Echinoidea: Spatangoida): Morphology and Evolution of the Intestinal Caecum

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Org Divers Evol (2014) 14:383–395 DOI 10.1007/s13127-014-0178-2

ORIGINAL ARTICLE

Rediscovery of an internal organ in heart urchins (Echinoidea: Spatangoida): morphology and evolution of the intestinal caecum

Alexander Ziegler

Received: 30 April 2014 /Accepted: 15 June 2014 /Published online: 9 July 2014 # Gesellschaft für Biologische Systematik 2014

Abstract A thorough understanding of the sea urchin Brisaster,andTripylaster, but not in the other seven (Echinodermata: Echinoidea) digestive tract anatomy is a schizasterid genera analyzed. The intestinal caecum is not prerequisite for the correct interpretation of physiological homologous to the sometimes equally named accessory struc- and biomechanical analyses focusing on the gut architecture ture present in the posterior digestive tract of other spatangoid of this ecologically important group of marine invertebrates. A taxa such as Echinocardium or Heterobrissus. Consequently, number of studies have addressed the general arrangement of the previously introduced term recto-intestinal caecum is here the sea urchin digestive tract, but accessory structures such as applied for this latter organ. No correlation could be found siphons and caeca have received less attention. Two studies between the absence or presence of the intestinal caecum and carried out to analyze the gut physiology of various marine any known biological or morphological characteristics of invertebrates briefly mentioned the presence of a previously schizasterid heart urchins. The distribution of the organ undescribed pouch in the posterior digestive tract of the heart among schizasterids supports a close relationship of the gen- urchin (Echinoidea: Spatangoida) species Brisaster latifrons. era Brisaster, Tripylaster, and selected species of Abatus. Dissections, histological, and magnetic resonance imaging data, as well as three-dimensional reconstructions corroborate Keywords Schizasteridae . Digestive tract . Sea urchin . these findings. The novel structure—here termed the intestinal Anatomy caecum—is suspended by a thin mesentery within a coil formed by the posteriormost part of the intestine. The kidney-shaped organ constitutes a derivative of the intestine, to which it is laterally connected through a narrow canal. In Introduction contrast to the sediment-packed main digestive tract, the intestinal caecum is filled with liquid and a flocculent mass. The Sea urchins (Echinodermata: Echinoidea) constitute a numer- organ’s histology is characterized by a thin connective tissue ically significant and ecologically important part of the inver- layer with only a small number of hemal lacunae and muscle tebrate macrofauna in many marine benthic habitats (Steneck fibers, as well as an inner simple columnar epithelium that 2013). Most echinoids are epifaunal organisms that ingest contains numerous dark-brown vacuoles. The intestinal plant material or scrape encrusting organisms off the different caecum is found exclusively among members of the types of substrate (De Ridder and Lawrence 1982). However, Schizasteridae (Spatangoida: Paleopneustina). Specifically, a derived taxon within sea urchins, the Irregularia, has devel- the organ is present in selected species of the genera Abatus, oped a range of infaunal lifestyles, leading to drastically altered modes of food intake (Mooi 1990). A major group of these irregular echinoids, the so-called heart urchins Electronic supplementary material The online version of this article (Irregularia: Spatangoida), significantly affects marine sedi- (doi:10.1007/s13127-014-0178-2) contains supplementary material, which is available to authorized users. ments through movement and feeding and has, therefore, attracted attention not only from paleontologists but also from * A. Ziegler ( ) sediment geologists and physiologists (Bromley and Asgaard Ziegler Biosolutions, Fahrgasse 5, 79761 Waldshut-Tiengen, Germany 1975; Plante et al. 1990; Kanazawa 1992; De Gibert and e-mail: [email protected] Goldring 2008). 384 A. Ziegler

Of particular importance for the comparison of biome- observed in the posterior part of the intestine of B. latifrons.In chanical and physiological data gathered from infaunal particular, the goal is to determine whether this structure is organisms are accurate descriptions of the morphology and homologous to the pouch found in the posterior digestive tract anatomy of the digestive tract. The gut of heart urchins con- of other spatangoid taxa, or whether it actually constitutes a sists of the following regions: buccal cavity, anterior esopha- separate organ. Furthermore, the available histological and gus, posterior esophagus, anterior stomach, posterior stomach, physiological data are compared to propose a potential role of intestine, and rectum (Holland and Ghiselin 1970; De Ridder this structure in the spatangoid digestive process. Finally, a and Jangoux 1993). Like most other irregular sea urchins, comparative analysis among selected spatangoid taxa is under- heart urchins lack an Aristotle’s lantern and, therefore, a taken to shed light on the evolutionary origin of the organ and pharynx, the gut region that passes through the echinoid to examine potential phylogenetic implications. feeding apparatus (Holland 2013). In spatangoids, all regions of the main digestive tract are packed with sediment ingested by the animal. In contrast, accessory structures such as si- Materials and methods phons or caeca remain free of sediment particles, suggestive of a specialized function (De Ridder and Jangoux 1982). Specimens One of these accessory structures, the gastric caecum, is connected to the anterior stomach and forms a thin-walled, Specimens of Brisaster latifrons were dredged from the mud- convoluted pouch (Hoffmann 1871). This often quite promi- dy surface sediment layer in water depths of about 200 m in nent organ can be found in most heart urchin species (Ziegler the central Puget Sound, WA, USA. Individuals of Abatus et al. 2010). The gastric caecum is filled with seawater (Rolet cavernosus, A. ingens, A. nimrodi,andA. shackletoni were et al. 2012) and is presumed to function in nutrient absorption collected near Davis station, Antarctica. The remaining spec- (Thorsen 1998; Rolet and De Ridder 2012). imens used in this study were obtained from the following An additional pouch, assigned different names over time, collections: Australian Antarctic Division (AAD, Kingston, has been described in selected spatangoid species (Koehler Tasmania, Australia), California Academy of Sciences 1883; Wagner 1903; Eichelbaum 1909). This highly contrac- Invertebrate Zoology (CASIZ, San Francisco, CA, USA), tile structure, present near the junction of intestine and rectum, Museum of Comparative Zoology (MCZ, Cambridge, MA, usually contains multiple nodules with a diameter of up to USA), Zoologisches Museum Berlin (ZMB, Berlin, Germany), 1 cm (De Ridder et al. 1985; De Ridder and Jangoux 1993). and Zoological Museum University of Copenhagen Mortensen While some observers hypothesized that these nodules con- Collection (ZMUC, København, Denmark). Further specimen stitute fecal matter (Koehler 1883; Anisimov 1982), more data were found in the literature. In addition, the database The recent studies have shown that they are in fact complex Echinoderm Files (Kroh et al. 2013) was queried for previously structures composed of several layers formed by bacterial uncited articles. Table 2 provides a list of specimens incorporated symbionts that surround large ingesta such as animal remains, into the present study. algal fragments, or pieces of wood (De Ridder et al. 1985; Brigmon and De Ridder 1998;DeRidderandBrigmon2003). Dissection and photography The results of a systematic morphological study suggest that this symbiosis can be found in a few distantly related Dissections were performed on freshly fixed and museum wet spatangoid taxa (De Ridder 1994). specimens under direct observation through a stereomicro- In addition to these two relatively well-known caeca, anoth- scope. The dissections were carried out by removing the aboral er pouch associated with the spatangoid digestive tract is briefly part of the test as well as all gonadal tissue. Photographs of mentioned in two studies that focus on the gut physiology of dissected specimens were taken using digital cameras. several invertebrate sediment burrowers, including the heart urchin Brisaster latifrons (Plante and Jumars 1992;Mayer Histology et al. 1997). As in the case of the abovementioned organ, these two studies assigned multiple different names to this novel Living specimens of B. latifrons were relaxed in 7 % MgCl2, structure. However, as was shown to be the case with other immersed in Bouin’s fixative, and dissected. To ensure pene- internal organs of sea urchins (Ziegler et al. 2009, 2010), the tration of the gut tissues, a hole was cut in the test of each usage of multiple synonymous terms for individual structures specimen before immersion in fixative. Digestive tract seg- by different authors over time (Table 1) may have prevented ments were excised and embedded in paraffin. Histological an unambiguous identification of digestive tract components. sections, 7.5 μm-thick, were cut on a microtome, mounted on In order to provide further insight into the morphological slides, and stained with Gomori’s trichrome stain. The sec- diversity of the spatangoid digestive tract, the aim of the present tions were inspected using a light microscope equipped with a study is to present the first detailed description of the pouch video camera. Rediscovery of an internal organ in heart urchins 385

Table 1 Trilingual list of terms assigned to accessory structures Language Term Reference encountered in the posterior digestive tract of heart urchins English Additional caecum De Ridder and Jangoux (1982) (Echinoidea: Spatangoida). Listed Caecum 2 Kaburek and Hilgers (1999) in alphabetical order Caecum of the small intestine Anisimov (1982) Digestive annex Schatt and Féral (1996) Hind caecum Mayer et al. (1997) Hindgut caecum Plante and Jumars (1992) Hindgut diverticulum Plante and Jumars (1992) Intestinal caecum De Ridder et al. (1985), Temara et al. (1991, 1993), De Ridder and Jangoux (1993), De Ridder (1994), Bromley et al. (1995), Schatt and Féral (1996), Warnau et al. (1998), Thorsen (1998, 1999), De Ridder and Foret (2001), De Ridder and Brigmon (2003), Ziegler (2008), Ziegler et al. (2010), Rolet et al. (2012) Intestinal cecum Brigmon and De Ridder (1998) Posterior caecum De Ridder and Jangoux (1982) Recto-intestinal caecum Buchanan et al. (1980) Second caecum Hyman (1955), Holland and Ghiselin (1970), Kaburek and Hilgers (1999), David et al. (2005) Second gut caecum De Ridder (1982) Small caecum De Ridder and Jangoux (1982) French Ampoule rectale Koehler (1914) Ampoule stercorale Koehler (1914) Appendice Koehler (1883) Boursouflement Léger (1897) Caecum intestinal De Ridder and Jangoux (1985), De Ridder (1987) Dilatation Koehler (1914) Diverticulum Koehler (1883) Poche Koehler (1914) German Blinddarm Eichelbaum (1909) Blindsack Eichelbaum (1909) Divertikel Wagner (1903), Eichelbaum (1909) Kleiner Blindsack Ludwig and Hamann (1904) Sackförmige Erweiterung Eichelbaum (1909) Zweiter Blinddarm Eichelbaum (1909) Zweites Divertikel Wagner (1903), Eichelbaum (1909)

Magnetic resonance imaging using the software ParaVision 4.0 (Bruker Biospin GmbH, Ettlingen, Germany). Information on specimen preparation An intact museum wet specimen of A. cavernosus (ZMB and specific MRI scanning parameters for echinoid museum 5854) was scanned using a high-field small animal magnetic specimens has been published elsewhere (Ziegler et al. 2008; resonance imaging (MRI) scanner equipped with a 7 T super- Ziegler and Mueller 2011; Ziegler 2012). conducting electromagnet (Pharmascan 70/16, Bruker Biospin GmbH, Ettlingen, Germany). Data were obtained Three-dimensional modeling using a shielded gradient set with a maximum gradient strength of 300 mT/m and a birdcage resonator with an inner Three-dimensional (3D) visualization of the MRI-scanned diameter of 38 mm. The scanning sequence used was a specimen of A. cavernosus was carried out using the software FLASHprotocolwith(3.12cm)3 field of view, (384)3pixel Amira 5.2 (Visage Imaging GmbH, Berlin, Germany). All matrix size, 6.7 ms echo time, 30 ms repetition time, 12 structures were manually segmented using the brush tool. averages, ca. 15 h scanning time, and (81 μm)3 voxel resolu- 3D surface rendering, smoothing, and VRML export of the tion. Data acquisition and reconstruction were carried out individual organ models were performed in Amira 5.2. The 386 A. Ziegler

Table 2 List of heart urchin (Echinoidea: Spatangoida) speci- Species Specimen information (locality; test length; source; figure in text) mens analyzed in the present study. All species belong to the Agassizia scrobiculata Valenciennes, Peru, Pacific Ocean; 25 mm; MCZ 3241; Fig. 3a family Schizasteridae, except for 1846 the outgroup taxa Agassizia Paleopneustes cristatus A. Agassiz, 1873 Cuba, Caribbean Sea; 120 mm; MCZ 7887; Fig. 3b (Prenasteridae), Paleopneustes Paleopneustes tholoformis Chesher, 1968 Caribbean Sea; 23.5–161 mm; Chesher (1968) (Paleopneustidae), and Pericosmus akabanus Mortensen, 1939 Gulf of Aqaba, Indian Ocean; 82 mm; ZMUC; Fig. 3c Pericosmus (Pericosmidae) Abatus agassizii Mortensen, 1910 South Orkney Islands, Southern Ocean; 29 mm; ZMUC; Fig. 3d South Orkney Islands, Southern Ocean; 41 mm; ZMUC; Fig. 3e Abatus cavernosus (Philippi, 1845) Kerguelen Islands, Indian Ocean; 28 mm; ZMB 5854; Fig. 1b–d Davis Station, Southern Ocean; 35 mm; fresh material; Figs. 1a, 3f Abatus cordatus (Verrill, 1876) Kerguelen Islands, Indian Ocean; 34 mm; ZMUC; Fig. 3g Kerguelen Islands, Indian Ocean; 41 mm; ZMB 5437 Abatus elongatus (Koehler, 1908) South Orkney Islands, Southern Ocean; 32 mm; ZMUC; Fig. 3h Abatus ingens Koehler, 1908 Davis Station, Southern Ocean; 32 mm; fresh material Abatus nimrodi (Koehler, 1911) Davis Station, Southern Ocean; 40 mm; fresh material Abatus shackletoni Koehler, 1911 Davis Station, Southern Ocean; 40 mm; fresh material Aceste ovata A. Agassiz & H. L. Preparis Island, Indian Ocean; 21 mm; Koehler (1914) Clark, 1907 Tasman Sea, Pacific Ocean; 21 mm; ZMUC Tasman Sea, Pacific Ocean; 22 mm; ZMUC; Fig. 3i Amphipneustes koehleri Mortensen, 1905 Elephant Island, Southern Ocean; 57 mm; ZMUC; Fig. 3j Amphipneustes lorioli Koehler, 1901 South Orkney Islands, Southern Ocean; 45 mm; ZMUC Amphipneustes similis Mortensen, 1936 Elephant Island, Southern Ocean; 36 mm, CASIZ 169959; Fig. 3k Elephant Island, Southern Ocean; 38.5 mm; CASIZ 169959 Brachysternaster chesheri Larrain, 1985 Elephant Island, Southern Ocean; 62 mm; ZMUC; Fig. 3l Gibbs Island, Southern Ocean; 63 mm; CASIZ 169930 Brisaster antarcticus Döderlein, 1906 Kerguelen Islands, Southern Ocean; 43 mm; ZMUC; Fig. 2b Heard Island, Southern Ocean; 52 mm; AAD HIMI H171 Brisaster capensis (Studer, 1880) Cape Peninsula, Atlantic Ocean; 48 mm; ZMUC; Fig. 3m Brisaster fragilis (Düben & Koren, 1844) Tromsö, Atlantic Ocean; 25 mm; ZMB 2766 Faroe Islands, Atlantic Ocean, 50 mm; ZMUC; Fig. 3n Brisaster latifrons (A. Agassiz, 1898) Hokkaido, Pacific Ocean; 48 mm; MCZ 4407; Figs. 3o, 4a Puget Sound, Pacific Ocean; 60 mm; fresh material; Fig. 2c–h Moira atropos (Lamarck, 1816) Virgin Islands, Caribbean Sea; 28 mm; ZMB 5491 St. Thomas, Caribbean Sea; 48 mm; ZMUC; Fig. 3p Ova canaliferus (Lamarck, 1816) Egypt, Mediterranean Sea; 36 mm; ZMUC; Fig. 3q Rovinj, Mediterranean Sea; 40 mm; Kaburek and Hilgers (1999) Marseille, Mediterranean Sea; 44 mm; Koehler (1883) Schizaster edwardsi Cotteau, 1889 Senegal, Atlantic Ocean; 42 mm; ZMUC; Fig. 3r AAD Australian Antarctic Divi- sion, fresh material private col- Schizaster lacunosus (Linnaeus, 1758) n/a; 20 mm; ZMUC lection (specimen not deposited), Triest, Mediterranean Sea; 35 mm; ZMB 4839 MCZ Museum of Comparative Tripylaster philippii (Gray, 1851) South Georgia, Atlantic Ocean; 39 mm; ZMUC Zoology, n/a data not available, ZMB Zoologisches Museum Ber- n/a; 44 mm; ZMB 3744 lin, ZMUC Zoological Museum n/a; 57 mm; MCZ 3235; Fig. 3s University of Copenhagen Tripylus cordatus (Koehler, 1912) Helmert Bank, Southern Ocean; 40 mm; ZMUC; Fig. 3t Mortensen Collection

interactive 3D PDF model was created using Adobe Acrobat Results 9.4 and Adobe 3D Reviewer 9.4 following the procedures described previously (Kumar et al. 2008, 2010; Ziegler et al. The data provided in the two physiological studies that briefly 2011). mention the presence of an additional accessory structure in Rediscovery of an internal organ in heart urchins 387 the spatangoid digestive tract (Plante and Jumars 1992;Mayer structure located near the junction of intestine and rectum in et al. 1997)werebasedonBrisaster latifrons, a species nested other spatangoid taxa such as Echinocardium Gray, 1825 or within the heart urchin family Schizasteridae Lambert, 1905 Heterobrissus Manzoni & Mazzetti, 1878 will be referred to (Spatangoida: Paleopneustina). Apart from reexamining as the recto-intestinal caecum (RIC). The reasons for this B. latifrons, a closely related genus—Abatus Troschel, choice of terminology will be given in the “Discussion” 1851—was studied as well, because observations of juvenile section. specimens had indicated the presence of a similar structure also in this schizasterid taxon (Schatt and Féral 1996). Gross morphology of the digestive tract of Abatus cavernosus Dissections of adult specimens of the brooding heart urchin and Brisaster latifrons Abatus cavernosus show that a conspicuous pouch can indeed be identified in the posterior part of the intestine (Fig. 1a, The morphology of the main digestive tract of A. cavernosus labeled “ic”). MRI data and 3D reconstructions of selected and B. latifrons is similar and consists of buccal cavity, anterior internal organs reveal the precise location of this pouch and posterior esophagus, anterior and posterior stomach, intes- (Fig. 1b, c, d, drawn in red). To avoid potential confusion tine, and rectum (Figs. 1d and 2a). In addition, a short primary with regard to terminology (Table 1), the pouch found in the siphon and two pouches are connected to the main digestive schizasterids B. latifrons and A. cavernosus will here be tract. The esophagus and rectum are slender structures, while referred to as the intestinal caecum (IC), while the caecal stomach and intestine are relatively wide. The primary siphon

Fig. 1 Gross morphology of the digestive tract of Abatus cavernosus. a revealing the location of the intestinal caecum. d Fronto-dorsal view with Dissected specimen with aboral half of test and gonadal tissue removed to all digestive tract elements made transparent except for the intestinal expose the digestive tract. Ambulacrum III facing upwards. b–d Various caecum. Click anywhere on this figure to open an embedded interactive views of a MRI-based 3D model showing selected structures of the 3D model (requires Adobe Reader version 9 or higher on all operating digestive tract. Blue main digestive tract, cyan gastric caecum, pink systems). Table 2 provides additional specimen information. ae anterior primary siphon, red intestinal caecum. b Aboral view. In this specimen, esophagus, as anterior stomach, bc buccal cavity, gc gastric caecum, ic the intestinal caecum is almost completely covered by the intestine and intestinal caecum, in intestine, pe posterior esophagus, ps posterior stom- rectum and therefore can be seen only partially. c Aboral view with main ach, re rectum, si primary siphon. The interactive content of this figure digestive tract made transparent to expose underlying structures, canbeviewedintheSupplementary Material 388 A. Ziegler

Fig. 2 Gross morphology and histology of the digestive tract of Brisaster. a Schematic fronto- dorsal view of the digestive tract of B. latifrons, modified after De Ridder et al. (1985). b Oral view of the digestive tract of B. antarcticus.Partofthe stomach removed (stippled line) to expose the intestinal caecum. Ambulacrum III facing upwards. c–h Histological sections through the posterior digestive tract of B. latifrons, their approximate locations indicated in (b). c Intestine. d Intestinal caecum. e Vacuoles inside the simple columnar epithelium of the intestinal caecum. f Part of the canal connecting intestinal caecum and intestine. g Mesenterial suspension of the intestinal caecum. h Rectum. Table 2 provides additional specimen information. ae anterior esophagus, bc buccal cavity, ca canal, ct connective tissue, ep epithelium, gc gastric caecum, ic intestinal caecum, in intestine, lu lumen of the digestive tract, me mesentery, nu nucleus, pe posterior esophagus, re rectum, si primary siphon, so somatocoel, va vacuole

extends across the body cavity from the junction of anterior and All regions of the digestive tract of A. cavernosus and posterior esophagus to about the midpoint of the stomach. A B. latifrons are filled with closely packed sediment, except secondary siphon is absent. The gastric caecum, a large con- for the two caeca and the primary siphon, which are filled with voluted pouch, is attached to the main digestive tract near the a transparent liquid. In addition, the IC contains a white- to junction of posterior esophagus and anterior stomach. brown-colored, flocculent mass. In the posterior third of the intestine, a kidney-shaped pouch (i.e., the IC) is in contact with the main digestive tract Histology of the posterior digestive tract regions of Brisaster through a short, narrow canal that connects to the lateral side of the intestine. The IC is held in place by a thin mesentery and Histologically, the gut of echinoids consists of the following is suspended horizontally within a coil of the digestive tract layers, from the gut lumen outwards (Hamann 1887): an inner formed by the posterior part of the intestine. In some speci- lining of simple columnar epithelial cells, a layer of connective mens, the IC was almost completely covered by the intestine tissue including hemal lacunae and inner longitudinal and outer and rectum (Fig. 1b). In A. cavernosus, the length of the main circular muscle fibers, and finally a squamous epithelium lining body of the IC was 6 mm, amounting to 17 % of the speci- the main coelomic cavity (or somatocoel). Figure 2b shows an men’s test length of 35 mm and 7 mm in a specimen of 28 mm oral view of the digestive tract morphology of Brisaster test length (25 %). In B. latifrons, the IC was 14 mm long in a antarcticus, here used to illustrate the approximate location of specimen of 48 mm test length (29 %) and 22 mm long in a the histological sections obtained from B. latifrons.Table3 specimen of 60 mm test length (37 %). provides a direct comparison between morphological and Rediscovery of an internal organ in heart urchins 389

Table 3 Comparison of morphological and histological data gathered on the posterior digestive tract of Brisaster latifrons and Echinocardium cordatum. Data for E. cordatum taken from De Ridder and Jangoux (1993)

Species Intestine Intestinal caecum (IC) Recto-intestinal Rectum caecum (RIC)

Contents B. latifrons Sediment, liquid Liquid, flocculent – Sediment, liquid matter E. cordatum Sediment, liquid – Nodules, sediment, Sediment, liquid liquid Inner epithelium B. latifrons Folded (dorsally), smooth Smooth, forming – Folded (ventrally) shallow protrusions E. cordatum Smooth – Folded Smooth Inner epithelial B. latifrons 8–15 μm25–30 μm – 10–15 μm cell height E. cordatum 30–40 μm – 40–90 μm15–30 μm Connective B. latifrons Well-developed, large number of Thin, small number of - Well-developed, large number tissue layer hemal lacunae, small number hemal lacunae and of hemal lacunae and muscle of muscle fibers, presence of muscle fibers fibers golden-brown granular material E. cordatum Well-developed, large number of – Well-developed, Well-developed, large number hemal lacunae, small number large number of of hemal lacunae and muscle of muscle fibers, presence of muscle fibers fibers, presence of iron-rich iron-rich granules and haemal granules lacunae histological findings for B. latifrons and similar data Presence of the intestinal caecum in heart urchins obtained for Echinocardium cordatum (Pennant, 1777). In most parts of the intestine of B. latifrons, the inner epithe- Based on the presence of the IC in A. cavernosus (Fig. 1)and lium has different arrangements on opposing sides of the gut: the B. latifrons (Fig. 2), taxon sampling was extended to include epithelium of the ventral half of the gut circumference is smooth, other members of the family Schizasteridae as well as the while it folds inwards at fairly regular intervals in the dorsal half representatives of further extant paleopneustine taxa, i.e., (Fig. 2c). However, the gut wall and epithelium are entirely Agassizia (Prenasteridae Lambert, 1905), Paleopneustes smooth in the most posterior part of the intestine, i.e., posterior (Paleopneustidae A. Agassiz, 1904), and Pericosmus to the junction of the IC and intestine. The epithelial cells are (Pericosmidae Lambert, 1905). about 8–15 μm tall throughout the intestine. This gut region The IC is absent in the outgroup taxa Agassizia (Fig. 3a), possesses a well-developed connective tissue layer filled with a Paleopneustes (Fig. 3b), and Pericosmus (Fig. 3c). The organ is golden-brown granular material, but only a few muscle fibers. also absent in the schizasterid genera Aceste (Fig. 3i), In contrast to the intestine, the inner epithelium of the IC is Amphipneustes (Fig. 3j, k), Brachysternaster (Fig. 3l), Moira smooth throughout, forming only shallow, irregularly distribut- (Fig. 3p), Ova (Fig. 3q), Schizaster (Fig. 3r), and Tripylus ed protrusions (Fig. 2d). The epithelial protrusions appear to be (Fig. 3t). associated with material contained within dark-brown, spheri- However, an IC can be found in members of the cal vacuoles (Fig. 2e). The inner columnar epithelial cells are genera Abatus (Fig. 3f, g), Brisaster (Fig. 3m–o), and about 25–30 μm tall. Only a small number of hemal lacunae Tripylaster (Fig. 3s). The IC is present in all analyzed and muscle fibers can be found within the connective tissue species of the genera Brisaster and Tripylaster.In layer of the IC. The golden-brown granular material observed Abatus, an IC is present in the species A. cavernosus in the connective tissue layer of the intestine is not present in the (Fig. 3f)andA. cordatus (Fig. 3g),butitisabsentinA. wall of the IC. The epithelium of the canal connecting the agassizii (Fig. 3d, e), A. elongatus (Fig. 3h), A. ingens, IC with the intestine is folded to a greater extent than A. nimrodi,andA. shackletoni. In specimens of the epithelium of the caecum itself (Fig. 2f). A narrow A. agassizii (Fig. 3d, e), sexual dimorphism with regard mesentery surrounds the caecum and canal (Fig. 2g). tothepresenceoftheICwasnotobserved. The rectum is characterized by an epithelium that forms None of the 26 species analyzed in this study had a RIC, wide folds in the cross-sectional plane (Fig. 2h). Epithelial although in particular Moira atropos (Fig. 3p)andOva cells in the rectum are about 10–15 μmtall.Theconnective canaliferus (Fig. 3q) showed prominent dilations in the tissue layer of the rectum is well-developed and contains posteriormost parts of the digestive tract. However, these numerous hemal lacunae and muscle fibers. dilations were located more lateral than the RIC, and the 390 A. Ziegler

Fig. 3 Absence and presence of the intestinal caecum in selected Paleopneustidae (b), and Pericosmidae (c). d–t Selected species of paleopneustine heart urchins (Spatangoida: Paleopneustina). Aboral the family Schizasteridae. Table 2 provides additional specimen views of partially dissected specimens. Ambulacrum III facing upwards. information. gc gastric caecum, ic intestinal caecum, in intestine, a–c Representatives of three outgroup taxa, i.e., Prenasteridae (a), re rectum. Scale bar = 1 cm characteristic nodules usually found inside this organ were of the spatangoid digestive tract (i.e., the RIC). He used also not present. the term “diverticulum” (Table 1) to describe the novel organ he discovered in Echinocardium flavescens (O. F. Müller, 1776). Since then, numerous studies have shed Discussion light on the morphology, histology, ultrastructure, and function of the RIC. In particular, the work performed Homology of posterior digestive tract structures by De Ridder and colleagues has provided fundamental in spatangoids insights into the nature of the symbiosis occurring within this digestive tract structure. However, the caecum Koehler (1883) was the first author to mention the mentioned in these reports differs in the following presence of an accessory structure in the posterior part points from the caecum described here (Fig. 4). Rediscovery of an internal organ in heart urchins 391

Fig. 4 Homology of selected accessory structures associated with the schematic drawing of the general digestive tract morphology of digestive tract of heart urchins (Echinoidea: Spatangoida). a Dissected spatangoids with a recto-intestinal caecum (right). Drawing is modified specimen of Brisaster latifrons (left) and schematic drawing of the from Wagner (1903). Primary and secondary siphons are not shown in the general digestive tract morphology found within all species of the genera schematic drawings. ae anterior esophagus, as anterior stomach, gc Brisaster and Tripylaster, as well as some species of the genus Abatus gastric caecum, ic intestinal caecum, in intestine, pe posterior esophagus, (right). b Dissected specimen of Heterobrissus niasicus (left)and ps posterior stomach, re rectum, ric recto-intestinal caecum

(1) The IC is suspended by a thin mesentery, while the RIC 1993). (6) The color of the IC ranges from white to brown is not (Koehler 1883; De Ridder and Jangoux 1993). (2) The (Figs. 1, 2,and3), while the RIC is often described as a dark- IC is connected to the intestine through a narrow, distinct colored or even completely black structure (Eichelbaum 1909; canal, while the RIC usually connects to the intestine through De Ridder et al. 1985). a broad opening (Wagner 1903; Eichelbaum 1909; De Ridder The two organs also differ significantly in their histological and Jangoux 1993). (3) The position of the IC relative to the characteristics (Table 3): (7) The IC is a non-contractile, intestine is always lateral, while the position of the RIC is smooth-walled pouch, while the RIC is a highly contractile predominantly aboral (Koehler 1883;Wagner1903; organ whose walls are usually thrown into numerous deep Eichelbaum 1909; De Ridder and Jangoux 1993). (4) The IC folds (Wagner 1903). (8) The inner epithelium of the IC is is filled with a transparent liquid and contains a flocculent smooth, while it is heavily folded in the RIC (Eichelbaum mass, while the RIC is predominantly filled with characteristic 1909; De Ridder and Jangoux 1993). (9) The inner epithelial nodules composed of ingested matter and bacterial symbionts cells are taller in the RIC than in the IC (Wagner 1903;De (De Ridder et al. 1985; Brigmon and De Ridder 1998). (5) The Ridder and Jangoux 1993). (10) The connective tissue layer is shape of IC and RIC differs significantly: the IC is a kidney- significantly more developed in the RIC than in the IC and shaped structure, while the RIC is variable in form and size contains a large number of muscle fibers (Eichelbaum 1909; (Koehler 1883; Eichelbaum 1909; De Ridder and Jangoux De Ridder and Jangoux 1993).

Table 4 Physiological parameters of digestive tract regions of the schizasterid Brisaster latifrons. Data from Plante and Jumars (1992)andMayeretal.(1997)

Esophagus Gastric caecum Stomach Intestine Intestinal caecum (IC) N

Redox potential Eh [mV] +305±148 +380±37 +249±120 +269±134 +337±81 2–4 pH 7.3±0 7.8±0.8 7.4±0 7.4±0.1 7.7±0.6 2–3

O2 concentration [μM] 0 21±29 0 0 2±3 2–4 Lipids [g/L] 0.1 –––– 1 Protease [μM/min] 0.1 0.9 0.2 0 2.1 2–14 Esterase [μM/min] 0.8 36.9 5.1 0.2 5.3 2–14 Lipase [μM/min] 0.7 3.1 0.8 0.2 1.2 2–14 Glucosidase [μM/min] 0 0.1 0.1 0 0 2–14 Contact angle [°] 64.3 36.5 32.5 83.6 74 2–14

Micelles Evidence of micelles in all gut regions 4 Low-molecular weight amino acids [mM] 4 13 11 1 22 2–14 High-molecular weight amino acids [mM] 1 7 3 1 23 2–14

Rectum not sampled. Mean values with standard deviation, where available 392 A. Ziegler

The IC is already present in post-metamorphic specimens (Schatt and Féral 1991; Hood and Mooi 1998; David et al. of A. cordatus (Schatt and Féral 1996), which suggests that the 2005), absence or presence of a gastric caecum (Ziegler et al. IC is a permanent organ that can be found in all specimens and 2010), as well as absence or presence of ferric phosphate in the at any given developmental stage of a species known to intestinal wall (Buchanan et al. 1980;DeRidderetal.1985). possess this structure. Furthermore, the gradual decline in Unfortunately, such data for schizasterids are scarce, in par- inner epithelial folding from intestine to caecum (Fig. 2f) ticular with regard to deep-sea taxa. suggests that the IC is, like the RIC, histologically a derivative The general morphology of the organ (i.e., kidney-shaped, of the intestine. The development of the IC could be explained flattened to rounded, narrow connecting canal, lateral position by a migration of intestinal cells, during or shortly after towards intestine, thin caecal wall, blind-ending, liquid-filled, metamorphosis, into the mesentery suspended in the coil of absence of sediment, mesenterial suspension, flocculent con- the posterior part of the intestine. tent)isindicativeofanexchangeofliquidbetweentheICand Based on the abovementioned evidence, it can be conclud- intestine. Furthermore, the histological properties (i.e., thin ed that the IC and RIC are in fact different structures, in turn requiring a differential terminology. Consequently, the IC is defined here as “a non-contractile pouch laterally connected to the posterior third of the spatangoid intestine through a narrow canal,” while the RIC is defined as “a contractile pouch aborally connected to the spatangoid digestive tract near the junction of intestine and rectum through a broad opening”. This terminology incorporates results presented in the present study as well as conclusions reached by Buchanan et al. (1980) and De Ridder and Jangoux (1993). The IC is also not homologous to the accessory digestive tract structure observed in two specimens of the deep-sea schizasterid Aceste ovata.Koehler(1914) briefly mentioned the presence of a relatively large, egg-shaped outgrowth located ventrally near the junction of intestine and rectum. However, dissections of two specimens belonging to the same species did not allow discerning this structure (Fig. 3i).

Potential role of the intestinal caecum in the spatangoid digestive process

While the function of the RIC has been extensively studied (Temara et al. 1991, 1993; Brigmon and De Ridder 1998; Warnau et al. 1998; Thorsen 1998, 1999; De Ridder and Brigmon 2003; Da Silva et al. 2006), almost nothing is known about the IC in this regard. Nonetheless, the anatomical results presented here, in combination with previously acquired physiological data (Table 4), permit developing an initial hypoth- esis about the potential role of the IC. As a first step to determine the possible function of this organ, an attempt was made to correlate the presence or Fig. 5 Occurrence of the intestinal caecum in heart urchins (Echinoidea: absence of the IC in some schizasterids with known biological Spatangoida). a An intestinal caecum is found only in the Schizasteridae and ecological characteristics of this group of heart urchins. (Spatangoida: Paleopneustina). b Distribution of the intestinal caecum among schizasterids. Both phylogenetic trees show the results of an However, the analysis failed to reveal any positive or negative analysis based on morphological characters (Stockley et al. 2005). The correlation with developmental mode (Emlet 1989), feeding three genera mentioned as incertae sedis were not part of the cited strategy (David et al. 2005), burrowing behavior (De Gibert phylogenetic study. Black-framed box genus analyzed in the present and Goldring 2008), sediment composition (David et al. study, black square hypothetical evolutionary event, black star intestinal caecum present in all species analyzed, black-framed star intestinal 2005), brooding behavior (Schatt and Féral 1991), test shape caecum present only in some of the analyzed species, cross fossil taxon, (Kanazawa 1992), bathymetrical or geographical distribution R! loss of structure Rediscovery of an internal organ in heart urchins 393 connective tissue layer, small number of hemal lacunae and while two other studies have shown that Abatus in general muscle fibers, smooth inner epithelium) as well as the avail- could be a paraphyletic clade (Madon-Senez and David 2001; able physiological data (i.e., neutral pH, positive redox poten- Diaz et al. 2012). In the light of these results, the distribution tial, low oxygen concentration, accumulation of amino acids, of the IC within the two taxonomically problematic genera presence of micelles, high enzymatic activity) could indicate a Abatus and Tripylus could constitute an additional informative secretion of digestive fluids into the IC. However, ultrastruc- morphological character. tural and physiological studies will be required to ascertain the Given the presence of the IC in A. cavernosus (Fig. 3f)and organ’s function and to understand its role in the complex A. cordatus (Fig. 3g), a close relationship of these two species digestive process of spatangoids. Species of Brisaster—a seems likely. Similarly, the fact that an IC is absent in all other taxon with a worldwide distribution that comprises several analyzed Abatus species as well as in Tripylus could indicate a shallow-water species—would constitute suitable candidates closer relationship of these taxa, thus rendering Abatus for further analyses. paraphyletic. However, it is certainly also possible that the IC was lost twice, i.e., once within Abatus and once in the Phylogenetic implications ancestor of Tripylus and the other, more derived, schizasterids (Fig. 5b). In this context, it is interesting to note that, with The IC is exclusively found in a subset of schizasterid regard to shape and size, the IC in A. cordatus (Fig. 3g) closely heart urchins (Fig. 3). Unfortunately, the taxon Schizasteridae resembles the organ seen in Brisaster and Tripylaster,while is in need of revision, and to date, no phylogenetic the IC found in A. cavernosus (Fig. 3f) is more rounded than analysis has been performed that would incorporate all kidney-shaped and also significantly shorter. Although only a 19 fossil and 20 extant genera assigned to this heart small number of individuals of this latter species was studied urchin family (Mortensen 1951;Chesher1966, 1972; so far, these observations could point towards a gradual re- Fischer 1966; McNamara and Philip 1980; Larrain 1985a, duction of the structure within the genus Abatus. b; De Ridder et al. 1992; Hood and Mooi 1998; David et al. 2005). However, broad phylogenetic analyses of sea urchins in general (Kroh and Smith 2010) and heart urchins in particular (Stockley et al. 2005) have placed the family Prenasteridae as sister to Schizasteridae, and a clade com- Conclusion posed of Paleopneustidae and Pericosmidae as sister to the previous two families (Fig. 5a). These four families are united The data presented here demonstrate that a further gut acces- in the Paleopneustina, sister to the more speciose heart sory structure—not homologous to any previously known urchin clade Micrasterina, which includes, for example, organ of the sea urchin digestive tract—is present in selected Echinocardium and Heterobrissus. heart urchin taxa. The results also show that sea urchin mor- Due to the lack of suitable wet material, not all extant phology has still not been surveyed comprehensively on the schizasterid genera could be included in the present analysis. organ level. Further morphological studies are therefore Nonetheless, the distribution of the IC strongly suggests bound to provide novel clues for analyses ranging from phys- that this organ evolved only once within Schizasteridae iological to phylogenetic aspects. A more thorough taxon (Fig. 5b). The IC thus seems to constitute a synapomor- sampling in combination with systematic analyses will help phy of a derived subset of schizasterids. However, the to shed further light on the diversity and evolution of sea most derived schizasterids such as Amphipneustes or urchin digestive tract and other internal structures. Brachysternaster are characterized by the absence of an IC, indicating a loss of this structure at some point in schizasterid evolution. Acknowledgments The present article is partially based on results Previous results of a comprehensive phylogenetic analysis obtained by Stephen B. Sampson—I am particularly grateful to him for based on morphological characters reveal a close relationship granting access to previously unpublished data. I would like to thank between Brisaster and Tripylaster (Hood and Mooi 1998). Ashley Miskelly, Rich Mooi, and Andrew Williston for their help with The presence of the IC in all analyzed species belonging to specimen collection, dissection, and photography. Ty Hibberd, Carsten Lüter, Jørgen Olesen, and Robert M. Woollacott provided generous these two genera supports this conclusion. A similarly close access to museum specimens. I am very grateful to Adam Baldinger relationship has been proposed for Abatus and Tripylus and Tom Schiøtte for their help with specimen handling and shipment. (Mortensen 1951; Fischer 1966; David et al. 2005), but mo- Georgy V. Mirantsev provided access to literature, and Susanne Mueller lecular and morphological analyses lead to conflicting hypoth- was instrumental in obtaining MRI data. I would like to thank John M. Lawrence and Andreas Ziegler for their comments on earlier versions of eses regarding the interrelationships between species belong- this manuscript. Additional comments by Chantal De Ridder and Andreas ing to these two genera: two studies have placed Tripylus Kroh helped to improve the text. I am particularly indebted to Gonzalo within Abatus (Féral and Derelle 1991; Féral et al. 1994), Giribet for his hospitality. 394 A. Ziegler

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