Naturwissenschaften (2013) 100:1187–1191 DOI 10.1007/s00114-013-1117-3

SHORT COMMUNICATION

Larval anatomy of the pterobranch Cephalodiscus gracilis supports secondarily derived sessility concordant with molecular phylogenies Thomas Stach

Received: 23 August 2013 / Revised: 31 October 2013 / Accepted: 3 November 2013 / Published online: 6 December 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Pterobranchs have been interpreted as “missing links” combining primitive invertebrate features with advanced vertebrate-like characteristics. The first detailed morphological description of an ontogenetic stage of a pterobranch, based on digital 3D-reconstruction at electron microscopic resolution, reveals a triploblastic animal with monociliated epithelia, an extensive coelomic cavity, a through gut with an asymmetrically developed gill slit but no signs of planktonic specializations, such as ciliated bands. Therefore, this crawling larva supports the hypothesis proposed in previous molecular phylogenetic studies that pterobranchs could be derived within enteropneusts rather than being “missing links”. Keywords Deuterostome evolution . Hemichordate . Dipleurula

lifestyle, feeding on small plankton with a crown of tentacles led to the hypothesis that pterobranchs were intermediate between invertebrates and fish-like vertebrates (Ax 2003). Recent molecular phylogenies challenged this view and regard pterobranchs as a highly specialized side branch within the deuterostome acorn worms (enteropneusts) (Cannon et al. 2009). However, there is currently no independent evidence to support this alternative hypothesis (Peterson et al. 2013). The anatomy of larval stages can be decisive in such questions (Thompson 1836; Nakano et al. 2013). If pterobranchs with gill slits were primitive within deuterostomes, a planktonic larval stage with similarities to dipleurula-type larvae would be expected. If on the other hand pterobranchs were the sister group of harrimaniid enteropneusts, a derived creeping larval stage would be hypothesized. Here, I present the first threedimensional reconstruction of an embryonic stage of the pterobranch Cephalodiscus gracilis based on complete serial sections at transmission electron microscopic resolution.

Introduction Within deuterostomes, the evolution of an actively swimming fish-like animal from marine invertebrates is highly controversial. Pterobranchs are a small group of deuterostomes that occupy a central role in these discussions. With a pair of gill slits, a dorsal brain, and a muscular postanal appendage, some adult pterobranch species possess features reminiscent of fishes. On the other hand, their sessile, typically invertebrate Communicated by: Sven Thatje Electronic supplementary material The online version of this article (doi:10.1007/s00114-013-1117-3) contains supplementary material, which is available to authorized users. T. Stach (*) Humboldt-Universität zu Berlin, Comparative Zoology, Berlin, Germany e-mail: [email protected]

Materials and methods Collection of specimen Coralline pebbles and cobbles with adult specimens of C. gracilis Harmer 1905 (Cephalodiscida, Pterobranchia) attached were collected from 1 to 2 m depth off the bridge of Castle Harbor Causeway, Bermuda (N32° 21′ 30.92″ W64° 42′ 22.802) in October 2008. The pebbles covered with seawater were brought to the laboratory and kept in sea tables with running ambient seawater. The actively crawling larva was found within the transparent tubes of C. gracilis. The tubes were carefully severed and the individual was transferred to the anaesthization solution consisting of 7 % MgCl2 for 5 min prior to processing for transmission electron microscopy (TEM).

1188

Transmission electron microscopy and 3D-reconstruction For TEM, most of the relaxation agent was removed and replaced with ice-cold primary fixative containing 2.5 % glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2), adjusted to an osmolarity of approximately 800 mosM by the addition of NaCl. Primary fixation was stopped after 45 min with three buffer rinses for 10, 15, and 20 min. Primary fixation was followed by 30 min of postfixation with 2 % OsO4 in sodium cacodylate buffer. Postfixation was stopped with three buffer rinses (15, 30, and 30 min) followed by two rinses with ddH2O (15 and 30 min). After dehydration through a graded series of ethanol, the specimen was embedded in Araldite for TEM. A complete series of transverse serial ultrathin sections (60–70 nm) of the individual was prepared using a diamond knife on a Reichert Ultracut S (Reichert Labtec, Germany). Sections were stained with 2 % uranyl acetate and 2.5 % lead citrate in an automatic stainer (Nanofilm Technologie GmbH, Göttingen) for TEM. TEM pictures were documented with a Philips CM120 BioTWIN (FEI, the Netherlands) electron microscope. Images were aligned using Photoshop CS3 software. Based on the resulting stack of images, a 3D model of the anatomy of the creeping larva was reconstructed in Amira 3.0 software (Mercury Computer Systems, Berlin).

Results The crawling larva of C. gracilis possesses three germ layers (Fig. 1): a uniformly monociliated ectoderm that is used in ciliary benthic gliding, a likewise monociliated endoderm that forms in principal a through gut with a gill slit, and the mesoderm that contains epithelial cells and muscle cells. No concentration of nerve cells and no sensory organs could be detected in the epidermis. At the posterior end slightly on the left ventral side, an epidermal protuberance probably marks the position of the prospective postanal stalk (Fig. 1(b), arrowheads). The endoderm forms a hollow through gut that begins on the anterior ventral side at the mouth opening (Fig. 1(a1)) and ends with the terminal anus. Slight constrictions demarcate three partitions corresponding probably to prospective pharynx, esophagus, and intestine. In front of the mouth opening, the gut bulges anteriorly (Fig. 1(a)), forming a comparatively large, hollow cavity anterior to the mouth. An additional single opening is present on the ventral side (Figs. 1(a3), 2a). The connection of the latter opening to the endodermal epithelium indicates that it is a simple gill opening on the ventral side slightly left to the ventral mid line of the body. On the right side, an incipient Anlage of the rightsided counterpart of the aforementioned gill slit is demonstrated (Fig. 2a). The mesoderm surrounds a single continuous coelomic cavity (Fig. 1(a, b)), traversed by cells differentiated

Naturwissenschaften (2013) 100:1187–1191

as muscle cells. The mesoderm surrounds the gut at the posterior third of the animal, stretches far more anteriorly on the ventral side, and connects to the epidermis precisely left and right to the area of the gill slit (Figs. 1 and 2). The complete series of sections in transmission electron microscopic resolution revealed no traces of ciliary bands typical for dipleurula-type larvae. An excretory system involving the leftsided anterior coelom, typical for dipleurula-type and cephalochordate larvae was also not detected.

Discussion Few studies have investigated developmental stages in the genus Cephalodiscus and the results are restricted by the limitations of light microscopy (Masterman 1900; Harmer 1905; Anderson 1907; Schepotieff 1909; Gilchrist 1915; John 1932; Dilly 2013). Light microscopy however does not allow for the unambiguous identification of extracellular matrix, which is the only reliable structural distinction of early ontogenetic germ layers (Bartolomaeus et al. 2009). Moreover, reports on the development of Cephalodiscus rely on material from several different species. Only once a developmental stage with a differentiated through gut was observed (John 1932) in C. nigrescens Lankester 1905. Recently, it has been proposed that possibly two different modes of sexual reproduction exist in Cephalodiscus species (Dilly 2013), and this hypothesis might explain the divergent reports in addition to species specificity. The larva described here is slightly asymmetric with the left gill slit being open, whereas the right seems to be incipient. This slight asymmetry corresponds well with the one described for the ontogeny of Saccoglossus kowalevskii (Agassiz 1873) recently (Kaul-Strehlow and Stach 2013) and fits within the general preponderance of left-sided asymmetries in deuterostomes (Spéder et al. 2007). These are very pronounced in echinoderms (Duboc et al. 2005) and larval cephalochordates (Conklin 1932; Stach 2000). While the correspondence with slight asymmetric development in the harrimaniid S. kowalevskii supports the conclusion of a closer relationship of Cephalodiscus species to harrimaniid enteropneusts, the recent detailed statistical description (Sato and Holland 2008) of fluctuating asymmetry (sensu Palmer and Strobeck 1986; antisymmetry of the authors) in adults of the pterobranch Rhabdopleura normani Allman, 1869, cautions against putting too much phylogenetic weight into the observation of slight asymmetries. The similarities in form of ciliated bands (Nielsen 2013), number of serotonergic cells in the apical organ (Hay-Schmidt 2000), enterocoelic origin of mesoderm (Nielsen 2012), and excretory system (Balser et al. 1993) substantiates the hypothesis that the last common ancestor of hemichordates and echinoderms possessed a planktonic larval stage that has been

Naturwissenschaften (2013) 100:1187–1191

1189

a4 r a

d

v

a3

l

ep

co

p

in co

a2

co

a1

a4 in gsol

ep co

ep in gspir

mo

gsil

co

co

a

*r gsol

gspor

a3

*l

ep

ep in dr a

co

p

l v

ep

cot

a2

gsol

ep

ep

in in l

50 µm mo

v

b

d p

a

a1

150 µm

*l

in

r

mo

*l *r

gsol

cot 150 µm

Fig. 1 a Semi-schematic representation of 3D-reconstruction with transmission electron micrographs (TEM; a 1 –a 4 ) of cross section through crawling larva of a pterobranch. Planes of cross sections indicated in scheme. b Digital 3D-reconstruction based on complete series of TEM sections. Top: oblique view, bottom: ventral view. a, anterior; co, coelom; cot, epithelium of coelom; d, dorsal; ep, epidermis; gs ol/il , left inner/

outer gill slit; gs por/pir, right prospective inner/outer gill slit; in, epithelium of intestine; l, left; mo, mouth; p, posterior; r; right; v, ventral; * l/r, left/ right coelomopore; single arrowhead, epidermal extension (site of prospective stalk). Colors: light blue, epidermis; light red, mesoderm, light green, epithelium of intestine, dark blue, extracellular matrix

termed dipleurula larva (Semon 1888). Moreover, similarities in the aforementioned structures with the exception of ciliated bands can be found in the planktonic larval stage of cephalochordates (Holland and Holland 1993; Stach 2000, 2002), suggesting that a planktonic larva was present in the

last common ancestor of deuterostomes. Figure 3 contrasts two phylogenetic hypotheses. One is based on the logically consistent application of cladistic principles applied to adult morphological traits (Fig. 3a; Ax 2003) whereas the other is the result of current molecular analyses (Fig. 3b; Cannon et al.

1190

Naturwissenschaften (2013) 100:1187–1191

in ep in gspir

gsil

*r

*l

gsol

gspor

*r

ep

co

co

ecm

co

ecm

ep

5 µm

40 µm

in ep in gspir

gsil

*r

*l

gsol

gspor

*r

ep

co

co

ecm

co

ecm

ep

5 µm

40 µm

b

-

-

* Enteropneusta

a

pl/i

gs

- Pterobranchia

Cyrtotreta

-

Pharyngotremata Stomochordata Deuterostomia

gs

Bilateria

Fig. 3 Two contrasting phylogenetic hypotheses of higher deuterostome taxa. a Phylogenetic hypothesis based on morphological characters of adults according to Ax. b Molecular phylogenetic hypothesis according to Vignettes depict representative larval stages. Several transformation events are mapped onto the respective cladogram. cb/d, crawling, benthic/direct development; pl/i, planktonic/indirect development; sb/d, semibenthic (with

sb/d cb/d cb/d

Pt

*

gs

a

pl/i

hi n

Ec

Pr

ot o

os

Ec

ot Pr

-

pl/i

od er m yc at ho de a H r ar rim idae an C iid ep ae ha R ha lod bd isc op ida le e ur id ae

pl/i

pl/i

ia

pl/i

m

cb/d sb/d

st o

cb/d

C

pl/i

to m ia hi no d er R ha m a bd op ta C le ep ur ha id ae lo H d is ar rim cid ae Pt an yc iid ho ae d C ho erid ae rd at a

pl/i

ecm, extracellular matrix; ep, epidermis; gs ol/il, left inner/outer gill slit; gs por/ pir, right prospective inner/outer gill slit; in, epithelium of intestine; * l/r, left/ right coelomopore; arrow, mesodermal cell; double arrowhead, termination of ecm. Colors: light blue, epidermis; light red, mesoderm, light green, epithelium of intestine, dark blue, extracellular matrix

da t

Fig. 2 a Higher magnification transmission electron micrograph of transverse section with gill slit. Plane of cross section indicated in scheme in Fig. 1(a3). b Higher magnification of transmission electron micrograph of right coelomopore, approximately 0.5 μm posterior to plane of section of Fig. 2a. Top: plain transmission electron micrograph. Bottom: transmission electron micrograph overlain with interpretative color code. co, coelom;

ho r

a

Hemichordata Ambulacraria Deuterostomia

Bilateria

b telotroch as remnant of planktonic stage)/direct development. The dagger indicates the suggested phylogenetic position of a recently described Cam, reduction of planktonic, brian fossil of tubicolous enteropneust. dipleurula-like larva; , origin of gill slits;

, modification of planktonic dipleurula-like larva; , reduction of gill slits

Naturwissenschaften (2013) 100:1187–1191

2009). On both hypotheses, larval traits and the decisive morphological character from Ax's hypothesis—absence versus presence of gill slits—are mapped. If the presence of a dipleurula-type larva in the last common ancestor of deuterostomes is accepted, the hypothesis based on adult morphology requires the independent reduction of such a planktonic larva in three different lineages. The molecular hypothesis on the other hand is more parsimonious in this respect, because it requires only a single reduction event in the stem lineage of Harrimaniidae plus Pterobranchia. While the molecular hypothesis requires an additional step in the reduction of gill slits compared to Ax's hypothesis, it is still more parsimonious if these two character complexes are considered. Moreover, the molecular hypothesis smoothly accommodates the recent finding of a fossil tubicolous enteropneust (Caron et al. 2013) from the Cambrian as the sister taxon of Pterobranchia. Thus, the fact that the anatomy of the embryo of C. gracilis lacks any trace of planktonic specializations is therefore consistent with the hypothesis that it evolved from the reduced larva of derived enteropneusts and therefore the sessile adults developed from free-living enteropneust-like animals. Acknowledgments I thank Katrin Braun, Ronny Vogler, and Benjamin Hebel for their help with the 3D-reconstruction. Funding of the DFG and BIOS is gratefully acknowledged.

References Agassiz A (1873) The history of balanoglossus and tornaria. Memoirs of the American Academy of Arts and Sciences. New Series 9:421–436 Anderson K (1907) Die Pterobranchier der schwedischen SüdpolarExpedition 1901–1903. Wiss Ergebnschwedischen Südpolarexpedition 5:1–122 Ax P (2003) Multicellular animals. Springer, Berlin Balser EJ, Ruppert EE et al (1993) Ultrastructure of the coeloms of auricularia larvae (Holothuroidea: Echinodermata): evidence for the presence of an axocoel. Biol Bull 185:86–96 Bartolomaeus T, Quast B, Koch M (2009) Nephridial development and body cavity formation in Artemia salina (Crustacea: Branchiopoda): no evidence for any transitory coelom. Zoomorphology 128:247–262 Cannon JT, Rychel AL, Eccleston H, Halanych KM, Swalla BJ (2009) Molecular phylogeny of hemichordata, with updated status of deepsea enteropneusts. Mol Phyl Evol 52(1):17–24 Caron J-B, Morris SC, Cameron CB (2013) Tubicolous enteropneusts from the Cambrian period. Nature 495:503–506 Conklin EG (1932) The embryology of amphioxus. J Morphol 54:69–151 Dilly PN (2013) Cephalodiscus reproductive biology (Pterobranchia, Hemichordata). Acta Zool. doi:10.1111/azo.12015 Duboc V, Röttinger E, Lapraz F, Besnardeau L, Lepage T (2005) Left-

1191 right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Dev Cell 9:147–158 Gilchrist JDF (1915) Observations on the cape Cephalodiscus (C. gilchristi, Ridewood) and some of its early stages. With an appendix by Sidney F. Harmer, Sc.D., F.R.S. J Nat Hist 16:233–246 Harmer SF (1905) The Pterobranchia of the Siboga Expedition with an account of other species. In: Weber M (ed) Siboga-Expeditie: uitkomsten op zoölogisch, botanisch, oceanographisch en geologisch gebied verzameld in Nederlandsch Oost-Indië 1899– 1900 aan boord HM Siboga onder commando van Luitenant ter Zee 1e kl GF Tydeman. E. J. Brill, Leyden, pp. 132 Hay-Schmidt A (2000) The evolution of the serotonergic nervous system. Phil Trans R Soc London B 267:1071–1079 Holland ND, Holland LZ (1993) Serotonin-containing cells in the nervous system and other tissues during ontogeny of a lancelet, Branchiostoma floridae. Acta Zool 74:195–204 John CC (1932) On the development of Cephalodiscus. ‘Discovery’ Report 6: 193–204 Kaul-Strehlow S, Stach T (2013) A detailed description of the development of the hemichordate Saccoglossus kowalevskii using SEM, TEM, Histology and 3D-reconstructions. Front Zool 10:53 Lankester E (1905) On a new species of Cephalodiscus (C. nigrescens) from the antarctic ocean. Proc Roy Soc London 76:400–402 Masterman AT (1900) On the further anatomy and budding process of Cephalodiscus dodecalophus. Trans R Soc Edinb 34:507–527 Nakano H, Lundin K, Bourlat SJ, Telford MJ, Funch P, Nyengaard JR, Obst M, Thorndyke MC (2013) Xenoturbella bocki exhibits direct development with similarities to Acoelomorpha. Nat Commun 4: 1537 Nielsen C (2012) Animal Evolution. Interrelationships of the living phyla. Oxford University Press, Oxford Nielsen C (2013) Life cycle evolution: was the eumetazoan ancestor a holopelagic, planktotrophic gastraea? BMC Evol Biol 13(1):171 Palmer AR, Strobeck C (1986) Fluctuating asymmetry: measurement, analysis, patterns. Annu Rev Ecol Syst 17:391–421 Peterson KJ, Su Y-H, Arnone MI, Swalla B, King BL (2013) MicroRNAs support the monophyly of enteropneust hemichordates. J Exp Zool B Mol Dev Evol 320:368–374 Sato A, Holland PWH (2008) Asymmetry in a pterobranch hemichordate and the evolution of left–right patterning. Dev Dyn 237:3634–3639 Schepotieff A (1909) Die Pterobranchier des Indischen Ozeans. Zool Jahrb (Abt Syst Ökol Geogr Tiere) 28:429–448 Semon R (1888) Die Entwicklung der Synapta digitata und ihre Bedeutung für die Phylogenie der Echinodermen. Jenaische Z Naturwiss 22:175–308 Spéder P, Petzoldt A, Suzanne M, Noselli S (2007) Strategies to establish left/right asymmetry in vertebrates and invertebrates. Curr Opin Genet Dev 17:351–358 Stach T (2000) Microscopic anatomy of developmental stages of Branchiostoma lanceolatum (Cephalochordata, Chordata). Bonn Zool Monogr 47:1–111 Stach T (2002) Minireview: on the homology of the protocoel in Cephalochordata and ‘lower’ Deuterostomia. Acta Zool 83:25–31 Thompson JV (1836) Natural history and metamorphosis of an anomalous crustaceous parasite of Carcinus maenas , the Sacculina carcini. Entomol Mag London 3:452–456