Evolutionary Morphology of the Organ Systems in Squat Lobsters and Porcelain crabs (Crustacea: Decapoda: Anomala): An Insight into Carcinization Jonas Keiler,* Stefan Richter, and Christian S. Wirkner Allgemeine & Spezielle Zoologie, Institut f€ ur Biowissenschaften, Universit€ at Rostock, Universit€ atsplatz, 2, Rostock 18055, Germany ABSTRACT Porcelain crabs (Porcellanidae) are one of three taxa within anomuran crustaceans (Anomala) which possess a crab-like body form. Curiously, these three lineages evolved this shape independently from true crabs (Brachyura) in the course of the evolutionary process termed carcinization. The entire pleon in porcelain crabs is flexed under the cephalothorax and the carapace is approximately as broad as long. Despite their crab-like habitus, porcelain crabs are phylogenetically nested within squat lobsters (Munidopsidae, Munididae, Galatheidae). With a pleon which is only partly flexed under the cephalothorax and a cephalothorax which is longer than it is broad, squat lobsters represent morphologically intermediate forms between lobster-like and crab-like body shapes. Carcinization has so far mostly been studied with respect to outer morphology; however, it is evident that internal anatomical features are influenced through this change of body shape too. In this paper, the situation in Galatheoidea is elucidated by adding more taxa to existing descriptions of the hemolymph vascular systems and associated structures and organs. Micro-computer tomography and 3D reconstruction provide new insights. Autapomorphic states of various internal anatomical characters are present in nearly all the studied species, also reflecting some degree of anatomical disparity found within Galatheoidea. The ventral vessel system of porcelain crabs differs distinctly from that of squat lobsters. The differences in question are coherent (i.e. structural dependent) with morphological transformations in the integument, such as the shortening of the sternal plastron, which evolved in the course of carcinization. Shifts in the gonads and the pleonal neuromeres are coherent with the loss of the caridoid escape reaction, which in turn is a consequence of carcinization. The arterial transformations, however, are minor compared to other instances of carcinization in anomuran crustaceans since the last common ancestor of squat lobsters and porcelain crabs was already “half carcinized”. J. Morphol. 276:1–21, 2015. VC 2014 Wiley Periodicals, Inc.

KEY WORDS: Anomura; Galatheoidea; Porcellanidae; hemolymph vascular system; evolutionary morphology

INTRODUCTION Porcelain crabs (Porcellanidae) are one of three taxa within anomalan crustaceans (Anomala) which evolved a crab-like body form independently from each other and independently from true crabs (Brachyura) in the course of the evolutionary process C 2014 WILEY PERIODICALS, INC. V

termed carcinization. As in other crab-like taxa, the entire pleon in porcelain crabs is flexed under the cephalothorax and the carapace is broader than it is long. Despite their crab-like habitus, porcelain crabs are phylogenetically nested within squat lobsters (Fig. 1, see text below). With a pleon which is only partly flexed under the cephalothorax and a cephalothorax which is longer than it is broad, squat lobsters can be seen as morphologically intermediate forms between lobster-like and crab-like body shapes (i.e., squat lobster are “half carcinized”). Traditionally, squat lobsters were subsumed as Galatheidae, which appeared to be paraphyletic, and have only recently been separated into three taxa: Galatheidae, Munididae and Munidopsidae (Ahyong et al., 2010). Porcelain crabs most probably evolved from a squat lobster-like ancestor, a hypothesis first posed at the beginning of the last century (Borradaile, 1907) and returned to by various subsequent researchers (Glaessner, 1960; Martin and Abele, 1986; McLaughlin and Lemaitre, 1997; Tudge, 1997; Schram, 2001). Recent cladistic analyses (Morrison et al., 2002; Dixon et al., 2003; McLaughlin et al., 2007; Chu et al., 2009; Schnabel et al., 2011; Reimann et al., 2011; Bracken-Grissom et al., 2013) have shown that Porcellanidae are closely related to or even nested within squat lobsters. Other analyses show that Galatheoidea are monophyletic, that is, consist of representatives of Munidopsidae,

Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: German Science Foundation (DFG); Contract grant numbers: WI 3334/1-2 and 3334/3-1. *Correspondence to: Jonas Keiler, Allgemeine & Spezielle Zoologie, Universit€ atsplatz 2, Rostock 18055, Germany. E-mail: [email protected] Received 21 March 2014; Revised 6 June 2014; Accepted 19 June 2014. Published online 22 August 2014 in Wiley Online Library ( DOI 10.1002/jmor.20311



Fig. 1. Different phylogenies of Galatheoidea based on combined molecular and morphological (Schnabel et al., 2011) and solely morphological data (Reimann et al., 2011) implying that Porcellanidae derived from a squat lobster-like ancestor.

Munididae, Galatheidae, and Porcellanidae (Schnabel et al., 2011; Reimann et al., 2011; BrackenGrissom et al., 2013). This is supported by the presence of a unique character combination of distinct seta types on the grooming limbs of all four taxa (Keiler and Richter, 2011). On the basis of molecular systematic analyses, Galatheidae and Porcellanidae constitute a clade which is the sister group to Munididae, while Munidopsidae take a basal position within Galatheoidea (Schnabel et al., 2011). Taking morphological data pertaining to the foregut into account, Munididae are basal and paraphyletic and Galatheidae are paraphyletic with respect to Porcellanidae (Reimann et al., 2011). Both hypothesized phylogenies (Fig. 1) necessarily imply that with regard to internal and external morphology, porcelain crabs had a squat lobster- or galatheidlike ancestor. The transformation from the last common ancestor of Galatheidae and Porcellanidae (LCA GP) into recent porcelain crabs is referred to as carcinization, the evolutionary shaping into a crab (Borradaile, 1916; McLaughlin and Lemaitre, 1997; Morrison et al., 2002; see also Scholtz, 2014 for a critical approach). In previous studies of carcinization, this process has been recognized as an evolutionary transformation concerning external features only (e.g., McLaughlin and Lemaitre, 1997; Tsang et al., 2011). However, it appears highly probable that inner organs and inner integumental structures (the endophragmal skeleton) were transformed at the same time (Keiler et al., 2013). Coherence (i.e., structural dependence sensu Richter and Wirkner, 2014) between changes in the integument in the course of carcinization and changes in inner organs such as the hemolymph vascular system has already been shown in a previous study of hermit crabs and king crabs (Keiler et al., 2013). Proceeding from this, we wanted to know whether similar coherence between carcinization and structural features of the hemolymph vascular system and other organs might also be present in porcelain crabs. To date, detailed investigations into the hemolymph vascular system (hvs) and associated organs of Galatheoidea have been limited to the squat lobster Galathea squamifera (Pike, 1947). Journal of Morphology

Only patchy information is available on the vascular and nervous systems of the porcelain crab Porcellana platycheles and the squat lobster Munida sp. (Bouvier, 1891; Pike, 1947), and the internal anatomy of representatives of Munidopsidae has not yet been studied at all. The present study takes a detailed comparative look at the hemolymph vascular system and associated structures and organs in squat lobsters and porcelain crabs, reconstructs morphological transformations and interprets them in an evolutionary context. It also adds more taxa to existing descriptions of the hemolymph vascular systems and associated structures and organs in Galatheoidea, while recently established techniques such as microcomputer tomography and computer-aided 3D-reconstruction provide new insights. Anatomical features are compared and our results discussed in the light of the squat lobster derivation of porcelain crabs and the evolutionary process of carcinization. What kind of transformations in the hemolymph vascular system and associated structures and organs in the ancestral lineage leading to recent porcelain crabs can be deduced by comparing recent Galatheidae, Munididae, Munidopsidae and Porcellanidae? Do anatomical correlations exist between the endophragmal skeleton and the inner organs? What effects did carcinization have on the internal morphology of these taxa? Which structures underwent changes as a result of carcinization?

MATERIAL AND METHODS Studied Species The following species were studied: Galatheoidea Samouelle, 1819 Munidopsidae Whiteaves, 1874 Munidopsis serricornis (Lov en, 1852) (North Sea near Bergen, Norway, coll. 2008) Munidopsis polymorpha Koelbel, 1892 (Lanzarote, Canary Islands, coll. 2011)

Munididae Ahyong et al., 2010 Munida sarsi Huus, 1935 (North Sea near Bergen, Norway, coll. 2008) Munida tenuimana Sars, 1872 (North Sea near Bergen, Norway, coll. 2008)

Galatheidae Samouelle, 1819 Galathea squamifera Leach, 1814 (Gulmarsfjord, Sweden, coll. 2011/2012) Galathea nexa Embleton, 1834 (North Sea near Bergen, Norway, coll. 2008) Galathea dispersa Bate, 1859 (North Sea near Bergen, Norway, coll. 2008)

Porcellanidae Haworth, 1825 Petrolisthes eriomerus Stimpson, 1871 (San Juan Island, WA, USA, coll. 2010) Pachycheles rudis Stimpson, 1860 (San Juan Island, WA, USA, coll. 2010)

CARCINIZATION IN GALATHEOIDEA Porcellana platycheles (Pennant, 1777) (Mediterranean Sea near Banyuls-sur-Mer, France, coll. 2009) Pisidia longicornis (Linnaeus, 1767) (Mediterranean Sea near Banyuls-sur-Mer, France, coll. 2009)

A full list of the studied specimens (which are catalogued and stored in the Zoological Collection of the University of Rostock) and a description of each of the methods applied are provided in Supporting Information Table S1. Resin Injection The acrylic casting resin Mercox 2-CL (Ladd Research, Williston, VT) or the polyurethane-based casting resin PU4ii (vasQtec, Zurich, Switzerland) was injected into the heart of specimens killed immediately before using CO2-saturated water. In a first step, the resin was mixed with the appropriate catalyst and placed in a 5-ml syringe (Luer Lock Solo, Braun, Melsungen, Germany) just before use. The resin in the syringe was injected via an injection cannula (diameter 0.3–0.6 mm, Sterican, Braun, Melsungen, Germany) through the carapace into the heart and the specimens were left for several minutes to allow the resin to polymerize and temper. Alternatively, micropipettes for injection (Hilsberg pipettes, diameter 1.0 mm, thickness 0.2 mm; pulled with a KOPF Puller 720) were filled using the syringe. The pipettes were placed on an adjustable instrument holder in a mechanical micromanipulator and made to pierce the intersegmental membrane between segments, thus penetrating the vascular system.

Fixation and Dehydration For lCT specimens (injected and noninjected) were fixed in Bouin’s or Karnovsky’s fluid or 4% glutar- or formaldehyde for several days, washed and then critical point (EMITECH K850, UK) or freeze dried (Alpha 1-4, Martin Christ, Osterode a.H., Germany; UniCryo MC2L, UniEquip, Munich, Germany).

Corrosion Casting Alternatively, injected specimens were macerated for 1–3 days by repeated baths in 10% potassium hydroxide at 50 C. For easier dissection, the cuticle was bleached in 5% hypochlorite for up to 24 h.

Micro Computer Tomography Dried or macerated specimens were mounted with hot glue on a specimen holder. X-ray imaging was performed with a

Phoenix Nanotom-180 (PhoenixjX-ray, GE Sensing & Inspection Technologies) high-resolution MicroCT system in high resolution mode using the program datosjx aquisition (target: Molybdenum, Mode: 0–1; performance: ca. 8–13 W; number of projections: 720–1440; detector timing: 1,000–3,000 ms; voxelsize ca. 10–30 mm). Using the software datosjx reconstruction a volume file was generated, and a stack of virtual sections exported using the software VGStudio Max (Volume Graphics, Heidelberg). Xradia MicroXCT-200 X-ray imaging system (Carl Zeiss Xray Microscopy Inc., Pleasanton, USA) at 20 KV and 4 W (10.0 scintillator-objective lens unit, 5–20 lm pixel size)

3D-Reconstruction 3D-reconstruction was performed using image stacks of virtual sections obtained by lCT. All reconstructions were created using the software Imaris 6.4.1 and 7.0.0 (Bitplane). The con-


tours of each studied organ (differentiated by gray scale values) were marked on the virtual cross sections with a polygon, and the polygons then used to calculate a surface model (rendering) of the organ (“create surface”). For illustrative purposes, the surface models of some organs were used to expose the volume region corresponding to the organ in question (“mask channel”) and depicted as “blend projection.” In a further optional step, an automatic surface model was calculated (“automatic creation”) to obtain a more realistic surface model of the organ.

Image Management All figure plates were arranged using Corel Graphics Suite X3 (Corel, Ottawa). Bitmap images were embedded into Corel Draw X3 files and digitally edited with Corel PhotoPaint X3.

RESULTS Both external and internal morphology differs in several regards between squat lobsters (Munidopsidae, Munididae, Galatheidae) and porcelain crabs (Porcellanidae), and to a minor extent between the studied species within these taxa. Notable differences between differently sized specimens of the same species were not found. By studying the anatomical structures in more than one specimen of each species we tried to take possible intraspecific variability into account which, if present, is mentioned. A short summary of anatomical features for each studied species is provided in Supporting Information Table S2. External Morphology Squat lobsters (Fig. 2F III,IV) have a carapace longer than it is broad, while porcelain crabs (Fig. 2F V,VI) possess a more rounded carapace which is approximately equal in length and width (proportions illustrated in Fig. 2F IV, VI). In both groups, the linea anomurica (fracture line during ecdysis; arrow) divides the branchiostegites horizontally (Fig. 2A,B). In the squat lobsters (Fig. 2A), the lower section of the branchiostegites (lbs) is about four times the height of the upper section (ubs) and extends posteriorly to the coxa of the fourth leg (arrowhead), while in the porcelain crabs (Fig. 2B) the lower is only 1.5 times the height of the upper section and extends to the coxa of the third leg only (arrowhead). In vivo, the pleon of all studied species is bent under the cephalothorax (Fig. 2C,G), though not to the same degree: in the squat lobsters, the fifth and sixth pleonal segments are not visible in dorsal view, and the overall shape of the body is oval (Fig. 2F IV). In the porcellanid species the fourth to six pleonal segments are hidden and the overall body shape is fairly circular (Fig. 2F VI). In correspondence to the general shape of the pleon, pleonal tergites, and pleurites together form a convex arch in Munida (Fig. 2G,H), though the pleurites are less bent in Galathea (Fig. 2A,I) and Munidopsis. In porcelain crabs, pleurites and tergites form a less convex shield (Fig. 2B,J). In all species, the third to Journal of Morphology

Fig. 2. External integumental structures (A-E, G-J: volume renderings). A and B: Lateral view of Galathea squamifera (A) and Petrolisthes eriomerus (B). Dashed and dotted lines indicate the different height ratios of the branchiostegites present in squat lobsters (A) and porcelain crabs (B). Arrow indicates linea anomurica; arrowhead indicates posterior extension of the lower branchiostegite (lbs). C–E: Ventral view of Pachycheles rudis (C), Munida sarsi (D), Porcellana platycheles (E); pleon virtually cut in D and E to show plastron (plas). Arrowheads show the distinct suture between third thoracic sternites and rest of plastron. F: Schematic drawings of the various different studied galatheoids: (I) Munidopsis serricornis, rostral region. (II) Munidopsis polymorpha, rostral region. (III) M. sarsi, pleon artificially unbent. (IV) G. squamifera. (V) P. eriomerus (Porcellanidae). (VI) P. platycheles, pleon artificially unbent. Dashed lines in III–VI indicate first pleonal segment hidden under carapace. Different relative ratios of carapace length to width in squat lobsters and porcelain crabs are shown as values beside red lines in IV and VI (width set to 1.0). Red square brackets show relative carapace length including rostrum. Scale varies between drawings. G: Lateral view of bent pleon in M. sarsi. H–J: Posterior view of bent pleon in M. sarsi (H), G. squamifera (I) and P. eriomerus (J). Dotted yellow lines indicate divide between tergites and pleurites. Scale bar 5 2 mm. lbs, lower branchiostegite; p1–p5, legs (pereiopods); pl, pleon; pl1–6, pleonal segments; plas, plastron; st3, third thoracic sternite; st8, eighth thoracic sternite; ubs, upper branchiostegite.

Fig. 3. Spatial relationships between the hemolymph vascular system and other internal organs (A-F: surface renderings). C, D, E, and F contain interactive 3D content in the PDF. Please click on each figure to activate the content and then use the mouse to rotate the objects. A–C: Petrolisthes eriomerus (female) in dorsal view (A), ventral view (B) and right-sided lateral view (C). Dashed square indicates thoracic bridge. D: P. eriomerus, male. Dorsal view. E: Munidopsis polymorpha, female. Dorsal view. F: Galathea dispersa, male. Dorsal view. G: Various patterns of gastric ceca (ce) in (I) Munidopsidae, (II) Munididae, (III) Galatheidae and Porcellanidae. Dotted circles indicate position of heart. Scale bar 5 2 mm. a2a, antennal artery; ag, antennal gland; ala, anterior lateral artery; am, anterior gastric muscle; ama, anterior gastric muscle artery; ao, anterior aorta; aoa, accessory optic artery; bl, bladder; br, brain; ce, cecum; cp, carapace; da, descending artery; dxm, dorsal pleonal extensor muscle; eda, epidermal artery; eso, esophagus; fa, fringe artery; g, gut; ga, gastric artery; goa, gonadal artery; h, heart; ha, hepatic arteries; hp, hepatopancreas; lpa, left pleonal artery; ov, ovaries; p1a–p5a, leg arteries; pa, posterior aorta; pm, posterior gastric muscle; pxm, pleonal extensor muscle; pya, pyloric artery; ra, rostral artery; rpa, right pleonal artery; stc, stomach; tm, thoracic muscles; tst, testes; vd, vas deferens; vv, ventral vessel; xm, external mandible adductor muscle.



Fig. 4. Diversity of endophragmal structures. A: Dorsal view of ventral vessel system (red; surface rendering) in Pachycheles rudis located within ventral portion of the cephalothorax (grey; volume rendering, virtually cut). Leg arteries (p1–5) run between colored endosternites (es) in the periphery. Note the significant distance between p4 and p5. B: Posterior portion of the cephalothorax (grey; blend projection) in Munida sarsi virtually opened to uncover the wide passage, formed by the thoracic bridge (tb) and endosternites 7/8, through which the ventral vessel (red; surface rendering) runs into the pleon. Arrowhead points at fused left arteries p1 1 2. C: P. rudis. Same situation as in B, but the passage is distinctly smaller. D–I: Various shapes of the anterior processes (magenta) of the transverse thoracic bridge (tb; volume renderings) in Munidopsis serricornis (D), M. sarsi (E), Galathea squamifera (F), Porcellana platycheles (G), Petrolisthes eriomerus (H) and P. rudis (I). Arrows point at the tip of each process. Scale bar 5 1 mm. da, descending arery; es, endosternites; p1–5, leg arteries; pl, pleon; pvv, posterior ventral vessel; tb, thoracic bridge.

seventh thoracic sternites are fused into a sternal plate, or plastron (Fig. 2D,E), which is roughly semioval in ventral view. Although fused to the plate, the third thoracic sternites are distinctly delimited by a suture (arrowheads, Fig. 2C–E). In the squat lobster species (Fig. 2D), the plastron has an almost straight posterior margin, while in the porcellanid species (Fig. 2E) the emargination is markedly semicircular. Internal Morphology Digestive system. In all studied species the stomach is situated dorsally in the anterior portion of the cephalothorax, while the gut extends Journal of Morphology

as a tube to the telson (Fig. 3A–F). The hepatopancreas fills most of the cephalothorax and is formed by two bunches of tubular diverticles which are connected to the pylorus on each side via a main duct. The hepatopancreas embeds the stomach posteroventrally and extends ventrally close to the ventral nerve cord (Fig. 3A–E). While in M. polymorpha, M. serricornis and M. sarsi, two unpaired ceca are present, they are absent in the Galathea and porcellanid species investigated (Fig. 3G III). The anterior cecum emanates dorsally from the pylorus. In M. serricornis and M. polymorpha, the root of the posterior cecum is located in the cephalothorax (Fig. 3G I), while in



Fig. 5.

M. sarsi it is located in the second pleonal segment (Fig. 3G II). The course of the ceca is variable between specimens.

Antennal glands. The antennal glands are located anterolaterally in the cephalothorax near the second antennae and are dorsally connected to Journal of Morphology



the antennal bladder (Fig. 3A–C). In M. polymorpha (Fig. 3E), the anterior lobes of the bladder are broad and cover the stomach anteriorly and ventrally while the posterior portion of the bladder lies under the carapax and covers the majority of the hepatopancreas. In M. sarsi and G. squamifera, the lobes of the bladders are thinner and extend less far posteriorly (not shown). In the porcelain crabs (Fig. 3A–C), the lobes of the bladders are even thinner and restricted to the anterolateral portion of the cephalothorax (confirmed in P. platycheles and P. eriomerus). Reproductive system. The gonads are paired and folded into two lobes located laterally to the stomach and dorsally on the hepatopancreas (Fig. 3A–D,F) (confirmed in M. sarsi, G. dispersa, P. eriomerus, P. platycheles, P. longicornis). The testes are connected to the coiled vasa deferentia (vd), which both run to the gonopores on the coxae of the eighth thoracic segment. In the Galathea (Fig. 3F) and Munida species, a transversal connection between the testes was not found, while in P. eriomerus (Fig. 3D), P. platycheles and P. longicornis, the vasa deferentia are tightly coiled and lie close together on the ventral nerve cord, encompassing the anterior portion of the gut. The ovaries are connected with each other below the heart, resulting in an H-shaped structure (confirmed in Galathea, P. eriomerus, P. platycheles). In the squat lobsters, the paired posterior lobes of the ovaries do not extend beyond the thorax. In contrast, in the porcellanid species, the lobes extend into the sixth segment of the pleon (confirmed in P. platycheles; Fig. 3A–C). While in the other squat lobsters and porcelain crabs numerous small eggs are released, M. polymorpha females release two large eggs only while up to two precursors (Fig. 3E) are present in each oviduct.

Endophragmal skeleton. The endophragmal skeleton is formed by endosternites and endopleurites, that is, cuticular invaginations located between two contiguous thoracic segments (Fig. 4A). In all species, a transverse connection or transverse thoracic bridge formed by several of the endosternites and endopleurites is present, spanning the ventral portion of the cephalothorax (Figs. 3C and 4B,C). The shape of the bridge differs between the studied species. In squat lobsters, endosternites 4/5, 5/6, and 6/7 and endopleurites 3/4 and 4/5 form the thoracic bridge (confirmed in M. sarsi and Galathea sp.). In porcelain crabs, the bridge is formed by endosternites 4/5, 5/6, 6/7, and 7 and endopleurites 3/4, 4/5, 5/6, and 6/7 (confirmed in P. rudis and P. platycheles). Anteriorly, either one or two anterior processes are present: two small anterior processes in Munidopsis (Fig. 4D) and a short unpaired process in Munida, Galathea and Porcellana (Fig. 4E–G). In Pachycheles and Petrolisthes (Fig. 4H–I), the unpaired process is broad at its base and has a long and slender tip. On the unpaired processes, a longitudinal medial ridge or anterior bifurcation is present. Furthermore, the endopleurites which form the thoracic bridge and endosternites 7/8 together form a small passage in porcelain crabs (Fig. 4C). In squat lobsters, this passage is distinctly wider (Fig. 4B). Ventral thoracic muscles. The ventral thoracic muscles are extrinsic muscles of the legs and are located directly above the plastron. In porcelain crabs, the ventral thoracic muscles of legs p2–p4 differ in orientation from those in squat lobsters. In squat lobsters, each pair of ventral thoracic muscles overlies the pair running into the preceding pair of legs (e.g., the muscles of p3 overlie those of p2; Fig. 5A,B). In porcelain crabs, however, each pair of ventral thoracic muscles is

Fig. 5. Spatial relations between organ systems of the ventral body side. A and B contain interactive 3D content in the PDF. Please click on each figure to activate the content and then use the mouse to rotate the objects. Color code: pink, ventral thoracic muscles; red, ventral vessel system; yellow, nervous system. A: Dorsal view of ventral thoracic muscles, ventral vessel system and nervous system in Munida sarsi (surface renderings). Neuropils are depicted in orange. Descending artery (da) merges with the ventral vessel at the level of the second leg arteries (p2a, arrow). Blue dashed line indicates posterior margin of plastron. B: Dorsal view of ventral thoracic muscles (surf. rend.), ventral vessel system (surf. rend.) and nervous system (volume rendering) in Munidopsis serricornis. Descending artery (da) merges with the ventral vessel at the level of the third leg arteries (p3a, arrow). C: Ventral side of pleon in M. sarsi (vol. rend. and surf. rend.). Cuticle virtually removed to show segmental arrangement of pleonal ganglia (pn2–6). D: Dorsal view of ventral thoracic muscles and ventral vessel system in the ventral portion of the cephalothorax in Petrolisthes eriomerus (virtually cut). Descending artery (da) merges with the ventral vessel at a point between the second and third leg arteries (p2a-p3a, arrow). Blue dashed line indicates posterior margin of plastron. E–G: Various courses of the ventral vessel system (surface renderings, lateral view) as represented in M. serricornis (E), Munidopsis polymorpha (F) and Pachycheles rudis (G). Dashed line represents course assumed as plesiomorphic condition. Arrowhead indicates the distinct bend in the descending artery (da) in porcelain crabs. H: Posterior side view of the ventral vessel system (surface rendering) and virtually cut cephalothorax (vol. rend.) in P. rudis uncovering the medial keel (mk) below the ventral vessel. Arrows point at ascending arteries from the proximal portion of the leg arteries. Asterisks indicate ascending arteries which supply the cephalothoracic ganglion. I: Cephalothoracic ganglion (cg) and fused pleonal ganglia (pn2–pn6) in Porcellana platycheles (vol. rend.). J–K: Schematic drawing of the ventral organs in squat lobsters (J; represented by Galathea) and porcelain crabs (K; represented by Pachycheles) in dorsal view. Color code: pink, ventral thoracic muscles; light blue, sternal plastron; dark blue, endosternites; red, ventral vessel system; yellow, neuropil areas of the ventral nerve cord. Scale bar 5 1 mm. br, brain; cc, circumesophageal connective; cec, circumesophageal commissure; cg, cephalothoracic ganglion; da, descending artery; eg, egg; ema, esophageal-mandibular artery; mp, ganglia of the mouthparts; p1–p5, legs; p1a–p5a, leg arteries; p1nv–p5nv, leg nerves; mk, medial keel; od, oviduct; on, optic neuropil; pfm, pleonal flexor muscles; pn1–pn6, pleonal neuromeres; pvv, posterior ventral vessel; tn4–tn8, thoracic neuromeres; up, uropod muscles; vnc, ventral nerve cord; vtm, ventral thoracic muscles; vv, ventral vessel.

Journal of Morphology



Fig. 6. Schematic drawings of the cephalothoracic ganglion (cg) and the pleonal ganglia (pg2–pg6) and their segmental position within the body. Note the varying degrees of condensation of the pleonal ganglia in porcelain crabs. In Pisidia and Petrolisthes, the sixth pleonal ganglion is located in the fifth pleonal segment, while in Pachycheles and Porcellana it is located between the eight thoracic and first pleonal segment. Note that the depicted order does not necessarily represent an evolutionary cline. The condition in Pisida, however, represents the plesiomorphic state within Porcellanidae (see text also). cg, cephalothoracic ganglion; pg4, fourth pleonal ganglion; pn1, first pleonal neuromere; pl1–pl6, pleonal segments.

overlaid by the preceding pair (e.g., the muscles of p2 overlie those of p3; Fig. 5D). Ventral nerve cord. In all species, the neuromeres of the mandibular, the first and second maxillar and the thoracic segments and the first pleonal neuromer are fused to form a cephalothoracic ganglion (Fig. 5A,B,I) located in the anterior portion of the cephalothorax (thoracic segments th3–th6). Anteriorly, the cephalothoracic ganglion is connected with the brain via the circumesophageal connectives (Fig. 5A). The paired neuropils of the anterior neuromeres lie close together (Fig. 5B), while the neuropils of the posterior neuromeres (tn4–tn8) lie at a greater distance from each other (Fig. 5B and Supporting Information S-Fig. 1). In the porcelain crabs, the distance between the thoracic neuromeres tn4–tn8 is markedly reduced, giving the cephalothoraic ganglion a more compact shape compared with that of the squat lobsters (see Fig. 5J,K; see also Supporting Information S-Fig 1). Posteriorly, the pleonal ganglia (pn2–pn6) are connected to the cephalothoracic ganglion via connectives (Fig. 5A,I). In the squat lobsters, the pleonal ganglia pn2–pn6 are clearly separated from the cg and from each other and are arranged in a segmental pattern, the last ganglion reaching into the sixth pleonal segment (Fig. 5C). In the porcelain crabs (P. platycheles, P. eriomerus, P. rudis) pleonal ganglia pn2–pn5 lie close together within the seventh thoracic to the first pleonal segment (Fig. 5I). While the last ganglion (pn6) in P. eriomerus lies

in more distance from the other ganglia and is located in the fifth pleonal segment, pn6 does not reach beyond the first pleonal segment in P. platycheles (Fig. 5I) as it is closely attached to the preceding ganglion. A schematic overview is provided in Figure 6. Circulatory System Hemolymph vascular system. All the organs mentioned above are supplied by the hemolymph vascular system. In general, within each family of studied species the hemolymph vascular systems are very similar, while differences between the families are apparent in vascular branching patterns and course. Significant differences in vascular morphology between families or species will be discussed below. The terminology used here is an extended version of that used for the descriptions of the hemolymph vascular systems of hermit crabs (Paguridae) and king crabs (Lithodidae) by Keiler et al. (2013). Heart (Fig. 7). The heart is located dorsally in the cephalothorax under the carapace and above the anterior portion of the gut, suspended in place by several ligaments (Fig. 7A). Dorsal ligaments extend between the heart and the carapace. Ventral ligaments extend between the two lateral margins of the heart and the inner side of the integument. The heart is enclosed in a hemolymph-filled space, the pericardial sinus (ps, Fig. 7B,D), which is framed laterally by the endopleurites of the endophragmal skeleton, ventrally Journal of Morphology



Fig. 7. Gross morphology of the heart in various galatheid species. A: Dorsal view of the heart in Pachycheles rudis (volume rendering). Dorsal ostia indicated by dashed circles. Arrows indicate the prominent dorsal margins to which the cardiac ligaments (cl) attach. B: Dorsal view of the heart (h) within the pericardial sinus (ps) in Munida sarsi (surface rendering). Dorsal ostia indicated by dashed circles. C: Lateral view of the heart (h) in M. sarsi and the arteries branching off it (surface rendering). Ostia on the left side are indicated by dashed circles. D: Anterolateral view of the heart within the pericardial sinus (ps) in M. sarsi showing the associated branchipericardial sinus carrying oxygenated hemolymph from the gills (surface rendering). E: Virtual horizontal section through the heart in M. sarsi showing the ventral intrinsic muscles strands (surface rendering). Arrows indicate the distinct pair of strands found in all studied species. F: Anterior view of the heart in Petrolisthes eriomerus showing the slightly concave ventral side and the convex dorsal side (volume rendering). Ventral ostia are indicated by dashed circles. G: Volume projection of an ostium with a pair of flaps (arrows) visible in P. rudis. H: Horizontal section through the heart in P. rudis to show the dorsal intrinsic muscle strands (volume rendering). I: Virtual transversal section through the heart in P. eriomerus. Scale bar 5 500 lm. ala, anterior lateral artery; ao, anterior aorta; bps, branchiopericardial sinus; cl, cardiac ligament; da, descending artery; g, gut; h, heart; ha, hepatic artery; myc, myocardial muscle strands; ov, ovaries; P5, fifth pereiopod; pa, posterior aorta; ps, pericardial sinus.

by the pericardial septum (Fig. 7I) and dorsally by the integument. The heart is almost pentagonal in horizontal profile. The ventral side is slightly concave and the dorsal markedly convex. The heart is equipped with three pairs of ostia, slit-like openings which are arranged transversely to the anteroposterior body axis (Fig. 7C). Two pairs are located dorsally (anterodorsal and posterodorsal ostia; Fig. 7A,B) and one pair is located ventrolatJournal of Morphology

erally (Fig. 7F). The ostia are equipped with a pair of lips each (Fig. 7G). Several muscle strands (myc) cross the lumen of the heart (Fig. 7E,H). While the overall shape of the heart is broadly symmetrical, most muscle strands appear to be arranged asymmetrically. However, in the ventral portion of the heart, some of the main strands are arranged pairwise (arrows, Fig. 7E; confirmed in M. sarsi, G. squamifera and P. rudis).



Fig. 8. Dorsal anterior arteries. E and F contain interactive 3D content in the PDF. Please click on each figure to activate the content and then use the mouse to rotate the objects. Color code: pink, anterior aorta; blue, anterior lateral artery; green, hepatic artery. A: Dorsal view of the cephalothorax in Munida sarsi. Arrows indicate arteries of the rostral spines. Surface renderings. B and C: Anterior aorta (pink) and brain (yellow) in M. sarsi. X-ray-dense tissue (neuropil) displayed in orange. Surface renderings. D: Volume rendering of cast of the left eye in M. sarsi. Arrows indicate side branches of the optic artery which supply the optic neuropils. E: Lateral view of the cephalothorax in M. sarsi. Surface renderings. F: Lateral view of the anterior aorta (ao) in Pachycheles rudis showing the anterior bend (arrow). Note the anteroventral course of the hepatic artery (ha). Surface renderings. G and H: Anterior aorta (pink; surface rendering) and brain (yellow; volume rendering) in Petrolisthes eriomerus. Asterisks indicate accessory brain arteries. I: Anterolateral view of the anterior aorta (surface rendering) in P. eriomerus showing the four emanating accessory brain arteries (asterisks). J: Schematic drawings of optic arteries in squat lobsters (I) and porcelain crabs (II). Scale bar 5 5 mm in A & E and 1 mm in B–D and F–I. a1a, antennular arteries; a2a, antennal arteries; a1nv/a2nv, antennal nerves; aba, accessory brain artery; aga, antennal gland artery; ama, anterior gastric muscle artery; aoa, accessory optic artery; ba, brain artery; bsl, branchiostegal lacunae; cec, circumesophageal commissure; eda, epidermal artery; epa, epistomal artery; fa, fringe artery; ga, gastric artery; h, heart; lba, lateral brain artery; maf, myoarterial formation; oa, optic arteries; pct, protocerebral tract; ra, rostral artery; rn, renal hemolymph network; sgl, arterial widening of sinus gland.

Journal of Morphology



Fig. 9. Schematic vascular trees of the anterior lateral arteries illustrating the various distinct patterns observed in Galatheoidea. Labels in circles represent the target organs or terminal arteries, respectively. Colors indicate recurring combinations of arteries. Distances between the nodes do not reflect the natural proportions (see Fig. 8A,E,F). a1 first antenna; a2 second antenna; aba, accessory brain artery; aga, antennal gland artery; ama, anterior gastric muscle artery; aoa, accessory optic artery; epa, epistomal artery; eda, epidermal artery; fa, fringe artery; goa, gonadal artery; ga, gastric artery; pya, pyloric artery; ra, rostral artery.

Artery systems. Five artery systems branch off the heart: the unpaired anterior aorta, the paired anterior lateral arteries, the paired hepatic arteries, the unpaired posterior aorta and the ventral vessel system. The latter consists of the vertical descending artery (da) and the horizontal ventral vessel (vv). The proximal portion of all these cardiac vessels lies within the pericard. Between the heart and each vessel there is a valve. Anterior aorta (Figs. 3A,D and 8). The anterior aorta emanates anteriorly from the heart, passes the posterior gastric muscles and runs in an anterior direction above the stomach just below the carapace into the cephalic region, passing the anterior gastric muscles on the way (Fig. 3A). Above the brain, the unpaired brain artery branches off and descends ventrally into the brain (Fig. 8B,C,G), where it turns anteroventrally and bifurcates. Both branches then turn in a posterior direction toward the olfactory lobes of the brain (confirmed in M. serricornis, M. sarsi, P. eriomerus, P. rudis; Fig. 8G). Directly posterior to the brain artery, a pronounced myoarterial formation is observable as a widening of the anterior aorta (Fig. 8C,E,F,H). The anterior aorta continues anteriorly and bifurcates into the optic arteries which run into the eye stalks to supply the sinus gland and the optic neuropils. (arrows, Fig. 8D; confirmed in M. sarsi). The course of the optic arteries differs slightly between the taxa. While they bifurcate horizontally at a 45 angle in squat lobsters (Fig. 8J I) Journal of Morphology

and run directly anteriorward (in a V-shape; confirmed in M. sarsi and G. squamifera), they carry out a posterior turn in porcelain crabs (Fig. 8J II) before continuing anteriorward into the eyestalks (in a W-shape; confirmed in P. platycheles, P. eriomerus and P. rudis). From the proximal part of the optic arteries posteroventrally directed branches (accessory brain arteries) emanate into the brain (Fig. 8G,H,I; confirmed in P. eriomerus and P. rudis). In porcelain crabs, the optic arteries are tightly attached to the anterior margin of the brain and are internalized into the protocerebral tract (Fig. 8G,H; confirmed in P. eriomerus and P. rudis). The anterior part of the anterior aorta in P. rudis differs from that in the other porcellanids in its exhibition of a distinct posteroventral bend (arrow, Fig. 8F). Anterior lateral arteries (Figs. 3A,D,E; 8A,E,F and 9). The anterior lateral arteries (ala) emanate from the heart directly laterally to the anterior aorta. They run (above the gonads) in an anterior direction at a lateral angle of approximately 45 and then follow an almost parallel course to the anterior aorta (Figs. 3A,D and 8A,E,F). Both anterior lateral arteries give rise to several laterally directed and variably arranged arteries which supply mainly the epidermis (eda, Fig. 8A,E) and the arteries supplying the gonads (goa, Fig. 3D). Medially, two arteries emanate: the pyloric artery (pya) and the gastric artery (ga) which split into several small branches which supply the stomach (Fig. 8A).


In the cephalic region, the same set of arterial branches (defined by their target organ or region) is present in all studied species (Figs. 3D, 8A,E,F, and 9; confirmed in M. serricornis, M. sarsi, P. eriomerus, P. rudis): Distolaterally, a branch (red, Fig. 9) turns laterally in a backward direction and gives off several smaller arteries which supply the external mandible adductor muscles (Fig. 3D). It then (as confirmed in P. eriomerus) continues in a posterior direction (fringe artery) along the branchiostegites. Anteriorly, a side branch runs into the second antennae (antennal artery). Ventromedially, a branch (antennal gland artery) runs into the antennal gland and branches there into numerous smaller arteries (rn, Fig. 8A,E). Dorsomedially, a branch (green, Fig. 9) emanates and bifurcates into the arteries which supply the rostrum (rostral artery) and the anterior gastric muscles (anterior gastric muscle artery; Fig. 3D). Distomedially, a branch (blue, Fig. 9) bifurcates into a dorsomedial and ventromedial vessel. The dorsomedial vessel branches off into each eye-stalk (accessory optic artery) and toward the brain (accessory brain artery). The ventromedial vessel branches off into the first antennae (antennular artery) and the epistome (epistomal artery) (Fig. 8E). The sequence of these arterial branches differs distinctly between squat lobsters and porcelain crabs (see Fig. 9) and the relative distances between the branches differ between the species. The rostral arteries also differ between the species due to differences in the shape of the rostrum. If present, lateral spines are supplied by side branches off the rostral arteries. In the Munida species, each rostral artery gives rise to a lateral vessel. These run first into the supraocular spine and on into the medial spine, where they anastomose into an unpaired vessel (Fig. 8A). Hepatic arteries (Figs. 3D, 7C and 8A,E,F). The hepatic arteries (ha) branch off directly beneath the anterior lateral arteries and continue their course anteroventrally (below the gonads; Figs. 3D and 7C), giving rise to several branches which supply the hepatopancreas (Fig. 8A,E,F). In some specimens of P. eriomerus and P. rudis, the hepatic arteries were found to anastomose below the pylorus. In one of these specimens (P. eriomerus), the right hepatic artery also anastomosed with the left mouthpart arteries of the ventral vessel (not shown). Posterior aorta (Figs. 3 and 10). The posterior aorta emanates from the posteroventral portion of the heart and runs above the gut in a posterior direction. The proximal section bifurcates into two equally sized main arteries, the left and right pleonal artery, which run parallel on each side of the gut (in females above the posterior lobes of the ovaries) toward the end of the pleon into the telson (Fig. 10). On the way, several lateral vessels (posterior lateral


arteries) emanate segmentally which mainly supply the pleonal muscles (dorsal extensor muscles and ventral flexor muscles). Several scattered vessels emanate medially from the pleonal arteries and supply the gut. The unpaired proximal section of the posterior artery differs in length between the studied species (Fig. 10A). In M. sarsi, G. squamifera, M. polymorpha, P. platycheles, P. longicornis, P. eriomerus and P. rudis, the point of bifurcation into the pleonal arteries is located in either the last thoracic or the first pleonal segment (Fig. 10A). In M. serricornis, in contrast, the bifurcation is located in the last pleonal segment, resulting in a long unpaired proximal section from which the posterior lateral arteries emanate to each side. Furthermore, the first and second posterior lateral arteries in M. serricornis have a common root (Fig. 10D) and the sixth pleonal segment (pl6) possesses an additional posterior lateral artery (pla6, Fig. 10F,G). Ventral vessel system (Figs. 3, 5, 11 and 12). The ventral vessel system is made up of the descending artery, the ventral vessel and its side branches and mainly supplies the ventral nerve cord, the legs (pereiopods p1–p5) and the mouthparts. The descending artery, which has the greatest diameter of all arteries branching off the heart, emanates from the right side of the posteroventral part of the heart, directly beside the root of the posterior aorta, and runs ventrally (Fig. 3C). In all studied specimens (see Table 1), the descending artery crosses the gut on the right (Fig. 3A,C,D). The descending artery proceeds ventrally, pierces the ventral nerve cord between the sixth and seventh thoracic neuromeres (tn6 1 tn7) and merges into the ventral vessel below. The merging point of the descending artery and the ventral vessel is directly under the transverse thoracic bridge (Fig. 4A). The ventral vessel gives rise, in a segmental pattern to each side, to the paired leg arteries (p1a–p5a) and to the arteries supplying the mouthparts (Fig. 11). It merges anteriorly into the esophageal-mandibular artery and posteriorly into the posterior ventral vessel that runs into the pleon. In squat lobsters, the posterior ventral vessel runs into the pleon directly, while in porcellanids it branches into several smaller vessels (Fig. 5D,G,H). The ventral vessel supplies the cephalothoracic ganglion (cg) via six ascending arteries which run between the neuromeres: four arteries emanate anterior to the descending artery, one posterior to it and one laterally from it (Fig. 5H; confirmed in M. sarsi, G. squamifera, P. rudis, and P. eriomerus). The merging point and course of the descending artery, the angle between the leg arteries and the shape of the posterior ventral vessel differ between the species. The descending artery merges with the ventral vessel either at the level of the second leg arteries (p2a, Fig. 5A), between second and third leg arteries (p2a-p3a, Fig. 5D,H) or at the Journal of Morphology



Fig. 10. Posterior aorta. C and F contain interactive 3D content in the PDF. Please click on each figure to activate the content and then use the mouse to rotate the objects. A: Schematic drawings of the posterior aorta (violet) in the various galatheoid taxa: (I) Munidopsis serricornis. (II) Munidopsis polymorpha. (III) Munida sarsi. (IV) Galathea spp. (V) Porcellanidae. Specimens are not drawn to scale. B and C: Posterior aorta (violet) in male M. sarsi and its spatial relationship to pleonal extensor muscle (pxm), gut (g) and gonads (blue). Surface renderings. Dashed lines indicate borders between pleonal segments (pl1–pl6). Arrowheads indicate posterior lateral arteries. Dashed circles indicate gonopores. D and E: Posterior aorta (violet) and gut (g) in M. serricornis (D) and M. sarsi (E). Surface renderings. Arrowhead points at split between first and second posterior lateral arteries (pla1 1 pla2). Arrow points at root of the posterior cecum. F: Posterior aorta in M. serricornis. G: Rear portion of the posterior aorta (surface rendering) of M. serricornis. The sixth pleonal segment (volume rendering) is shaded in blue. Scale bar 5 2 mm. ce, posterior cecum; g, gut; h, heart; lpa, left pleonal artery; pla1–6, posterior lateral arteries; pxm, pleonal extensor muscle; rec, rectum; rpa, right pleonal artery; t, telson; ta, telsonal artery; tst, testes; up, uropod; upa, uropodal artery; vd, vas deferens.

Journal of Morphology

CARCINIZATION IN GALATHEOIDEA TABLE 1. Various patterns of the ventral vessel system Species

Course da

Merging point da-vv

Branching of pvv

Munidopsis serricornis Munidopsis polymorpha Munida sarsi Munida tenuimana Galathea dispersa Galathea sp. Galathea squamifera Petrolisthes eriomerus Pachycheles rudis Porcellana platycheles Pisidia longicornis

R (3) R (7) R (13) R (2) R (1) R (2) R (6) R (12) R (3) R (12) R (2)

p3 p3 p2 ? p3 p3 p3 p2-p3 p2-p3 p2-p3 ?

Not branched Not branched Not branched ? Not branched Not branched Not branched Branched Branched Branched ?

In all galatheoid species (number of specimens in parentheses), the descending artery (da) runs along the right side (R) of the gut. The merging point of the descending artery with the ventral vessel (vv) differs relative to the roots of the leg artery (p2-p3).

level of the third leg arteries (p3a, Fig. 5B; see also Table 1). Despite these differences, the merging point of the descending artery and the ventral vessel is located roughly between the fourth and


fifth thoracic segments, taking the ventral thoracic muscles as a point of reference. In the Galathea and Munida species, the descending artery takes a slightly curved anteroventral course before piercing the ventral nerve cord (Fig. 5E). In the porcellanid species, it describes a much more distinct curve, running ventrally, then bending anteriorly about 90 and continuing just above the ventral nerve cord before piercing it (Fig. 5G). Furthermore, the ventral vessel in porcelain crabs runs above a dorsally directed median keel located anteriorly to the posterior emargination of the plastron, resulting in a distinct posterior rise (Fig. 5G,H). The keel is much less pronounced in squat lobsters. The course of the descending artery in the two Munidopsis species is variable. However, the descending artery in all studied females of M. polymorpha (n 5 5) describes, uniquely, an inverted L, running steeply ventrally toward the ventral nerve cord, then, at about the level of the fifth leg arteries, turning sharply anteriorly by about 90 and bypassing the huge eggs in the ovaries (Fig. 5F).

Fig. 11. Mouthpart arteries (surface renderings). B, E, and F contain interactive 3D content in the PDF. Please click on each figure to activate the content and then use the mouse to rotate the objects. A: Munidopsis polymorpha. B: Galathea squamifera. C: Pachycheles rudis. D: Petrolisthes eriomerus. E: P. eriomerus. F: Porcellana platycheles. A-D, anteroventral view. E-F, posterolateral view. Scale bar 5 1 mm. br, brain; cec, circumesophageal commisure; cg, cephalothoracic ganglion; ea, esophageal artery; ema, esophageal-mandibular artery; es, endosternites; eso, esophagus; md, mandibular artery; mx1-2, maxillar arteries; mxp1-3, maxillipedal arteries; p1a–p4a, leg arteries; scg, scaphognatite artery; stc, stomach.

Journal of Morphology



Fig. 12. Various examples of the variable patterns of mouthpart arteries found in squat lobsters (Munida, Galathea) and porcelain crabs (Petrolisthes, Pachycheles, Porcellana). Colored lines indicate specific arteries (red 5 esophageal mandibular artery (ea1md); green 5 arteries to first and second maxillipeds, which emanate close together; blue 5 artery of the third maxillipeds). Dashed lines indicate variable course within the respective pattern. eso, esophagus; md, mandible; mx1-2, maxillae; mxp1–3, maxillipeds; p1, first leg (pereiopod 1).

Before the leg arteries run into their respective leg, they proceed between the cuticular walls formed by the endosternites and endopleurites (Fig. 4A). The pattern of the leg arteries is variable and was asymmetrical in some specimens (Fig. 5A). The asymmetry was most marked in the Munida species and lowest in porcelain crabs. However, in most specimens, the pattern was fairly symmetrical. The angles at which the leg arteries (p1a–p5a) emanate and the distances between the leg artery pairs differ between squat lobsters and porcelain crabs. In squat lobsters, the (symmetrical) leg arteries emanate successively at roughly equally intervals (Fig. 5J). The arteries of p1 emanate at about 45 anteriorly, of p2 at about 90 and of p3–p5 at about 30–45 posteriorly. In porcelain crabs, leg arteries p1a–p4a emanate close together, while p5a emanates some distance posteriorly (Figs. 3B, 4A, and 5H,D,K). The arteries of p1 emanate at about 30 anteriorly, p2–5 at about 30–45 posteriorly. From the proximal portion of the leg arteries p1a–p4a, a pronounced branch (bra) runs dorsally and supplies the thoracic muscles attached to the thoracic bridge (Fig. 5H; confirmed in G. squamifera and P. rudis). Arterial supply to the mouthparts (Figs. 11 and 12). In the cephalic region, the ventral vessel ascends at an angle of 30–45 (Fig. 4E–G) and terminates in the mouthpart arteries. The mouthpart arteries originate from the anterior part of Journal of Morphology

the ventral vessel. In all species, the ventral vessel bifurcates anteriorly at the level of endosternites 3/4, giving off successive side branches to the maxillipeds (mxp1–3) and the first and second maxillae (mx1-2; Fig. 11C). Medially between these two branches (Fig. 11B,C) or asymmetrically from the left (Fig. 11D) or right (Fig. 11A) branch, the unpaired esophageal-mandibular artery (ema) emanates and then bifurcates immediately posterior to the circum-esophageal commissure (cec, Fig. 5A). While present in all other species, no unpaired esophageal-mandibular artery was observed in M. serricornis, where the mandibles and esophagus are supplied by the two lateral branches that supply both pairs of maxillae and the three pairs of maxillipeds (not shown). The branching pattern of the mouthpart arteries is highly variable between and within the studied species in other ways as well (Fig. 12). The roots of the arteries to mxp1 and mxp2 generally lie close together (confirmed in M. sarsi, G. squamifera, P. eriomerus, P. rudis, P. platycheles), but in some specimens of these species these arteries actually have a common root. The roots of the arteries of the third maxillipeds (mxp3; blue line in Fig. 12) are also subject to interspecific variation and intraspecific variability, with the situation in P. platycheles (Fig. 11D) differing distinctly from that in the other species, where the mxp3 arteries emanate from the proximal portion of the p1 arteries (Fig. 11). Variation is also observed in the root of the esophagealmandibular artery (red line in Fig. 12).


Hemolymph lacunar system (Figs. 7 and 8). After leaving the vascular system, hemolymph is channeled (back) to the heart via lacunae and sinuses (hls, hemolymph lacunar system). The most distinct sinuses are the branchiopericardial (or branchiocardiac) sinuses (Fig. 7D), which channel hemolymph from the paired gills (pleurobranchs) to the pericardial sinus. A large lacunar mesh (branchiostegal lacunae) is found between the epidermal layers of the branchiostegites covering their posterior and posterolateral regions (Fig. 8A,E) (confirmed in M. sarsi). This network is connected to the pericardial sinus via channel-like sinuses (not shown). DISCUSSION Our investigations reveal that the internal anatomy of squat lobsters and porcelain crabs differs in more aspects than previously known, and, surprisingly, that within squat lobsters the internal anatomy is far more disparate than one would expect from the rather uniform outer morphology of this group. Distinct anatomical differences were even discovered within munidopsid squat lobsters, as they were also within porcelain crabs. What information can be extracted from these new data to elucidate the evolution of Galatheoidea in general and of porcelain crabs and carcinization in particular? To be able to answer these questions, we deduce the anatomical ground pattern of Galatheoidea (represented by the last common ancestor of all galatheoids, LCA G) and of the (squat lobster-like) last common ancestor of Galatheidae (or if Galatheidae are paraphyletic, that branch which is closest to Porcellanidae) and Porcellanidae (LCA GP). The anatomical transformations toward Recent porcelain crabs are reconstructed and compared with those in other crab-like decapods in order to shed light in the transformation pathways involved in carcinization. The Anatomical Ground Pattern of Galatheoidea (LCA G) and Comparison to Other Decapods Habitus. The LCA G can be assumed to have featured a squat lobster-like habitus, differing in this respect from most noncarcinized decapods (“macrurans”) which have a more developed and straight pleon and which (with the exclusion of spiny and slipper lobsters) do not possess a broad plastron. The more flattened pleon in squat lobsters still permits the caridoid escape reaction (“tail flipping”). Squat lobsters thus represent what could be regarded as a morphologically intermediate state between “macrurans” and porcelain crabs and might therefore be termed “half carcinized.” Nervous system. The LCA G is assumed to have possessed segmentally arranged pleonal ganglia pg2–pg6 extending into the sixth pleonal seg-


ment, the plesiomorphic condition found in macruran decapods (see Bouvier, 1889). In macruran decapods, however, the first pleonal neuromere is distinctly separate from the preceding neuromeres of the cephalothorax, and the fusion of the first pleonal ganglion with the cephalothoracic ganglion has been suggested as a synapomorphy of Anomala and Brachyura (forming together Meiura; see Scholtz and Richter, 1995). Artery systems. In all studied Galatheoidea, five artery systems branch off from the heart—a condition which is represented in the LCA G and a situation found in all other decapods (see Keiler et al., 2013 and references therein), and therefore plesiomorphic for the LCA G. Differences, however, are evident in the course and branching pattern of these artery systems. Anterior aorta. The V shape of the optic arteries in squat lobsters represents the plesiomorphic condition since it is also found in pagurid hermit crabs and lithodid king crabs (Keiler et al., 2013). Anterior lateral arteries. The particular sequence of side-branches emanating from the anterior lateral arteries in squat lobsters is also present in hermit crabs and king crabs (Keiler et al., 2013, Fig. 5) and therefore is a plesiomorphy which was present in the LCA G. Hepatic arteries. In their anteroventral course, the hepatic arteries of galatheoids correspond with the hepatic arteries found in other decapods such as brachyuran crabs (McGaw, 2005, p. 23, Fig. 4a) or spiny lobsters (Belman, 1975, p. 296, Fig. 1) and represent a plesiomorphy in the LCA G. Posterior aorta. Most galatheoid species exhibit a proximal (more anterior) bifurcation into the two pleonal arteries in the eighth thoracic or first pleonal segment, a condition we suggest to be part of the LCA G. The posterior aorta of Munidopsis serricornis, however, is an unpaired vessel which does not bifurcate until the sixth pleonal segment, corresponding to the unpaired aorta in caridean shrimps (Brody and Perkins, 1930; Pillai, 1965). Nevertheless, because of the general distribution of the proximal bifurcation in other anomalan taxa (Keiler et al., 2013), we suggest that the condition in M. serricornis is derived and the proximal bifurcation is plesiomorphic. Ventral vessel system. The descending artery in all studied galatheoid species crosses the gut along the right side (also reported for Petrolisthes japonicas by Imafuku, 1993). This was present in the LCA G. Its anteroventral course in Galathea and Munida (Fig. 5E) reflects the condition found in other decapods (Pillai, 1965; McGaw and Reiber, 2002; McGaw and Stillman, 2010; Keiler et al., 2013), thus is plesiomorphic, and is also considered to be part in the LCA G. The presence of a long, nonbranching posterior ventral vessel in squat lobsters also was present in the LCA G since it is present in most other reptant Journal of Morphology



decapods (Baumann, 1921; Claus, 1884; Belman, 1975; Pearson, 1908). Taken together, the arterial pattern and the pattern of pleonal neuromeres in the LCA G correspond in a number of major aspects with those in most noncarcinized decapods, which suggest that no major transformations have taken place with regard to these features. In other words, all these characters represent plesiomorphies for the Galatheoidea. The Anatomy of the Last Common Ancestor of of Galatheidae and Porcellanidae (LCA GP) There are no major differences between the LCA G and the LCA GP. The absence of a posterior cecum (Fig. 3G), however, is a potential synapomorphy of Porcellanidae and Galatheidae and supports the hypothesis of a close relationship between the two (see introduction). The absence of a posterior cecum thus reflects the condition of the LCA GP while a posterior cecum was present in the LCA G. The unpaired proximal portion of the posterior aorta (Fig. 10A) in porcelain crabs is longer than in Galathea and, since it corresponds with a long portion in Munida and Munidopsis, might reflect the condition in the LCA G. The short portion is an autapomorphy of Galathea. Apart from that, the LCA GP was probably Galatheidae-like (represented by Galathea). Carcinization and Possible Effects on Internal Anatomy Carcinization in porcelain crabs is evidently characterized by the broadening of the cephalothorax, or in other words the carapace became broader than it is long (compare Figs. 2F IV and VI), and by a drastic bending of the pleon. This, in turn, effected the posterior concave emargination and thus the shortening in the midline of the plastron. The branching pattern of the anterior lateral arteries and the course of the optic arteries also changed in the lineage leading to porcelain crabs, and the antennal bladder decreased markedly in size. Because these internal anatomical transformations in the dorsal portion of the cephalothorax do not correlate with the external anatomical changes described above, though, we interpret them to have evolved independently from the process of carcinization and do not discuss them in detail here. Both the bending of the pleon and the shortening of the plastron did, however, apparently affect the internal anatomy of porcelain crabs (which therefore evolved in coherence with the external changes), and this will be discussed below. Transformations in the Pleon—Musculature and Gonads Porcelain crabs evolved a flattened pleon enabling them to carry it completely under the cephaJournal of Morphology

lothorax. This was associated with a reduction in the size of the pleonal muscles, which in turn led to the loss of the caridoid escape reaction and resulted in the evolution of new escape strategies (Wasson et al., 2002). The reduction in the pleonal muscles made the elongation of the ovaries into the pleon possible, whereas in the LCA GP the ovaries (and testes) were restricted to the thorax. Remarkably, the testes in Porcellanidae are restricted to the cephalothorax [Fasten’s (1917) statement that the testes in Petrolisthes eriomerus are located in the pleon is not correct]. Transformations in the Pleon—Arteries and Nervous System The pleon of porcelain crabs was also the site of arterial and neuronal transformations. In the lineage leading to porcelain crabs, the posterior ventral vessel dissolved into numerous small vessels that terminate in the anterior pleonal segments. In squat lobsters, this vessel does not branch. The posterior ventral vessel in porcelain crabs correlates with an anterior position of the pleonal ganglia. The shift is most prominent in Porcellana platycheles, where the last pleonal ganglion does not extend beyond the first pleonal segment (see also Bouvier, 1889 and Pike, 1947). Whether or not the branching of the posterior ventral vessel and the shifting of the ganglia appeared coherently (or at least at the same time) is a question that cannot be resolved. The pleonal ganglia in the porcelain crab Pisidia longicornis only exhibit a slight anterior shift (the last ganglion pg6 reaching into the fifth pleonal segment), and, as is the case in squat lobsters (see Fig. 5, see also Bouvier, 1889), are arranged segmentally and situated at distinct intervals from each other (see Fig. 6; see also Bouvier, 1891). However, the condition of the posterior ventral vessel in Pisidia is unknown. On the basis of the position of the ganglia, the situation in Pisidia probably corresponds to the porcellanid ground pattern. The anterior shift in the pleonal ganglia is seemingly coherent with the loss of the caridoid escape reaction. This assumption is supported by the situation in brachyuran crabs, in which the caridoid escape reaction has also been lost and all pleonal ganglia are fused with the cephalothoracic ganglion (Bouvier, 1891; Harzsch and Dawirs, 1993). This implies that the distinct anterior shift in all the pleonal ganglia did not appear until after the evolutionary transformation into the porcelain crab habitus, meaning that it was not simultaneous (and thus not coherent) with the carcinization process in Galatheoidea. Nevertheless, the crab like habitus (and the loss of the caridoid escape reaction) seems to be a predisposition for the anterior shift in the pleonal ganglia in porcelain crabs.


Transformations in the Cephalothorax Porcelain crabs appear to have evolved the concave emargination at the posterior margin of the plastron together with the shortening of the midline in response to the appearance of the sharp bend in the pleon (compare Figs. 5J and K) and a reduction in the surface available for muscle attachment. The medial keel (see Fig. 5G,H) may have evolved to provide additional attachment surface to compensate this loss. Conversely, the presence of the keel forces the ventral vessel to take a slightly more dorsal course, which is what gives the ventral vessel system in porcelain crabs its unique shape (Fig. 5G). The shortening of the midline of the plastron in porcelain crabs might also have been the cause of the anterior shift in and compaction of the neuromeres of the cephalothoracic ganglion, which in turn are coherent with the shift in the roots of the leg arteries of p2–p4 (comp. Fig. 5J and K). Although the transverse thoracic bridge is present in all galatheoids, it differs in shape between the species. The several shapes of the anterior process of the bridge probably evolved independently from the changes in the shape of the cephalothorax in the course of carcinization. The passage below the thoracic bridge, however, does seem to have been affected by carcinization (i.e., the concave emargination of the plastron). In porcelain crabs it is narrowed by the additional fusion of endopleurites and endosternites (compare Figs. 4B and C) and possibly subjects the ventral vessel to a structural constraint by separating the arteries of the fourth and fifth pereiopods, forcing the ventral vessel to travel a longer distance between the roots of these arteries (see Fig. 4A). The only other case within Galatheoidea in which endophragmal structures constrain the arterial pattern presumably evolved within porcelain crabs. The pattern of the arteries of the third maxillipedes in P. platycheles is unique in that they emanate from the cheliped arteries (see also Bouvier, 1891). The situation in P. platycheles might be coherent with a posterior shift in the knob-like endosternites 2/3 which caused them to protrude between endosternites 3/ 4 and displace the roots of the mouthpart arteries to a more posterior position (compares Fig. 11E and F). Although the rear leg arteries are not fused in porcelain crabs, their pattern corresponds with the pattern of the ventral vessel in most brachyuran crabs, in which the roots of leg arteries p4a and p5a are shifted anteriorly and fused together (McGaw and Reiber, 2002). A similar condition is present in king crabs, in which leg arteries p2a– p4a are fused (McGaw and Duff, 2008; Keiler et al., 2013). In all these three instances a plastron with a posterior emargination is present, a feature evolved in the course of carcinization


(apparently resulting from the bend in the pleon) and one which clearly constitutes a structural constraint for the ventral vessel system. The hemolymph vascular system has previously been discussed in the context of carcinization in pagurid hermit crabs and lithodid king crabs, where dramatic evolutionary transformations in the ventral vessel system were correlated with changes in the endophragmal skeleton (endosternites constraining the course of the leg arteries, etc.; Keiler et al., 2013). Despite the distinctness of the transverse thoracic bridge, the endophragmal skeleton in squat lobsters and porcelain crabs corresponds with that in lithodid king crabs in its broad plastron and paired endosternites which lie, medially, a good distance apart from each other (Keiler et al., 2013). The pattern of the ventral vessel system, however, differs significantly between porcelain crabs and lithodid king crabs and reflects their different evolutionary pathways toward a crab-like form. Taken together, the arterial transformations that happened in the pathway from the LCA GP to Recent porcelain crabs are only minor, which can be ascribed to the fact that the endosternites differ less distinctly between squat lobsters and porcelain crabs than between pagurid hermit crabs and lithodid king crabs since squat lobsters are already “half carcinized.” Missing “Guard Rails” Facilitate Variability The variability of the ventral vessel is markedly high in Galatheoidea. As shown for pagurid hermit crabs and king crabs, there is a strong correlation between the shape of the endophragmal skeleton and the potential for interspecific variation and intraspecific variability (Keiler et al., 2013). The narrow endophragmal structures in Paguridae effectively serve as “guard rails” and represent a structural constraint with regard to the ventral vessel system. The more open space provided by the endophragmal skeleton (and other internal structures), the higher the potential seems to be for interspecific arterial variation and intraspecific arterial variability, as seen in Lithodidae (Keiler et al., 2013). A spacious endophragmal skeleton as found in Lithodidae is present in all galatheoids. It is surprising that the variability of the leg arteries is especially pronounced in the studied specimens of Munida and not in the porcellanid species, since leg arteries p1a–p4a emanate closer together in porcellanids, actually increasing the potential for a common branch. The variable arteries supplying the mouthparts seem to underlie similar principles. The lack of a nonvariable and conserved arterial branching pattern within the taxa indicates that a specific route of the hemolymph supply in these cases is probably physiologically less important and rarely under selection pressure. Journal of Morphology



CONCLUSIONS Squat lobsters and porcelain crabs possess morphologically corresponding artery systems. However, apomorphic states of various internal anatomical characters are present in nearly all the studied species, i.e. a high anatomical disparity can be found within Galatheoidea. As a consequence of carcinization, porcelain crabs lost the caridoid escape reaction, and this is apparently coherent with a posterior elongation of the ovaries. Furthermore, carcinization and the loss of the caridoid escape reaction seem to be predispositions for the anterior shift in the pleonal ganglia observed in porcelain crabs. Even taking into account a high level of interspecific variation and intraspecific variability, the ventral vessel system differs between squat lobsters and porcelain crabs. The differences in question are coherent with external morphological transformations, such as the shortening of the sternal plastron, which evolved in the course of carcinization. The arterial transformations that took place in porcelain crabs were minor compared to those which occurred in other carcinized anomuran crustaceans, since the last common ancestor of squat lobsters and porcelain crabs was already “half carcinized.” Studies into other crab-like taxa within Brachyura and Anomala will reveal more about the process of carcinization and its putative effects on internal morphology. ACKNOWLEDGMENTS The authors wish to thank the late Christoffer Schander (Bergen Museum, Bergen), who sadly passed away during the preparation of this manuscript, for giving us the opportunity to obtain decapod material on one of his marine biology courses at the Espegrend biological station (University of Bergen). The authors thank the Laboratoire Arago (Sorbonne University, Banyuls-surMer) and Friday Harbor Laboratories (University of Washington, San Juan Island) for providing lab space and decapod material. Martin Fritsch (University of Vienna) is thanked for helping us collecting porcelain crabs in Banyuls-sur-Mer. Particular thanks go to Greg Staude (Friday Harbor Labs) for his kind help with information on species localities. The authors would also like to thank Elena Mateo (Cabildo Lanzarote) and Agustin Aguilar (Gobierno de Canarias) for helping issuing the permissions for collecting M. polymorpha and the Government of Lanzarote and Reserva de la Biosfera for funding the I. International Workshop for Marine and Cave Meiofauna in Lanzarote where M. polymorpha was collected. The workshop was organized and funded by Katrine Worsaae and Alejandro Martinez Garcia (University of Copenhagen). We are grateful to Christoph Noever (University of Bergen) for providing some of the squat lobster material. Also, the authors thank the staff Journal of Morphology

of the Electron Microscopy Centre (University of Rostock) for assistance with critical point drying. Grateful thanks go to Fred M€oke (Plant Physiology Department, University of Rostock) and his colleagues for technical help with freeze drying and Peter Michalik and his colleagues (Zoological Institute, University of Greifswald) for providing their microCT for some scans. Torben G€opel is thanked for his contribution to the reconstruction of the hvs of Munida. Lucy Cathrow helped to improve the English of the text. Finally, the authors thank their colleagues at the Zoological Institute of the University of Rostock, Joachim Haug (LMU Munich) and an anonymous reviewer for comments and suggestions which improved the manuscript. LIETERATURE CITED Ahyong ST, Baba K, McPherson, E, Poore, GCB. 2010. A new classification of the Galatheoidea (Crustacea: Decapoda: Anomura). Zootaxa 2676:57–68. Baumann H. 1921. Das Gef€ aßsystem von Astacus fluviatilis (Potamobius astacus L). Z Wiss Zool Abt B 118:246–312. Belman BW. 1975. Some aspects of the circulatory physiology of the spiny lobster Panulirus interruptus. Mar Biol 29: 295–305. Borradaile LA. 1907. On the classification of decapod crustaceans. Ann Mag Nat Hist 19:457–486. Borradaile LA. 1916. Crustacea. Part II. Porcellanopagurus: An instance of carcinization. Natural History Report. Zoology 3: 111–126. Bouvier EL. 1889. Le syste`me nerveux des crustac es d ecapodes et ses rapports avec l’appareil circulatoire. Ann Sci Nat Zool 7:73–106. Bouvier EL. 1891. Recherches anatomiques sur le syste`me art eriel des crustaces d ecapodes. Ann Sci Nat Zool 7:197–282. Bracken-Grissom HD, Cannon ME, Cabezas P, Feldmann RM, Schweitzer CE, Ahyong ST, Felder DL, Lemaitre R, Crandall KE. 2013. A comprehensive and integrative reconstruction of evolutionary history for Anomura (Crustacea: Decapoda). BMC Evol Biol 13:128. Brody MS, Perkins EB. 1930. The arterial system of Palaemonetes. J Morphol 50:127–142. Chu KH, Tsang LM, Ma KY, Ng PKL. 2009. Decapod Phylogeny: What can protein-coding genes tell us? In: Martin JW, Crandall KA, Felder DL editors. Crustacean Issues, Vol. 18: Decapod Crustacean Phylogenetics. Boca Raton: CRC Press. pp 89–99. Claus C. 1884. Zur Kenntnis der Kreislauforgane der Schizopoden und Decapoden. Arbeiten aus dem Zoologischen Institute der Universit€ at Wien und der Zoologischen Station in Triest 5:1–48. Dixon CJ, Ahyong ST, Schram FR. 2003. A new hypothesis of decapod phylogeny. Crustaceana 76:935–975. Fasten N. 1917. Male reproductive organs of Decapoda, with special reference to Puget Sound Forms. Puget Sound Marine Station Pub 1:284–307. Glaessner MF. 1960. The fossil decapod Crustacea of New Zealand and the evolution of the order Decapoda. NZ Geol. Surv Palaeont Bull 31:1–60. Harzsch S, Dawirs RR. 1993. On the morphology of the central nervous system in larval stages of Carcinus maenas L. (Decapoda, Brachyura). Helgol€ ander Meeresunters 47:61–79. Imafuku M. 1993. Observations on the internal asymmetry of the sternal artery and the cheliped asymmetry in selected decapod crustaceans. Crustacean Res 22:35–43. Keiler J, Richter S. 2011. Morphological diversity of setae on the grooming legs in Anomala (Decapoda: Reptantia)

CARCINIZATION IN GALATHEOIDEA revealed by scanning electron microscopy. Zool Anz 250: 343–366. Keiler J, Richter S, Wirkner CS. 2013. Evolutionary Morphology of the Hemolymph Vascular System in Hermit and King Crabs (Crustacea: Decapoda: Anomala). J Morphol 274: 759–788. Martin JW, Abele LG. 1986. Phylogenetic Relationships of the Genus Aegla (Decapoda, Anomura, Aeglidae), with Comments on Anomuran Phylogeny. J Crustacean Biol 6:576–616. McGaw IJ. 2005. The decapod crustacean circulatory system: A case that is neither open nor closed. Micros Microanal 11: 18–36. McGaw IJ, Reiber CL. 2002. Cardiovascular system of the blue crab Callinectes sapidus. J Morphol 251:1–21. McGaw IJ, Duff SD. 2008. Cardiovascular system of Anomuran crabs, Genus Lopholithodes. J Morphol 269:1295–1307. McGaw IJ, Stillman JH. 2010. Cardiovascular system of the Majidae (Crustacea: Decapoda). Arthropod Struct Dev 39: 340–349. McLaughlin PA, Lemaitre R. 1997. Carcinization in the Anomura - fact or fiction? I. Evidence from adult morphology. Contrib Zool 67:79–123. McLaughlin PA, Lemaitre R, Sorhannus U. 2007. Hermit crab phylogeny: A reappraisal and its “fall-out”. J Crustacean Biol 27:97–115. Morrison CL, Harvey AW, Lavery S, Tieu K, Huang Y, Cunningham CW. 2002. Mitochondrial gene rearrangements confirm the parallel evolution of the crab-like form. Proc Roy Soc Lond B Bio 269:345–350. Pearson J. 1908. Cancer. LMBC Mem typ Br mar PI anim 16: 1–209.


Pike RB. 1947. Galathea. LMBC Mem typ Br mar PI anim 34: 1–138. Pillai RS. 1965. The Circulatory System of Caridina laevis Heller. Crustaceana 8:66–74. Reimann A, Richter S, Scholtz G. 2011. Phylogeny of the Anomala (Crustacea, Decapoda, Reptantia) based on the ossicles of the foregut. Zool Anz 250:316–342. Richter S, Wirkner, CS. 2014. A research program for Evolutionary Morphology. J Zoolog Syst Evol Res (in press). Doi: 10.1111/jzs.12061. Schnabel KE, Ahyong ST, Maas EW. 2011. Galatheoidea are not monophyletic—Molecular and morphological phylogeny of the squat lobsters (Decapoda: Anomura) with recognition of a new superfamily. Mol Phyl Evol 58:157–168. Scholtz G, Richter S. 1995. Phylogenetic Systematics of the Reptantian Decapoda (Crustacea, Malacostraca). Zool J Linn Soc Lond 113:289–328. Scholtz G. 2014. Evolution of crabs—history and deconstruction of a prime example of convergence. Contrib Zool 83: 87–105. Schram FR. 2001. Phylogeny of decapods: moving towards a consensus. Hydrobiologia 449:1–20. Tsang LM, Chan TY, Ahyong ST, Chu KH. 2011. Hermit to king, or hermit to all: multiple transitions to crab-like form from hermit crab ancestors. Syst Biol 60:616–629. Tudge CC. 1997. Phylogeny of the Anomura (Decapoda, Crustacea): Spermatozoa and spermatophore morphological evidence. Contrib Zool 67:125–141. Wasson K, Lyon BE, Knope, M. 2002. Hair-trigger autotomy in porcelain crabs is a highly effective escape strategy. Behav Ecol 13:481–486.

Journal of Morphology

Evolutionary morphology of the organ systems in squat lobsters and porcelain crabs (crustacea: Decapoda: Anomala): an insight into carcinization.

Porcelain crabs (Porcellanidae) are one of three taxa within anomuran crustaceans (Anomala) which possess a crab-like body form. Curiously, these thre...
2MB Sizes 0 Downloads 5 Views