cuticle autofluorescence signal (green). (e) Blend projection of phalloidin-signal (light grey). In (c, d, e), extrinsic appendage muscle precursors are reconstructed and highlighted. Medial muscle precursors are shown in yellow and orange, lateral extrinsic precursors are shown in blue and light blue. Abbreviations: A1–T4 appendage anlagen of respective segments, lmp-mx1, lmp-mx2, lmp-t1 longitudinal muscle precursors of the respective segments; md-m, mx1-m, mx2-m, t1-m medial extrinsic appendage muscle precursors of the respective segments, md-l, mx1-l,mx2-l, t1-l medial extrinsic appendage muscle precursors of the respective segments. Scale bars are 100 μminallpanels.

the muscle precursors [st], [a2-l], [md-l], [lmp-t1]and assigned to a novel semaphoront Gf VI. Formation of a [lmp-post]. In Gf III, the first postnaupliar ganglion an- lateral extrinsic appendage muscle precursor [a1-l], extrin- lagen are present, namely in the first and second maxilla sic appendage muscle precursors of the first maxilla and segment [mx1-g], [mx2-g]. Furthermore, muscle precur- first thoracopod [mx1-I], [t1-l], medial extrinsic muscle sors [a1-m], [md-m], [mx2-l], [lmp-mx2] arise and offset precursors of the second maxilla, and the first thoracopod of segmentation [FS] is recorded. Gf P2 represents an [mx2-m], [t1-m] could not be observed during develop- intermediate stage between Gf III and Gf IV and is char- ment. Since these features are reported for the adult acterized by the appearance of a ganglion anlage in the (Table 2, Additional file 1), we assign them to a final first thoracopod segment [t1-g]. In Gf IV, the appendage semaphoront Gf HAT, together with the hatching event bud of the sixth pleopod arises [P6], as well as the muscle [HAT]. precursors [a2-m], [lmp-mx1]and[lmp-p6]. External Sicyonia ingentis: The initial semaphoront Si EN13 rep- morphology of Gf V is shown in Figure 4c. Developing resents the embryo at 13hpf (13 h post fertilization) which nervous tissue of the pleon is shown in Figure 4d. [p6-g] is shows distinct buds of the first antenna, the second Jirikowski et al. EvoDevo 2015, 6:4 Page 15 of 30 http://www.evodevojournal.com/content/6/1/4

Table 2 Overview of event data taken from literature Taxon Literature Literature Event abbreviation (same species) (related species) Artemia sp. [75][84] [NGA], [mx1-g], [mx2-g], [t1-g], [NEA], [PPN] [84] [A1/A2], [Md], [st], [a1-m], [a1-l], [a2-m], [a2-l], [md-m], [md-l], [NGA] Stomatopoda [67] [Mx1], [Mx2], [T1], [P6], [FS], [st], [a1-m], [a2-m], [a2-l], [md-m], [md-l], [mx1-m], [lmp-mx1], [mx2-l], [lmp-mx2], [lmp-t1], [lmp-p6], [lmp-post], [HAT] [82] [NGA], [mx1-g], [mx2-g], [t1-g], [NEA], [PPN], [85][a1-l], [mx1-I], [mx2-m], [t1-m][t1-l], [60] [p6-g] Dendrobranchiata - - [76] [A1/ A2], [Md], [st], [a1-m], [a1-l], [a2-m], [a2-l], [md-m], [md-l], [mx1-m], [mx1-l], [mx2-m], [mx2-l], [t1-l], [t1-m], [HAT] [86] [P6], [FS], [lmp-p6] Caridea [67] [A1/A2], [Md], [Mx1], [Mx1], [Mx2], [T1], [P6], [FS], [st], [a1-m], [a2-m], [a2-l], [md-m], [md-l], [mx1-m], [mx1-l], [lmp-mx1], [mx2-m], [mx2-l], [lmp-mx2], [t1-m], [t1-l], [lmp-t1], [lmp-p6], [lmp-post], [HAT] [87][a1-l] Astacidea [67] [FS], [st], [a2-l], [md-m], [md-l], [mx1-m], [mx1-l], [lmp-mx1], [mx2-m], [mx2-l], [lmp-mx2], [t1-m], [t1-l], [lmp-t1], [lmp-p6], [lmp-post], [HAT] [69] [A1], [A2], [Md], [Mx1], [Mx2], [T1], [P6] [80] [NGA], [Mx1-g], [Mx2-g], [t1-g] [88][a1-m], [a1-l], [a2-m] Mysidacea [67] [A1/ A2], [Md], [Mx1], [Mx1], [Mx2], [T1], [FS], [st], [a2-m], [a2-l], [md-m], [md-l], [mx1-m], [mx1-l], [lmp-mx1], [mx2-m], [mx2-l], [lmp-mx2], [t1-m], [t1-l], [lmp-t1], [lmp-p6], [lmp-post], [HAT] [89][a1-m], [a1-l] [90] [NEA] Amphipoda [67][st], [a2-m], [a2-l], [md-m], [md-l], [mx1-m], [mx1-l], [lmp-mx1], [mx2-m], [mx2-l], [lmp-mx2], [t1-m], [t1-l], [lmp-t1], [lmp-p6], [lmp-post], [HAT], [FS] [81] [A1/ A2], [Md], [Mx1], [Mx2], [T1], [P6] [83] [NGA], [mx1-g], [mx2-g], [t1-g] [PPN] [91][a1-m], [ a1-l] [90] [NEA] The table gives an overview of literature sources which were used to determine developmental timing of specific events that could not be determined from our investigations, for each taxon used in the analysis. Event abbreviations are used as in Table 1. antenna, and the mandible [A1], [A2], [Md] (Additional and second thoracopod appear [Mx1], [Mx2], [T1] file 1). In the next semaphoront Si En15 (15hpf), the first (Figure 4f). Si N4 (nauplius stage 4) shows anlagen of muscle precursors become detectable in the second an- postnaupliar ganglia (Figure 4g) in the first and second tenna segment [a2-l]. In Si EN17 (17hpf), anlagen of the maxilla segment [mx1-g], [mx2-g], already showing com- protocerebral, deutocerebral, tritocerebral, and mandible missures. The same semaphoront also shows longitudinal ganglia appear [NGA] (Figure 4e). Additional muscle pre- muscle precursors, extending from within the first maxilla cursors arise in the first antennal and the mandibular segment into the telson anlage. [lmp-mx1], [lmp-mx2], segment [a1-m, a1-l, md-l]. The remaining naupliar ap- [lmp-t1], and [lmp-post] are therefore assigned to Si N4. pendage muscle primordia [a2-m], [md-m] appear Si N5 (nauplius stage 5) shows ganglion anlagen in the in semaphoront Si En20 (20hpf). Si EN23 (23hpf)ischar- first and second thoracopod segment [t1-g], (Figure 4h), acterized by distinct nerve fiber bundles forming the as well as musculature associated with the postnaupliar circumesophageal ring, the presence of the nauplius eye appendages which we identify as extrinsic appendage primordium [NEA] and the posterior pioneer neurons muscle precursors [mx1-m], [mx1-l], [mx2-m], [mx2-l], [PPN]. Si N1 (nauplius stage 1) hatches from the egg [t1-m], [t1-m]. In the second protozoea stage, Lv Z2 of membrane [HAT]. In Si N3 (nauplius stage 3), appendage L. vannamei segmentation of the trunk is complete, mean- buds of the fist maxilla, second maxilla, first thoracopod, ing all segmental mesoderm anlagen must have been Jirikowski et al. EvoDevo 2015, 6:4 Page 16 of 30 http://www.evodevojournal.com/content/6/1/4

Table 3 Rank table of developmental sequences Neocaridina heteropoda:Theegg nauplius stage, sema- Event number Abbreviation A.f. G.f. S.i. N.h. P.f. N.i. P.h. phoront Nh EN, shows first antennal, second antennal, 1 [A1/ A2] 2 1 1 1 1 1 1 and mandibular buds [A1], [A2], [Md], but no clear sign of 2 [Md] 2 1 1 1 1 3 1 differentiating nervous tissue (Figure 5a). Anti-acetylated α-tubulin staining data for Nh I could not be obtained. An 3 [Mx1] 7 3 7 3 2 3 2 additional semaphoront Nh I+ is described here which 4 [Mx2] 7 4 7 3 2 3 2 shows intermediate limb morphology, between Nh I and 5 [T1] 6 4 7 3 2 3 2 Nh II:[Mx1],[Mx2],[T1],(Figure5b).Nh I+ also shows a 6 [P6] n.a. 7 11 7 6 5 5 differentiated circumesophageal nerve ring, an anlage of 7 [FS] n.a. 5 10 4 4 3 4 the nauplius eye, and posterior pioneer neurons with 8[st ]1472256elongate longitudinal neurite bundles (Figure 5c,d). Since the anlagen of the naupliar ganglia must be formed be- 9[a1-m]25349911 tween Nh EN and Nh I+, we assign the event [NGA] to 10 [a1-l] 2 10 3 9 9 9 11 Nh I. Because of the advanced morphology of the posterior 11 [a2-m]2742978pioneer neurons and longitudinal neurite bundles, [PPN] is 12 [a2-l]2424278also assigned to semaphoront Nh I while [NEA] is assigned 13 [md-m]2542258to Nh I+. Nh II shows distinct buds of the third thoraco- 14 [md-l]2432269pod (Figure 5e). The full set of mesodermal segment an- lagen is present [FS]. Ganglion anlagen are present in the 15 [mx1-m]7895768 first and second maxilla segment and in the first and sec- 16 [mx1-l] 7 10 9 5 2 7 9 ond thoracopod segments [mx1-g], [mx2-g], [t1-g]. The 17 [lmp-mx1]778536- first maxilla segment shows developing commissures 18 [mx2-m] 9 10 9 5 7 6 8 (Figure 5f). Semaphoront Nh III shows intersegmental fur- 19 [mx2-l]8595578rows throughout the entire trunk (Figure 5g). Also more 20 [lmp-mx2]-58434- postnaupliar ganglia show differentiated commissures. However, the complete set of ganglion anlagen of the trunk 21 [t1-m] 7 10 9 5 7 7 8 is not yet present, though continuous longitudinal neurite 22 [t1-l] 7 10 9 5 7 7 8 bundles bilaterally connect the posterior pioneer neurons 23 [lmp-t1]7484346to the anterior ganglia (Figure 5h). Semaphoront Nh IV 24 [lmp-p6] n.a. 7 10 5 5 6 9 shows fully developed ganglia, in the most posterior trunk 25 [lmp-post]-4843-- segments, including a sixth and an additional seventh pleo- 26 [NGA] 2 2 3 2 2 4 3 mere ganglion anlage (Figure 5i). Therefore, we assign the feature [p6-g] to an intermediate semaphoront between 27 [Mx1-g] 7 5 8 4 3 5 3 Nh III and Nh IV: Nh III +. Nh IV shows appendage buds 28 [Mx2-g] 7 5 8 4 3 5 3 in the sixth pleon segment [P6]. The next semaphoront 29 [T1-g] 7 6 9 4 4 6 3 Nh HAT hatches from the egg envelope. Throughout 30 [P6-g] n.a. 9 10 6 6 8 7 embryogenesis of N. heteropoda,eveninfreshlyhatched 31 [NEA] 3 2 5 3 - - - individuals, lateral extrinsic muscle precursors of the first 32 [PPN] 5 2 5 2 2 - - antenna [a1-l] were not observed. They are assigned to a final semaphoront Nh VI. 33 [HAT] 4 10 6 8 8 2 10 Procambarus fallax forma virginalis: Pf EN represents The table shows rank values assigned to the events coded for heterochrony analysis. Events are ordered by number and sorted into groups as in Table 1 and the egg nauplius stage, with first antennal, second antennal, Table 2. Abbreviations of events are given in brackets. Rank values specify the and mandibular buds [A1], [A2], [Md] (Additional file 1). position of the respective event in the event sequence for each of the investigated In semaphoront Pf I, the embryo shows appendage buds species. Rank values range from 1 to 11. Minus signs are given where the respective feature is not present in the species of interest. Non applicable events of the first and second maxillae, the first thoracopod as are marked ‘n.a.’. Abbreviations: A.f. (A. franciscana, Branchiopoda), G.f.(G. falcatus, well as anlagen of the naupliar ganglia [Mx1], [Mx2], [T1], Stomatopoda), S.i. (S. ingentis, Dendrobranchiata), N.h.(N. heteropoda,Caridea),P.f. (P. fallax forma virginalis,Astacidea),N.i. (N. integer,Mysidacea),P.h.(P. hawaiensis, [NGA]. Also posterior pioneer neurons [PPN] (Figure 6a) Amphipoda). Event abbreviations are used as in Table 1. and a set of cephalic muscle precursors [st], [a2-l], [md-m], [md-l], [mx1-l], appear. Pf II shows distinct appendage formed [FS]. In a related species, Penaeus monodon,gan- buds down to the fifth thoracomere. Here early anlagen of glion anlagen are present in the entire trunk [p6-g] at this ganglia in the first and second maxilla segments are de- stage. [P6], [FS], [lmp-p6], and [p6-g] are assigned to this scribed [mx1-g], [mx2-g] (Additional file 1), as well as the semaphoront. In the second mysis stage, Lv M2 append- longitudinal muscle precursors [lmp-mx1], [lmp-mx2], age anlagen are present in the sixth pleomere [P6]. [lmp-t1], [lmp-post]. These features are followed by the Jirikowski et al. EvoDevo 2015, 6:4 Page 17 of 30 http://www.evodevojournal.com/content/6/1/4

appearance of appendage buds of ganglion anlagen in the [PPN], [NEA], [lmp-post] were neither observed nor are first thoracomere [t1-g] and offset of segment formation they reported for adult semaphoronts. They are coded [FS] at Pf AS6 (AS06 stage 6, V06 45%). In Pf III, ganglion as absent. anlagen of the most posterior trunk segment are still Parhyale hawaiensis: In the first semaphoront Ph E1, lacking, but continuous longitudinal neurite bundles are the first antennal, second antennal, and mandibular buds already present (Figure 6b). Two muscle precursors are [A1], [A2], [Md] appear. Semaphoront Ph E2 shows ap- formed: [mx2-l]and[lmp-p6]. Presence of the ganglion an- pendage buds in the first maxillary, second maxillary, and lagen in pleomere six [p6-g] is assigned to semaphoront Pf first thoracopod segments [Mx1], [Mx2], [T1]. Appear- AS8, (Figure 6c) which also shows presence of appendage ance of the naupliar, first maxillary, second maxillary, and buds in pleomere six [P6]. In Pf VI, further muscle precur- first thoracic ganglion anlage [NGA], [mx1-g], [mx2-g], sors are formed: [mx1-m], [mx2-l], [t1-m], [t1-l]. Hatching [t1-g], is reported for a corresponding semaphoront of the juvenile [HAT] is assigned to semaphoront Pf HAT. (S3 early) of the amphipod Orchestia cavimana [83] and is [a1-m], [a1-l], and [a2-m] were assigned to a final sema- assigned to semaphoront Ph E3. These ganglion anlagen phoront Pf V. The anlage of a nauplius eye [NEA] was appear rapidly in anterior posterior progression, with a never observed and is also not reported for adult crayfish. slight gap between naupliar and postnaupliar segments. It is coded as absent. However, since these events all occur within a single stage Neomysis integer: The first semaphoront Ni En repre- and since single events do not coincide with different sents an embryonic semaphoront with slender anlagen of events of our series, we assign them to one semaphoront the first and second antenna [A1], [A2], but no mandible and treat them as simultaneous in the sequence. In sema- bud (Figure 7a). It is followed by the hatching event phoront Ph E4, the germ band shows the full number of [HAT], assigned to semaphoront Ni HAT which again is adult mesodermal segment anlagen [FS]. Appendage buds followed by the first nauplioid stage, Ni Naup-d in the sixth pleon segment are seen in semaphoront Ph (Figure 7b). This semaphoront shows appendage buds of E5 [P6]. In Ph I, the first muscle precursors appear: [st], the mandibles, first and second maxillae and first thoraco- [lmp-t1]. Ganglion anlagen of the ventral nerve cord are pods [Md], [Mx1], [Mx2], [T1], as well as the complete set visible down to the third pleon segment (Figure 8c). The of mesodermal segment anlagen [FS]. The following sema- sixth pleomere shows early ganglion anlagen in semaphor- phoront Ni I shows external segmentation of the complete ont Ph II [p6-g], (Figure 8d). Ganglia of the sixth trunk (Figure 7c,e), as well as ganglion anlagen in the nau- pleomere show differentiated commissures in semaphor- pliar segments [NGA], (Figure 7d), as well as longitudinal ont Ph III (Figure 8e). This semaphoront also shows muscle precursors in the second maxilla segment and the several muscle precursors: [a2-l], [md-m], [md-l], first thoracic segment: [lmp-mx2]and[lmp-t1]. Ni II [mx1-m], [mx2-m], [mx2-l], [t1-m], [t1-l]. Further muscle shows stomodeal muscle precursors [st] and precursors of precursors arise in semaphoront Ph IV:[md-l], [mx1-l], the medial mandible muscles [md-m]. Presence of the dif- [lmp-p6]. Semaphoront Ph V is excluded from our sema- ferentiated circumesophageal nerve ring formed by the phoront series, as it does not introduce novel features. naupliar ganglia, as well as anlagen of the first postnaupliar The next semaphoront Ph HAT is characterized by hatch- neuromeres down to the second maxilla segment [mx1-g], ing from the egg envelope. The final semaphoront Ph VI [mx2-g], can be seen (Figure 7f). These neuromeres, as is assigned features that are not observed throughout de- well as more posterior ones show differentiated commis- velopment but are reported for the adult. [a1-m], [a1-l]. sures and connectives in the next semaphoront Ni III [lmp-mx1], [lmp-mx2], [lmp-post], [PPN], and [NEA] are (Figure 7g). [t1-g] is assigned to this semaphoront, as well neither observed during embryogenesis nor described for as the remaining longitudinal trunk muscle precursors adult Amphipoda. They are coded as absent. [lmp-mx1], [lmp-p6], and [md-l], [mx1-m], [mx2-m]. Artemia franciscana: Af EN1 (Na2)ischaracterized Semaphoront Ni IV shows further muscle precursors of by first appearance of stomodeal muscle precursors [st] the cephalic and first thoracic segments [a2-m], [a2-l], (Additional file 1). Af EN2 (Na3) shows first appearance [mx1-l], [mx2-l], [t1-m], [t1-l], but no ganglion anlagen in of naupliar appendage buds, naupliar ganglia, as well as pleomere six (Figure 8a). [p6-g] is assigned to an inter- the naupliar appendage muscle primordia [A1], [A2], mediate semaphoront Ni IV+, as semaphoront Ni V [Md], [NGA], [a1-m], [a1-l], [a2-m], [a2-]l,[md-m], [md- possesses a sixth (and seventh) neuromere showing con- l]. We specify a third embryonic semaphoront Af EN3,to nectives and lateral nerves with advanced differentiation which we assign the appearance of the nauplius eye (Figure 8b). Formation of the first antennal muscle precur- [NEA], according to published data (Additional file 1). Af sors [a1-m], [a1-l] was not recorded during embryonic and HAT is characterized by hatching from the egg mem- larval development. These features are assigned to a final brane. The first larval semaphoront Af I possesses poster- semaphoront Ni VI. Posterior pioneer neurons, a nauplius ior pioneer neurons [PPN]. Af II is characterized by eye anlage, and posterior longitudinal muscle primordium appendage buds in the first thoracic segment [T1] Jirikowski et al. EvoDevo 2015, 6:4 Page 18 of 30 http://www.evodevojournal.com/content/6/1/4

(Figure 9a). Appendage buds in the first and second max- considerably differing heterochrony rates between germ illa segments are not yet found at this stage (Figure 9b). layers, namely 1.12 in the ectoderm and 1.72 in the Formation of ganglion anlagen in the first maxilla, second mesoderm. maxilla, and first thoracopod segments, [mx1-g], [mx2-g], In the following, the ancestral developmental sequences [t1-g], are assigned to a novel semaphoront Af III.Here and heterochronies represented in the PGi superconsensus also first and second maxillary appendage buds appear tree are presented. [Mx1], [Mx2], as well as medial and lateral extrinsic ap- pendage muscle precursors and longitudinal muscle pre- The branchiopod/malacostracan last common ancestor cursors of the trunk [mx1-m], [mx1-l], [t1-m], [t1-l], [lmp- The analysis revealed an ancestral sequence for the bran- mx1], [lmp-t1] (Figure 9c). In the next semaphoront Af IV, chiopod/malacostracan clade in which the naupliar ap- a lateral extrinsic muscle precursor in the second maxilla pendage buds [A1/A2], [Md], the naupliar ganglion segment [mx2-l] has emerged (Figure 9d). A small medial anlagen [NGA], and the nauplius eye anlage [NEA] are extrinsic muscle precursor of the second maxilla [mx2-m] formed in the egg nauplius phase.However,thisphase is seen in semaphoront Af V (Figure 9e). A longitudinal lacks a large part of the naupliar myogenic events: [md-m], muscle precursor of the second maxilla segment is not ob- [a2-m], [a1-l], and [st] (Figure 11). The hatching event served at any time in development. We code [lmp-mx2]as [HAT] occurs only after the formation of the postnaupliar absent for A. franciscana. Furthermore, all features that re- appendage buds [Mx2] and [T1]. The postnaupliar ap- late to the sixth pleon segment [P6], [lmp-p6], [p6-g], also pendage buds and ganglion anlagen appear with significant [lmp-post] and offset of segment formation [FS] are coded delay to their counterparts in the naupliar segments. Fur- as absent, because these features are applicable only to thermore, the postnaupliar appendage buds do not appear Malacostraca. in anteroposterior progression in the reconstructed bran- chiopod/malacostracan LCA sequence. Buds of the second Heterochrony analysis and ancestral developmental maxilla [Mx2] are formed first, followed by the first thora- sequences copod bud [T1] and the first maxilla bud [Mx1]. The single runs of PGi provided pseudoconsensus trees with lengths of 101, 100, 98, and a mean tree length of Eumalacostraca 99.67. Heterochrony in the eumalacostracan tree is ex- PGi analysis shows several heterochronic changes in the tensive, with an average of 7.12 event changes per eumalacostracan stem lineage. [a2-l]and[md-l]are branch. This represents approximately 22% of the listed shifted out of the naupliar phase (Figure 11). The egg nau- events. The superconsensus tree including ancestral and plius phase of the eumalacostracan LCA comprises only terminal sequences, as well as listed heterochronic naupliar appendage bud formation [A1/A2, Md] and for- events and bootstrap support is shown in Additional file mation of naupliar ganglion anlagen [NGA]. Furthermore, 3. For all events, heterochrony rates were calculated [NGA] is shifted late within the egg nauplius phase.For- (Figure 10). Formation of the first and second antenna mation of the mandibular muscle precursors [md-m]is buds [A1/A2], the extrinsic appendage muscle precur- shifted late while formation of the first maxilla bud [Mx1], sors of the first thoracopod [t1-m], [t1-l], the posterior the first thoracic ganglion anlage [t1-g], the stomodeal pioneer muscle strand [lmp-post], the ganglion anlagen muscle precursors [st], and the longitudinal muscle pre- of the first and second maxilla segment [mx1-g], [mx2- cursor of the first thoracic segment [lmp-t1] are shifted g], and the posterior pioneer neurons [PPN] do not early. The hatching event [HAT] is shifted to the end of show any heterochrony (Figure 10a). In the case of ex- the sequence. Even though formation of the nauplius eye trinsic appendage muscle precursor formation, the lat- anlage [NEA] and posterior pioneer neurons [PPN] are no eral muscle precursors show a higher heterochrony rate longer part of the egg nauplius phase (which is defined by than the medial muscle precursors. Comparison of seg- the absence of any postnaupliar events), they still occur ment specific heterochrony rates (Figure 10b) shows that near the beginning of the sequence. This position is not al- myogenic events in the naupliar segments (A1, A2, Md) tered on the branches leading to Caridoida and Decapoda. have been altered in evolution significantly more often than appendage bud and ganglion formation. The first Caridoida and second maxilla segments show no heterochrony in In the caridoid stem lineage formation of the first maxilla neurogenesis but slightly higher rates of heterochrony in bud [Mx1], the sixth pleopod bud [p6] and the longitudinal appendage bud formation than in myogenesis. Between muscle precursor [lmp-p6] are shifted late while formation tissue types the mean heterochrony rates differ strongly, of the second antenna muscle precursor [a2-m]isshifted as reflected by mean rates of 1.32, 0.91, and 1.72 changes early. The egg nauplius phase of the reconstructed caridoid per event for epidermis (appendage buds), nervous tissue LCA consists only of naupliar appendage bud formation and musculature, respectively (Table 4). This results in [A1/A2, Md]. The first maxilla bud [Mx1] is still formed Jirikowski et al. EvoDevo 2015, 6:4 Page 19 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 10 Comparison of heterochrony rates. Box graph showing heterochrony rates of all events in the superconsensus tree. The heterochrony rate represents the total number of heterochronic shifts (early and late) for a specific event, multiplied with its mean bootstrap value. The color code for the different classes of events is given at the bottom. (a) Heterochrony rate for all events in the data set. (b) Heterochrony rate of events with clear segment affiliation, sorted from anterior to posterior (A1, A2, Md, Mx1, Mx2, T1, P6) and compared for the three different tissue types (appendage buds, ganglion anlagen, and muscle precursors). The heterochrony rates shown here for muscle precursors represent mean values of all myogenic events recorded for the respective segment. Events that are affiliated with the telson (PPN, lmp-post) are also included. Abbreviations for events in (a) are used as listed in Table 1. In (b), the body segments are labeled with the abbreviations of the corresponding appendage: A1 first antenna, A2 second antenna, Md mandible, Mx1 first maxilla, Mx2 second maxilla, T1 first thoracopod, and P6 sixth pleopod. after the second maxilla bud [Mx2] while postnaupliar gan- Decapoda glia maintain their anteroposterior progression. Formation In the lineage leading to the decapod LCA, two naupliar of appendage buds [P6], ganglion anlagen [p6-g], and lon- myogenic events have shifted early [md-m] and [a2-m] gitudinal muscle precursors [lmp-p6] are now concen- (Figure 11). The egg nauplius phase comprises naupliar trated near the end of the sequence. appendage bud formation [A1/A2, Md], the naupliar Jirikowski et al. EvoDevo 2015, 6:4 Page 20 of 30 http://www.evodevojournal.com/content/6/1/4

Table 4 Comparison of heterochrony rates between germ layers and tissue types Germ layer Tissue type Events Mean heterochrony rate Mean heterochrony rate of germ layer Ectoderm Epidermis 6 1.32 1.12 Neural tissue 7 0.91 Mesoderm Muscle tissue 18 1.72 1.72 The table shows event numbers and heterochrony rates for the three tissue types from which the events were coded: Epidermis, muscle tissue, and nervoustissue,as well as the germ layers the tissues originate from (ectoderm, mesoderm). The heterochrony rate of an event is the event-specific number of heterochronic changes shown by PGi for the entire superconsensus tree, multiplied by the mean bootstrap value for the event. Tissue-specific heterochrony rates were calculated by forming the mean heterochrony rate per event for all events of epidermis neural tissue or muscle development. Mean heterochrony rates of the germ layers were calculated from these values. neural events [NGA, NEA], and the majority of naupliar Peracarida myogenic events [st], [a2-l], [md-l], [md-m]. It is The reconstructed ancestral sequences of the caridoid and followed by the simultaneous formation of the first max- peracaridan LCA, as well as the developmental sequence illary, second maxillary, and first thoracic appendage of P. hawaiensis are shown in Figure 13, and the respective buds. The egg nauplius phase appears extensive in the changes of event positions are indicated. In the lineage sequence, due to the simultaneous occurrence of [Mx1, leading to Peracarida, the nauplius eye anlage [NEA] and Mx2, T1]. However, the late position of [Mx2] was not the posterior pioneer neurons [PPN] are lost. Formation revealed as heterochrony by the analysis, but results of extrinsic appendage muscle precursors of the second from the mean rank calculation for the superconsensus antenna [a2-l] and the mandible [md-l] are shifted to a tree. The posterior longitudinal muscle precursor later position while the formation of the longitudinal [lmp-post] is given as novelty for Decapoda by the ana- muscle precursor [lmp-mx2] and the sixth pleopod bud lysis. Nevertheless, in the decapod LCA sequence, the [P6] are shifted to an earlier position. In the peracarid postnaupliar longitudinal muscle precursors [lmp-mx1], LCA sequence, the egg nauplius phase now only consists [lmp-mx2], [lmp-t1], and [lmp-post] occur simultan- of epidermal appendage bud formation. It is followed by eously before offset of segment formation [FS] and for- rapid formation of the postnaupliar appendage buds. The mation of the postnaupliar appendage muscle precursors first part of the developmental sequence is dominated by [mx1-m, mx1-l, mx2-m, mx2-l, t1-m, t1-l]. formation of appendage buds and ganglion anlagen which all occur in strict anteroposterior progression. The major- S. ingentis (Dendrobranchiata) ity of myogenic events is concentrated in the second half Reconstructed ancestral event sequences of Decapoda, of the sequence and shows no trace of an anteroposterior Pleocyemata, and the terminal developmental sequence gradient in development. of S. ingentis are given together with the heterochronic changes in Figure 12. In the lineage leading to S. ingen- P. hawaiensis (Amphipoda) tis, formation of the first antennal muscle precursors P. hawaiensis shows loss of the longitudinal muscle pre- [a1-m, a1-l] and the hatching event [HAT] are shifted cursors [lmp-mx1]and[lmp-mx2] in the first and second early. Formation of the stomodeal muscle precursor [st] maxilla segments. Formation of extrinsic appendage is shifted to a later position after the egg nauplius phase. muscle precursors [md-m], [md-l], and [mx1-l] is shifted The simultaneous formation of postnaupliar appendage late while formation of the first thoracic and sixth pleon buds [Mx1], [Mx2], [T1], followed by postnaupliar longi- ganglion anlagen [t1-g] and [p6-g] are shifted to earlier tudinal muscles and postnaupliar extrinsic appendage positions. As a result, appendage bud and ganglion anlage muscles is retained in the lineage leading to S. ingentis. formation are even more concentrated at the beginning of Only formation of the first thoracic ganglion anlage the sequence. A temporal gap between formation of nau- [t1-g] is delayed. pliar and postnaupliar features is shown only for append- age buds, but not for neurogenic or myogenic events. Pleocyemata In the pleocyemate stem lineage formation of the second Discussion antennal muscle precursor [a2-l], the naupliar ganglion Tissue-related evolution of developmental timing anlagen [NGA] and the nauplius eye anlage [NEA] are Studies on developmental genetics of the fruit fly shifted to a later position. The nauplius eye anlage Drosophila melanogaster [98] and experimental develop- [NEA] is therefore formed only after the egg nauplius mental studies on P. hawaiensis [99] suggest that the phase as a result. Offset of segment formation [FS] and ectoderm has a strong regulatory influence on the devel- formation of the longitudinal muscle precursor in the opment of the mesoderm in Arthropoda, but not vice sixth pleon segment [lmp-p6] are shifted to an earlier versa. This does not necessarily imply that heterochro- position. nies within the ectoderm are unlikely. Fritsch & Richter Jirikowski et al. EvoDevo 2015, 6:4 Page 21 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 11 (See legend on next page.) Jirikowski et al. EvoDevo 2015, 6:4 Page 22 of 30 http://www.evodevojournal.com/content/6/1/4

(See figure on previous page.) Figure 11 Heterochronic changes and ancestral developmental sequences for major malacostracan nodes. Simplified phylogram of Malacostraca with ancestral developmental sequences from the PGi superconsensus tree. Sequences are shown as columns of downward pointing arrows. Each arrow represents a different semaphoront containing a single event or a group of simultaneous events (abbreviations listed in Table 1). The position of Leptostraca is indicated by a dotted line. Ancestral ontogenetic sequences of the branchiopod/malacostracan, the eumalacostracan, the caridoid, and the decapod LCA are shown. Events are color-coded corresponding to Figures 3 and 10a. Heterochronic changes are indicated by horizontal arrows that use the same color code as the respective events. The egg nauplius phase is indicated by brackets. A symbolic nauplius drawing marks the terminal taxa, which develop nauplius larvae. Arrows with a ‘plus sign’ mark events that are interpreted as new evolutionary acquisitions on the respective branch. Arrows with a ‘minus sign’ indicate evolutionary loss of a feature. Abbreviations: ENP egg nauplius phase. Event abbreviations are listed in Table 1.

[12] describe several instances of intraectodermal het- and therefore would have been more likely eliminated erochrony in evolution of Branchiopoda. Yet tracing the by selection, than timing alterations in mesoderm develop- evolutionary history of eumalacostracan, developmental ment. Of course this explanation is based only on timing by PGi in our study showed that the different tis- cross-species comparison of timing patterns and not sue types (epidermis, nervous system, musculature) have on experimental investigations of the developmental taken different evolutionary paths depending on the systems. Nevertheless, our findings support the hypoth- germ layer they originate from. The mean heterochrony esis that heterochronic change of muscle development rates of ectoderm and mesoderm development that were played a major role in evolutionary loss and reacquisi- calculated from the results of PGi analysis differ strongly tion of the nauplius larva. (Table 4). Ectodermal development is generally less af- fected by heterochrony in malacostracan evolution than Evolution of naupliar developmental patterns in mesodermal development. Almost no heterochronic Malacostraca events appear in the naupliar region if only the ectoder- In arthropod development, commonly the material of a mal development is considered. Heterochrony of muscle variable number of anterior segments is laid down in a dif- precursor formation is far more extensive in the first an- ferent manner than following segments that are added pos- tennal, second antennal, and mandibular segments than teriorly during development. This is reflected by the neural development and formation of appendage buds process of short germ development in embryogenesis or (Figure 10b). This suggests that the divergent evolution anamorphic postembryonic development [100], and refer- of developmental timing between mesodermal and ecto- ences therein and represents a condition of the arthropod dermal tissues reflects a modular property of the crust- ground pattern. A naupliar developmental pattern, mean- acean developmental system as would be expected due to ing that the material of the first antennal, second antennal, findings from genetic and experimental developmental and mandibular segments is formed (more or less) simul- biology [98,99]. taneously before the posteriorly following segments, can be Reconstruction of the ancestral developmental sequence understood as a specialized form of the arthropod develop- by computational heterochrony analysis with PGi suggests mental pattern. The plesiomorphic condition for crusta- that the naupliar pattern, known as ‘egg nauplius stage,’ ceans (or Tetraconata) including extinct representatives of was present in the last common ancestor of Eumalacos- the stem lineage (called Crustacea sensu lato in [101]) was traca as combination of epidermal and neurogenic, but a ‘head’ larva with functional first antennae followed by not muscle developmental patterns. In the last common three pairs of appendages. A larva bearing three appendage ancestor of Caridoida and Peracarida, the reconstructed pairs—the nauplius—is considered apomorphic for crown- sequence shows only an ‘epidermal egg nauplius’.Forma- group Tetraconata [102] (Pancrustacea) (Eucrustacea or tion of naupliar ganglion anlagen, however, occurs only Crustacea sensu stricto in [101]) [17]. Our analysis reveals slightly later in the sequence. The persistence of the very an ancestral developmental sequence for the branchiopod/ early timing of epidermal and neural naupliar events in malacostracan clade with an extensive egg nauplius phase. these lineages could be explained as the result of a devel- However, the reconstructed sequence is not fully compat- opmental constraint that limited the plasticity of develop- ible with a developmental mode comprising a free- mental timing in ectodermal development compared to swimming nauplius larva because some naupliar myogenic mesodermal development. This constraint would repre- events are formed only after the egg nauplius phase and sent a modular property of the developmental regulatory hatching occurs only after of the second maxilla and first system patterning the naupliar ectoderm, similar to the thoracopod bud are formed. Yet a large number of post- observations on other arthropods [98,99]. Evolutionary al- naupliar developmental events, such as formation of post- terations of timing in ectodermal development of the nau- naupliar ganglion anlagen and muscle precursors, occur pliar segments would thus have had a stronger impact on after hatching which is in line with nauplius larva forma- the developmental outcome and viability of the organism tion. Since a nauplius larva is predominant throughout the Jirikowski et al. EvoDevo 2015, 6:4 Page 23 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 12 (See legend on next page.) Jirikowski et al. EvoDevo 2015, 6:4 Page 24 of 30 http://www.evodevojournal.com/content/6/1/4

(See figure on previous page.) Figure 12 Heterochronic changes and ancestral developmental sequences within Decapoda. Phylogeny of Malacostraca as in Figure 11, but showing only Decapoda. Ancestral ontogenetic sequences reconstructed by heterochrony analysis using PGi are shown for the decapod and the pleocyemate LCA, as well as the terminal sequence of S. ingentis (Dendrobranchiata). Abbreviations: ENP egg nauplius phase. Event abbreviations are listed in Table 1. remaining crustacean taxa, we suggest that this was also The lineage leading to Dendrobranchiata represents a the condition in the last common branchiopod/malacostra- change in developmental mode and evolution of a free- can ancestor and that the late position of many naupliar swimming nauplius larva. Compared to the eumalacos- events is an artifact caused at this node by the exten- tracan stem lineage where the nauplius larva was lost, sive variation in the data set. S. ingentis shows only few (five) heterochronies, of which The developmental sequence of the malacostracan only three are relevant for the reacquisition of the ground pattern could not be reconstructed, because Lep- nauplius larva: early shift of the two muscle precursors tostraca, the sister group of Eumalacostraca according to of the first antenna and early shift of the hatching event. the phylogeny of Richter & Scholtz [28], is not present These changes were sufficient for the reacquisition of a in our taxon sampling. Embryogenesis of the leptostra- free-swimming nauplius larva because the other neces- can Nebalia bipes has been described [52,53,103] but sary events were already in place in the developmental unfortunately not sufficiently to integrate this species sequence of the decapod ground pattern. This refers to into the analysis. It is known however that N. bipes lacks the naupliar appendage bud formation and formation of free-swimming larval phases. Also, an egg nauplius stage naupliar ganglia, which constitute the ectodermal egg in appendage morphogenesis is described for this spe- nauplius stage in embryogenesis, as well as the early po- cies. The developmental pattern of the malacostracan sitions of naupliar myogenic events that are the result of last common ancestor can therefore not be expected to heterochronic shifts in the lineages leading to Decapoda. differ much from the eumalacostracan last common an- Therefore, our results support the hypothesis formulated cestor in these respects. by Scholtz [67] that an embryonic egg nauplius served as The eumalacostracan last common ancestor, according a prerequisite for the secondary evolution of the dendro- to heterochrony analysis with PGi, possessed a develop- branchiate nauplius larva. mental sequence with late position of the hatching event Within Decapoda, in the lineage leading to Pleocye- and thus major postnaupliar developmental events oc- mata, an egg nauplius pattern in ectodermal develop- curring in embryogenesis. The late shift of the hatching ment has been retained, together with a set of naupliar event to the end of the sequence, the early shift of post- muscle precursors in the egg nauplius phase, according naupliar muscle precursor, ganglion anlagen, and ap- to our results. This pattern differs only minimally from pendage bud formation suggests that in the lineage the decapod ground pattern. Yet timing of naupliar myo- leading to Malacostraca a change of ontogenetic mode genesis is altered within the Pleocyemata in the lineages took place and the nauplius larva was lost. Our analysis leading to N. heteropoda and P. fallax forma virginalis. suggests that only formation of naupliar appendage buds We should note that both species are direct developers, and naupliar ganglia remained part of the egg nauplius which is a derived condition within decapods. Yet the re- phase. The egg nauplius stage [67,69] was thus likely sults of PGi suggest that the pleocyemate ground pattern restricted to ectodermal tissues already in the eumala- is reminiscent of the decapod ground pattern and that costracan ground pattern. alterations to myogenesis have occurred only within the Along the branches leading from the eumalacostracan to group. Adding taxa with a more basal phylogenetic the caridoid and to the decapod last common ancestor, position and a zoea-like larva in future studies can be ex- comparatively few heterochronies are recovered by our pected to uncover a similar condition. analysis. Formation of two naupliar muscle precursors is It should be noted that the other malacostracan taxon shifted to an earlier position while naupliar appendage bud with a nauplius larva, the Euphausiacea, is not repre- formation and formation of naupliar ganglia are retained sented in our study. Following the phylogeny used here, close to the beginning of the sequence. The developmental Euphausiacea are the sister group of Neocarida. Another sequence reconstructed for the ground pattern of Deca- popular phylogenetic hypothesis places Euphausiacea poda shows an extensive egg nauplius phase, comprising and Decapoda together in a monophylum called Eucar- all naupliar ectodermal events and formation of part of the ida [104,105]. Thus mapping timing data on these two naupliar muscle precursor group. Thus naupliar myogenic alternative hypotheses could improve our understanding events must have been added to the egg nauplius stage in of malacostracan phylogeny and clarify whether a nau- development before the emergence of a nauplius larva in plius larva evolved once or twice within Malacostraca. It the evolution of Decapoda. is unlikely however that inclusion of Euphausiacea will Jirikowski et al. EvoDevo 2015, 6:4 Page 25 of 30 http://www.evodevojournal.com/content/6/1/4

Figure 13 (See legend on next page.) Jirikowski et al. EvoDevo 2015, 6:4 Page 26 of 30 http://www.evodevojournal.com/content/6/1/4

(See figure on previous page.) Figure 13 Heterochronic changes and ancestral developmental sequences within Peracarida. Phylogeny of malacostraca as in Figures 11 and 12. Only the caridoid LCA, the peracarid LCA, and the terminal sequence of P. hawaiensis are shown. Abbreviations: ENP egg nauplius phase. Event abbreviations are listed in Table 1. significantly change the reconstructed developmental segment are formed early in the sequence reconstructed mode in the eumalacostracan or malacostracan ground for the eumalacostracan last common ancestor, suggesting pattern, as this depends on the position of other not that functionality of the trunk did not precede differenti- included taxa, such as Leptostraca, Amphionidacea, ation in the posterior pleon segments, and consequently and Syncarida. that direct development rather than a zoea-like larva constituted the developmental mode. We point out that Evolution of a zoea-like larva the conclusions on this early node should be treated with In Malacostraca, both ectodermal and mesodermal tissues, caution because variation in the data set is extensive and from the second maxillary to the sixth pleonal segment, also because we cannot rule out the possibility of bias due are formed sequentially by proliferation of stem-like cells to the strong representation of direct development in (ectoteloblasts and mesoteloblasts) in the posterior growth the analysis. zone [61,62,106,107]. This mechanism leads to an observ- For the last common ancestor of Decapoda, a zoea-like able anteroposterior gradient of segment differentiation in larva as hatching stages appears well supported. In the de- the germ band. We have recently described a growth zone velopmental sequence of the decapod ground pattern, ap- independent muscle precursor ‘lmp-post’ which likely pendage bud formation and formation of the longitudinal plays a crucial role in development of a zoea-like larva muscle precursor in the sixth pleonal segment occur close [67,73]. lmp-post is formed in the telson and extends an- to the end of the sequence. Offset of segment formation teriorly while at the same time longitudinal muscle precur- also occurs late while the anterior postnaupliar appendage sors form in the anterior postnaupliar segments. This way buds are formed simultaneously and just after the egg nau- a continuous longitudinal muscle strand across all trunk plius phase. Also all anterior longitudinal muscle precur- segments is formed, even before the full set of trunk seg- sors and lmp-post are formed simultaneously. The same is ments is differentiated. Zoea-like larval forms share a true for the extrinsic appendage muscle precursors of the functional, movable trunk, consisting of the thoracic or anterior postnaupliar segments. In the lineage leading to pleonal segments and a paddle-shaped telson which can Dendrobranchiata, timing of postnaupliar events relevant perform extension and flexion movements, and actively to zoea-like larva formation remains nearly unchanged participate in swimming, e.g., by performing tail flip es- (with the exception of [t1-g]). The evolution of the novel cape reactions. Activity of [lmp-post] allows trunk func- developmental mode of Dendrobranchiata, involving the tionality before the posterior trunk segments are fully novel larval stages metanauplius, protozoea and mysis developed, as is the case in zoea-like larvae. Further com- stages, from an ancestral condition with a more extensive mon features of zoea-like larva development in terms of embryonic period did not depend on changes in develop- developmental timing are rapid formation of appendages mental timing of the analyzed morphogenetic events. It is in an anterior set of postnaupliar segments (first and sec- the predisplacement of the hatching event that makes the ond maxillae, thoracopods), late formation of pleonal seg- actual difference between larval and embryonic develop- ments [P6], [P6-g], [lmp-p6] (with the exception of the ment, while the sequence in which appendage buds, gan- stomatopod pseudozoea), and late offset of segment for- glion anlagen, and muscle precursor are generated mation in the germ band [FS]. Event data for the second remains largely unchanged. Certainly, acceleration of dif- thoracic to the fifth pleonal segment was not analyzed be- ferentiation processes which follow the formation of ap- cause the data could not be acquired for a sufficient pendage, ganglion, or muscle anlagen in the segments of a amount of species and semaphoronts. viable free-swimming larva must be assumed for the evo- The scenario reconstructed by PGi suggests that a lution of the dendrobranchiate ontogenetic mode. In the zoea-like larva likely evolved independently in the lineages lineage leading to Pleocyemata, [FS] and [lmp-p6]are leading to Stomatopoda and Decapoda and that the euma- shifted to earlier positions. Both changes point toward loss lacostracan last common ancestor developed directly. The of zoea-like larva formation, but the majority of relevant posterior longitudinal muscle primordium [lmp-post] events is still in place. Both pleocyemate representatives which we consider a necessary feature for zoea-like larval (N. heteropoda and P. fallax forma virginalis) develop dir- motility was acquired twice independently in the lineages ectly and lack larval stages. Therefore, we consider early leading to Stomatopoda and Decapoda and was not part placement of [FS] and [lmp-p6] as bias toward direct de- of the eumalacostracan ground pattern. The appendage velopment in our data set, not necessarily as part of the bud and longitudinal muscle precursor of the sixth pleonal pleocyemate ground pattern. Jirikowski et al. EvoDevo 2015, 6:4 Page 27 of 30 http://www.evodevojournal.com/content/6/1/4

Evolution of developmental timing in Peracarida epidermal egg nauplius phase and a late position of the Peracaridan development is derived in many respects hatching event. This may be an artifact of insufficient relative to the malacostracan ground pattern, because of taxon sampling. However, Mysidacea show a unique tim- the advanced mode of brood care that is autapomorphic ing pattern of appendage bud development with the first to this group [20,28,108-110]. In Peracarida, females and second antennal bud being formed clearly before possess a ventral brood pouch (marsupium), in which the mandible bud, early offset of segment formation, and eggs are reared. Nauplius or zoea-like larvae are not finally late formation of naupliar ganglia and muscula- found in peracarids. Changes to the developmental se- ture. These observations suggest that the developmental quence in the peracarid stem lineage comprise loss of pattern found in Mysidacea is not homologous to the the nauplius eye and the posterior pioneer neurons, as egg nauplius pattern of Eumalacostraca. well as late shift of several naupliar and early shift of postnaupliar events. In the ancestral peracaridan devel- Conclusions opmental sequence, naupliar and anterior postnaupliar Our reconstruction of developmental sequence evolu- appendage buds are formed rapidly at the beginning of tion of Malacostraca revealed that development of mus- the sequence, while naupliar ganglia are formed late. culature has played a crucial role in evolutionary Offset of segment formation and formation of the pos- transitions between larval and embryonic development. teriormost pleonal appendage bud occur early while the The following conclusions can be drawn from our analysis majority of muscle precursors are formed late. These of heterochrony: properties of the developmental sequence do not re- semble zoea-like developmental timing patterns. The  The eumalacostracan last common ancestor has condition in the caridoid ground pattern is difficult to retained the developmental timing pattern of interpret in terms of developmental mode. The late nauplius larva formation in epidermal appendage position of pleonal events suggests zoea-like larva for- development and neurogenesis, but not in mation but the posterior longitudinal muscle primor- myogenesis. The ontogenetic mode using a nauplius dium [lmp-post] is not formed in the sequence. The larva was replaced most likely by direct development question whether direct development might be plesio- in the lineage leading to the Malacostraca by delay morphic for Peracarida can therefore not be answered in naupliar muscle development. at this point.  Secondary evolution of the dendrobranchiate Within Peracarida, most likely development was con- nauplius larva involved only little heterochronic sistently adapted to efficient formation of juvenile body change, because the major features of naupliar morphology after the advent of the new developmental development were present already in the decapod mode, which was constrained by the specialized mode of last common ancestor. The transition relied on early maternal brood care. In P. hawaiensis (Amphipoda) the shift of naupliar muscle precursors. adaptation to efficient formation of juvenile body struc-  According to our analysis, convergent evolution of a ture is intensified. Here the events of the six anterior zoea-like larva in the stomatopod and decapod segments appear in an order corresponding to the tis- lineage is more likely than a zoea-like larva in the sues they belong to: appendage buds, followed by gan- eumalacostracan last common ancestor. glion anlagen, followed by muscle precursors. Together  The developmental sequence of the peracarid last with the early offset of segment formation, this suggests common ancestor has lost the larva-related timing a strong acceleration of morphogenesis in this lineage, patterns in embryogenesis. Developmental timing which resulted in more rapid anteroposterior progres- was likely adapted to efficient formation of juvenile sion of segment formation and an earlier onset of tissue body structure under the constraint of specialized differentiation. brood care within the Peracarida. In Mysidacea, an inert larval stage hatches, and re- mains in the marsupium, a situation we call pesudodir- Some key taxa of Malacostraca have not been sampled ect development. The hatchling is termed ‘nauplioid’ here: Leptostraca, Anaspidacea, Bathynellacea, and [110,111]. The name is suggestive of a cryptic larval Euphausiacea. Also inclusion of additional event data, stage related to a nauplius larva. Also the early hatching considering the thoracic and pleonal segments, more ad- event, presence of a solid cuticle with setation, and vanced stages of tissue differentiation, or the formation of intramarsupial molting to the ‘postnauplioid’ stage sug- external cuticular structures would contribute to a more gest that a part of an ancestral larval developmental pro- detailed picture of malacostracan developmental evolu- gram is still active in Mysidacea. The evolutionary tion. Such investigations have the potential to further clar- scenario reconstructed with PGi suggests that the mysid ify the evolutionary history of malacostracan development, sequence is derived from an ancestor with only an butthisislefttofuturestudies. Jirikowski et al. EvoDevo 2015, 6:4 Page 28 of 30 http://www.evodevojournal.com/content/6/1/4

Additional files Authors’ contributions Immunohistochemistry and microscopy were carried out by GJ, as well as sequence coding and PGi analysis. GJ drafted the manuscript. SR and CW Additional file 1: Event sequence overview for all investigated taxa. collected material of G. falcatus and critically revised the manuscript. All three For each of the investigated taxa, a matrix is given in form of a spreadsheet, authors conceived the project and approved the final version of the in which the events are listed against the specified semaphoronts. manuscript. Specificationsofsemaphorontsusedinourdatasetaregiveninboldinthe upper row. Stage specifications from the literature are given in italics (second row). Rank values are also shown. Event categories, event abbreviations Acknowledgements (brackets), and event numbers are given in three columns on the left. Previous work on P. fallax forma virginalis and collection of G. falcatus material Furthermore, the literature source used to code the respective events is was funded by the German science foundation (DFG grant Ri837/8-1 to SR, given. If an event occurs for the first time in a specific semaphoront, the Wo1461/1-1 to CW). We are particularly grateful to Luke Harrison for providing respective field is marked with one of the following abbreviations or symbols: and updating PGi, as well for patiently advising us in its use and giving ‘figure number’ (events that are coded from observations made in the present important theoretical input. We thank Carolin Haug and Joachim Haug for fruitful study and depicted in the specified figure), ‘x’ (events that are coded from discussions on crustacean developmental evolution and Steffen Harzsch for our observations but not depicted), ‘-’ (the event is not observed), ‘n’ sharing expertise on malacostracan neurogenesis. Furthermore, we thank Phillip (the event is not applicable), ‘[reference number]’ (the event is coded from Hertzler for providing original confocal image stacks of S. ingentis for the given literature source), ‘scientists name, personal communication’ reexamination. Caterina Biffis is thanked for providing further expertise on (if the event is coded following personal communications). dendrobranchiate nervous system development. We also thank William Browne for sharing knowledge of nervous system development in P. hawaiensis. Finally Additional file 2: Nexus file for analysis of heterochrony. Nexus file GJ thanks Martin Stegner, Jonas Keiler, and Martin Schwentner for providing containing the rank-matrix and the tree topology. The data is formatted for input and support during preparation of the manuscript. use with PGi under R’s ape package. A rank value is coded for every event shown in Table 1. This yields a matrix of 7 × 33 cells. Since PGi cannot Author details handle values with multiple digits, ranks 10, 11, and 12 were coded as letters 1Universität Rostock, Institut für Biowissenschaften, Allgemeine und Spezielle a, b, and c, respectively. Absent data is coded as ‘z’ by convention. The tree Zoologie, Universitätsplatz 2, 18055 Rostock, Germany. 2Humboldt-Universität topology from [28] is included in the nexus file, in a simplified form, which zu Berlin, Institut für Biologie, Vergleichende Zoologie, Philippstr. 13, Haus 2, includes only the seven investigated taxa. 10115 Berlin, Germany. Additional file 3: PGi superconsensus tree. Superconsensus tree generated by PGi-analysis from pseudoconsensus trees of three independent Received: 26 September 2014 Accepted: 20 January 2015 runs with algorithm parameters set to 100 cycles of selection per node, 200 Published: 11 March 2015 sequences per cycle of selection, a maximum of 100 ancestral developmental sequences to be retained at each node and ‘semi-exhaustive’ pseudoconsensus References setting with a limit of 3,000 evaluated solutions of equal score. Developmental 1. Gould SJ. Ontogeny and phylogeny. Cambridge: Belknap Press of Harvard sequences in the superconsensus tree are shown using only the event University Press; 1977. p. 209–404. numbers (shown in Table 1). Simultaneous events are combined by brackets; 2. McNamara KJ, McKinney ML. Heterochrony, disparity, and macroevolution. subsequent events are separated by a comma between brackets. The Paleobiology. 2005;31:17–26. reconstructed ancestral developmental sequences are shown as plain text in 3. Alberch P. 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