Comparative internal anatomy of Staurozoa (Cnidaria), with functional and evolutionary inferences

General body anatomy

The internal anatomy of nine species of stalked jellyfishes is described below (Figs. 2–58; Table 2). For each species we included detailed information on general body plan, peduncle and septa, gonads and gametoducts, intertentacular lobules, and claustrum (when applicable). The muscular system, manubrium and gastric radial pockets, gastric filaments, perradial and interradial anchors/primary tentacles, ostia, arms delimitation, white spots of nematocysts, batteries of nematocysts, internal subumbrellar layer of nematocysts, secondary tentacles, and pad-like adhesive structures were comparatively analyzed. Therefore, the figures used to illustrate these structures (Figs. 5–7, 9–13, 15–17, 24 and 32) will be mentioned in the descriptions independently of the species.

Suborder Myostaurida Miranda, Hirano, Mills, Falconer, Fenwick, Marques & Collins, 2016

• Family Haliclystidae Haeckel, 1879

• Genus Haliclystus Clark, 1863

• Haliclystus tenuis Kishinouye, 1910 (Figs. 2E, 2F; 3; 4; 5M, 5Q, 5V; 6; 8; 9A–9I; 10A–10F; 11A–11H; 12A–12C; 13C; 14; 15I–15Q; 17N–17Q; 21A; 57; 58A, 58C and 58D)

Basal region formed by pedal disk of peduncle (stalk) with increased surface area due to invaginations (Figs. 3A and 3B). Peduncle with four perradial chambers (delimited by gastrodermis), alternating with four interradial longitudinal muscle bands (epitheliomuscular cells) embedded in mesoglea (Fig. 4); chambers and muscles developed throughout peduncle (Figs. 4A–4C) except at pedal disk. Perradial chambers fusing at junction of peduncle and calyx (Figs. 4D–4G). Gastrodermis envelops interradial longitudinal muscles at basal region of calyx (Fig. 4H), defining four interradial gastric septa: one thin layer of mesoglea surrounded by two layers of gastrodermis (Figs. 4I and 4J). Four infundibula (peristomal pits) funnel-shaped with blind end, delimited by epidermis, deeply developed down to base of calyx, widening apically, with broad apertures on subumbrella (Figs. 2F and 4J). Gastrovascular cavity without claustrum. At base of infundibula, interradial longitudinal muscle becomes compressed and flattened, being V-shaped in cross section apically (as in other species examined, e.g., Figs. 5C, 5D, 5G, 5H, 5K and 5L), progressively dividing into two adradial bands toward the arms (Figs. 11A and 11B). Adjacent septal gastrodermis merge defining four perradial regions and dividing gastrovascular cavity (Figs. 6B–6D). Fusion of septal gastrodermis forms basal region of manubrium and gastric radial pockets (perradial pockets), i.e., central part of gastrodermis of each septum joins forming four-sided manubrial gastrodermis while lateral parts of adjacent septa join forming gastric radial pockets (Fig. 6). Similarly, each infundibular epidermis also progressively merges apically: central part of each infundibular epidermis becomes manubrial epidermis, and epidermis of adjacent infundibula forms epidermis of gastric radial pockets (Figs. 6E–6N). Therefore, each gastric radial pocket is formed by fusion of gastrodermis and epidermis of adjacent septa, and manubrium is formed by fusion of all four septa (Fig. 6). Four gastric radial pockets laterally separated from each other by interradial septa (Figs. 6L and 6N); gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (Figs. 10A–10F); each gastric radial pocket connected to main gastrovascular cavity. Manubrium (Fig. 3H) internally defined by gastrodermis, externally by epidermis (Figs. 6O and 6P). Gastric filaments composed of one layer of mesoglea surrounded by gastrodermis, formed by lateral evaginations of gastrodermal layer of septa at base of manubrium, concentrated at perradii (as in other species examined; Fig. 7). Gonads (Figs. 3F–3H) with approximately six rows of vesicles (follicles), which are serial gastrodermal evaginations at lateral regions of interradial septa, gastric radial pockets and arms (Fig. 8); vesicles of same gastric radial pocket formed by gastrodermis of two different interradial septa (two adjacent septa) (Fig. 8B). Vesicles composed of an internal layer of gastrodermis, mesoglea, an external (subumbrellar) layer of epidermis, and inner gonadal content (gastrodermal origin) (Figs. 8A, 8D and 8E). Female specimen analyzed (Fig. 8) with ovarian vesicles; gonadal content composed of two main layers: peripheral layer with immature oocytes in different developmental stages (Figs. 8C and 8D), internal layer with mature oocytes with scattered yolk granules (Figs. 8C and 8D). Mature oocytes surrounded by cells of gastrodermal origin (probably follicle cells), leading to gastric radial pockets through fusion of these cells with gastrodermis of the ovarian vesicles (Figs. 8D–8M), forming gametoducts (Figs. 8N and 8O). Cilia often associated with gametoducts. Anchors (rhopalioids) (Figs. 3D and 3E) hollow, with hollow stem as evaginations of body surface. Eight large anchors, each located between adjacent arms at calyx margin (Figs. 3D and 3E), four perradial (Figs. 9A–9I) and four interradial (Figs. 10A–10F). Gastrodermis of perradial anchors directly connected to gastrodermis of gastric radial pockets through stem (Figs. 9A–9I). At interradial regions, septa prevent direct connection of gastrodermis of interradial anchors with gastrodermis of calyx (Fig. 10A), but at margin of calyx, small ostia connect adjacent gastric radial pockets along margin of calyx, allowing gastrodermis of anchors to be contiguous with gastrodermis of calyx (gastric radial pocket) (Figs. 10B–10F). Anchors without nematocysts. Each gastric radial pocket extending throughout calyx margin, apically continuing into two adradial arms and respective tentacular clusters (Figs. 11A–11H). Subumbrellar epidermis (continuous with epidermis of infundibula) marginally merges with exumbrellar epidermis, dividing gastric radial pockets at origin of arms (Figs. 11A–11H). Eight bands of longitudinal muscles running between calyx base and arms, each band toward each one of eight arms (Figs. 11B–11H), then becoming thinner diffuse muscle bundles toward secondary tentacles (as in other species examined; Figs. 5Q–5V). Eight sections of coronal muscle (Fig. 3E) at calyx margin, each between adjacent arms (as in most of species examined; Figs. 5 and 11). Each arm with two bands (perradial and interradial) of coronal muscle (Figs. 5Q, 11G and 11H). Perradial and interradial white spots of nematocysts on subumbrella (Fig. 3G), between a layer of epidermis and gastrodermis, internally composed of peripheral layer of nematoblasts, and central mature nematocysts (Figs. 12A–12C). Epidermal thickening at central region of white spots of nematocysts (Fig. 12C). Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Fig. 13C). Distal exumbrellar end of arms with “U-shaped” space, a platform connecting arm with secondary tentacles, defined by gastrodermis and a thick layer of mesoglea (Fig. 14). Continuous layer of internal unorganized nematocysts visible in subumbrellar epidermis, from base of infundibula, passing through gastric radial pockets, arms, to tips of secondary tentacles (as in most of species examined; Figs. 15 and 16). Internal layer of nematocysts continuous with groups of nematocysts at tentacular base, in epidermis, with different sizes and types, also unorganized (as in other species examined; Fig. 16). Secondary hollow tentacles (Fig. 3I) composed of two parts, knob and stem (Fig. 17N). Secondary tentacles without pad-like adhesive structures. Each stem with inner layer of gastrodermis, and external layer of epidermis; epidermis with longitudinal muscles extending throughout tentacular stem (Fig. 17O). Nematocysts found at different regions of epidermis of stem of secondary tentacles (as in other species examined; Fig. 16Q). Tentacular knob with a thin layer of gastrodermis and a thick layer of epidermis with an external row of organized nematocysts (Figs. 17P and 17Q). Nematocysts also found at internal region of knob, among supporting cells of epidermis.

• Genus Manania Clark, 1863

• Manania uchidai (Naumov, 1961) (Figs. 2I, 2J; 5A, 5B, 5N; 7I; 12D, 12E; 13A, 13B; 17K–17M; 18; 19; 20; 21B; 22; 23; 24L–24N; 57; 58B, 58E)

Basal region formed by pedal disk of peduncle with increased surface area due to invaginations (Figs. 18F and 18G). Peduncle with one chamber (delimited by gastrodermis) and four interradial longitudinal muscle bands (epitheliomuscular cells) embedded in mesoglea, near the base (Figs. 19A–19F). Single chamber progressively divided into four chambers, at median region of peduncle, alternating with four interradial longitudinal muscle bands (Figs. 19G–19R). Perradial chambers do not merge at junction of peduncle and calyx, unlike other stauromedusae (Figs. 19S, 19T, 20A and 20B). Instead, claustra are defined below complete connection of perradial chambers (Figs. 20C–20G). Union of lateral projections of adjacent interradial septa forming claustra (Figs. 20D–20F), tissues composed of central layer of mesoglea surrounded by gastrodermis, dividing gastrovascular cavity (Figs. 20G and 20H). Four accessory radial pockets delimited (separated from main gastrovascular cavity) by claustra (Figs. 20F–20M). Four infundibula funnel-shaped with blind end, delimited by epidermis, deeply developed down to base of calyx, widening apically, with broad apertures on subumbrella (Figs. 19T, 20C, 20G and 20H). At base of infundibula, interradial longitudinal muscles remain intramesogleal (Fig. 21B), being progressively (toward manubrium) compressed, flattened, and divided into two thin bands (Figs. 20I, 20L and 20M). Adjacent septal gastrodermis merge again, dividing gastrovascular cavity once more, forming four principal radial pockets and manubrium (Figs. 20H–20M), similarly (and homologous) to formation of gastric radial pockets in all staurozoan species without claustrum such as H. tenuis (Fig. 6). Central part of gastrodermis of each septum joins forming four-sided manubrial gastrodermis, while lateral parts of adjacent septa join forming principal radial pockets (as in the formation of gastric radial pockets of H. tenuis, Figs. 6B–6D). Similarly, each infundibular epidermis progressively merges apically: central part of each infundibular epidermis becomes manubrial epidermis, and epidermis of adjacent infundibula forms epidermis of principal radial pockets (as in H. tenuis Figs. 6E–6G, 20I and 20J). Principal radial pockets (Figs. 20J–20M) are true gastric radial pockets, composed of same structures as gastric radial pockets in stauromedusae without claustrum (Fig. 6) and associated with gonads (and gametoduct) (Fig. 20M). Therefore, M. uchidai has eight radial pockets: four accessory radial pockets, directly associated with chambers in peduncle, anchors and arms; and four principal radial pockets, associated with manubrium and gonads (Figs. 20J and 20L). Four accessory and principal radial pockets separated by claustra (Figs. 20L and 20M). Four accessory radial pockets laterally separated from each other by interradial septa (Fig. 20L); accessory radial pockets directly connected only by means of small interradial ostia at margin of calyx (as in the gastric pocked of H. tenuis, Figs. 10A–10F). Manubrium internally defined by gastrodermis, externally by epidermis. Gastric filaments similar to those described for H. tenuis (as in other species examined; Fig. 7), and associated with principal radial pockets. Gonads (Figs. 18H–18J) in principal radial pockets, not organized in vesicles (Fig. 22). Gonadal content restricted to one layer between gastrodermis and epidermis of septa (Figs. 22B, 22D and 22G). Gonadal layers of same principal radial pocket formed by lateral tissue of two different interradial septa (two adjacent septa) (Figs. 20H–20K). Male specimen analyzed (Fig. 22), with spermatocytes adjacent to gastrodermis of principal radial pockets (internal), and spermatozoa adjacent to epidermis of principal radial pockets (external) (Figs. 20M and 22G). Spermatozoa divided into different sacs, delimited by cells of gastrodermal origin, which are connected to gastrodermis of principal radial pocket, forming gametoduct (Figs. 22E, 22I–22N). Cilia often associated with gametoduct. Anchors (Figs. 18C–18E) hollow, with hollow stem, as evaginations of body surface (Fig. 5N). Eight small anchors, each anchor located between adjacent arms at calyx margin, four perradial and four interradial, (Figs. 18C–18E). Internal organization similar to H. tenuis, with interradial ostia (Figs. 9A–9I, 10A–10F). Anchors connected to accessory radial pocket. Nematocysts present in knobbed remnant of primary tentacles at tip of anchors (Figs. 5N and 18E). Coronal muscle entire at calyx margin, and external (exumbrellar) in relation to anchor (Figs. 18C and 18D). Each accessory radial pocket extending throughout calyx margin, apically continuing into two adradial arms and respective tentacular clusters. Internal organization of arms similar to H. tenuis (Figs. 11A–11H). Perradial white spots of nematocysts on subumbrella, associated with gonads (Figs. 18H–18J, 20K–20M), between epidermis and gastrodermis of principal radial pockets, with internal organization (Figs. 12D and 12E) similar to H. tenuis. Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Figs. 13A and 13B). Distal end of arms with intertentacular lobules, a structure between adjacent secondary tentacles delimited by gastrodermis and one central layer of mesoglea (Figs. 17M and 23). Outermost secondary tentacles with pad-like adhesive structures (epidermal thickening) (Figs. 24L–24N). Continuous layer of internal unorganized nematocysts in subumbrellar epidermis not clearly recognizable. Secondary hollow tentacles composed of two parts, knob and stem, with organization (Figs. 17K and 17L) similar to H. tenuis. At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section (Fig. 17K).

• Family Lucernariidae Johnston, 1847

• Genus Lucernaria Müller, 1776

• Lucernaria quadricornis Müller, 1776 (Figs. 7G; 16D–16H; 17D–17F; 25; 26; 27; 28; 57)

Basal region formed by pedal disk of peduncle with increased surface area due to invaginations (Fig. 25C). Peduncle with one chamber (delimited by gastrodermis), and four interradial gastric septa: each septum consisting of mesoglea surrounded by gastrodermis and an internal longitudinal muscle band (epitheliomuscular cells) embedded in mesoglea (Figs. 26E and 26F). Organization of infundibula (Fig. 26H) similar to H. tenuis. Gastrovascular cavity without claustrum. At base of infundibula, muscle becomes compressed and flattened, progressively divided into two thin bands apically (as Lucernaria sainthilairei; Figs. 5C and 5D). Organization of manubrium and gastric radial pockets similar to H. tenuis (Fig. 6). Four gastric radial pockets laterally separated from each other by interradial septa; gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (as in other species examined; Figs. 10M–10U); each gastric radial pocket connected to main gastrovascular cavity. Organization of gastric filaments (Fig. 7G) similar to those of H. tenuis. Vesicles of gonads not clearly defined (Fig. 25H), with irregular shape: gametes located between a layer of gastrodermis and epidermis of septa, and this layer can be more or less wavy (Figs. 27A–27C). Vesicles of same gastric radial pocket formed by gastrodermis of two different interradial septa (two adjacent septa). Female specimen analyzed, with no evident regionalization of mature and immature oocytes (Fig. 27), although gametoduct is recognizable (Figs. 27D–27I). Oocytes surrounded by cells of gastrodermal origin (probably follicle cells), leading to gastric radial pockets through fusion of these cells with gastrodermis of the ovarian vesicles (Figs. 27D–27I), forming a gametoduct (Fig. 27I). Cilia often associated with gametoduct (Figs. 27G–27I). Anchors absent (Fig. 25). Arms paired at interradii (Figs. 25A, 25D and 25E), with internal organization similar to H. tenuis (Fig. 11). Eight sections of coronal muscle at calyx margin, each between adjacent arms. Organization of longitudinal and coronal muscles in arms similar to H. tenuis (as in other species examined; Figs. 5 and 11). Perradial and interradial white spots of nematocysts on subumbrella, with internal organization similar to H. tenuis (Figs. 12A–12C). Distal end of arms with intertentacular lobules, a structure between adjacent secondary tentacles delimited by gastrodermis and a central layer of mesoglea (Figs. 17D, 17E and 28). Secondary tentacles of arms without pad-like adhesive structures (Figs. 25D, 25F and 25G). Continuous layer of internal unorganized nematocysts visible in subumbrellar epidermis, from base of infundibula, passing through gastric radial pockets, arms, to tips of secondary tentacles (as in most of species examined; Figs. 15 and 16). Internal layer of nematocysts continuous with groups of nematocysts at tentacular base, in epidermis, with different sizes and types, also unorganized (Figs. 16D–16H). Secondary hollow tentacles composed of two parts, knob and stem, with organization similar to H. tenuis (as in other species examined; Fig. 17). At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section (Fig. 17F).

• Lucernaria bathyphila Haeckel, 1879 (Figs. 17G, 17H; 29; 30; 31; 32N–32W; 33; 57)

Internal anatomy similar to Lucernaria quadricornis. Peduncle with one chamber (delimited by gastrodermis), and four interradial gastric septa: each septum consisting of mesoglea surrounded by gastrodermis and an interradial longitudinal muscle band (epitheliomuscular cells) embedded in mesoglea (Fig. 30). Gastrovascular cavity without claustrum. Four gastric radial pockets laterally separated from each other by interradial septa; gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (as in other species examined; Figs. 10M–10U); each gastric radial pocket connected to main gastrovascular cavity. Organization of gastric filaments similar to those of H. tenuis (as in other species examined, Fig. 7). Vesicles of gonads not clearly defined (Figs. 29G, 29H, 31A–31C), with irregular shape: gametes located between a layer of gastrodermis and epidermis of septa, and this layer can be more or less wavy (Figs. 31A–31C). Vesicles of same gastric radial pocket formed by gastrodermis of two different interradial septa (two adjacent septa). Specimen analyzed probably an immature male and spermatozoa could not be distinguished (Fig. 31). Gametoduct clearly recognizable: cells of gastrodermal origin connected to spermatocytes, leading to gastric radial pockets through fusion of these cells with gastrodermis of vesicles (Figs. 31F–31Q). Cilia often associated with gametoduct (Fig. 31Q). Anchors absent (Fig. 29E). Eight sections of coronal muscle at calyx margin, each between adjacent arms. Organization of longitudinal and coronal muscles in arms similar to H. tenuis (as in other species examined; Figs. 5 and 11). Perradial and interradial white spots of nematocysts on subumbrella (Fig. 29H), with internal organization similar to H. tenuis (Figs. 12A–12C). Aperture of white spots at subumbrellar epidermis clearly recognizable in its central thicker region (Figs. 32N–32W): a pore divides subumbrellar epidermis and adjacent layer of mesoglea into two regions in a longitudinal section, allowing an outflow to central mature nematocysts (Figs. 32Q–32W). Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Fig. 29C), similar in internal organization to other species examined (Fig. 13). Distal end of arms with intertentacular lobules (Fig. 33). Secondary tentacles of arms (Figs. 29E, 29F and 29H) without pad-like adhesive structures. Continuous layer of internal unorganized nematocysts in subumbrellar epidermis, from base of infundibula, passing through gastric radial pockets, arms, to tips of secondary tentacles (as in most of species examined; Figs. 15 and 16). Internal layer of nematocysts continuous with groups of nematocysts at tentacular base, in epidermis, with different sizes and types, also unorganized. Organization of secondary tentacles (Figs. 17G and 17H) similar to H. tenuis. At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section (as in L. quadricornis; Fig. 17F).

• Lucernaria sainthilairei (Redikorzev, 1925) (Figs. 5C, 5D; 12I; 16J–16L; 17I, 17J; 34; 35; 36; 37; 57)

Internal anatomy similar to Lucernaria quadricornis. Peduncle with one chamber (delimited by gastrodermis), and four interradial gastric septa: each septum consisting of mesoglea surrounded by gastrodermis and an internal longitudinal muscle band (epitheliomuscular cells) embedded in mesoglea (Fig. 35). Gastrovascular cavity without claustrum. Four gastric radial pockets laterally separated from each other by interradial septa; gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (as in other species examined; Figs. 10M–10U); each gastric radial pocket connected to main gastrovascular cavity. Organization of gastric filaments similar to those of H. tenuis (as in other species examined, Fig. 7). Vesicles of gonads not clearly defined, with irregular shape (Fig. 36): gametes located between a layer of gastrodermis and epidermis of septa, and this layer can be more or less wavy (Figs. 36A–36D). Vesicles of same gastric radial pocket formed by gastrodermis of two different interradial septa (two adjacent septa). Specimen analyzed probably an immature male; spermatozoa and gametoduct could not be recognized (Fig. 36). Anchors absent (Figs. 34A, 34B and 34F). Eight sections of coronal muscle at calyx margin, each between adjacent arms. Organization of longitudinal and coronal muscles in arms similar to H. tenuis (as in other species examined; Figs. 5 and 11). Perradial and interradial white spots of nematocysts on subumbrella (Figs. 34B and 34F), with internal organization similar to H. tenuis (Fig. 12I). Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Fig. 34D), similar in internal organization to other species examined (Fig. 13). Distal end of arms with intertentacular lobules (Fig. 37). Secondary tentacles of arms without pad-like adhesive structures (Fig. 34). Continuous layer of internal unorganized nematocysts in subumbrellar epidermis, from base of infundibula, passing through gastric radial pockets, arms, to tips of secondary tentacles (as in most of species examined; Figs. 15 and 16). Internal layer of nematocysts continuous with groups of nematocysts at tentacular base, in epidermis, with different sizes and types, also unorganized (Figs. 16J–16L). Organization of secondary tentacles (Figs. 17I and 17J) similar to H. tenuis. At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section (as in L. quadricornis; Fig. 17F).

Suborder Amyostaurida Miranda, Hirano, Mills, Falconer, Fenwick, Marques & Collins, 2016

• Family Kishinouyeidae Uchida, 1929

• Genus Calvadosia Clark, 1863

• Calvadosia corbini (Larson, 1980) (Figs. 5I–5L, 5O, 5P, 5T, 5U; 7A–7F; 10P–10U; 12J–12M; 13H–13M; 15R, 15S; 16I; 17A–17C; 24A–24K; 38; 39; 40; 41; 57)

Peduncle with a broad basal pedal disk (Figs. 38A–38C). Base of peduncle with four perradial chambers (delimited by gastrodermis), with numerous evaginations (Figs. 39C–39H). Gradual connection of four perradial chambers, defining one central gastric chamber, and delimiting four interradial gastric septa toward apical region (Figs. 39E, 39F and 39I). Peduncle without interradial longitudinal muscles (Figs. 39I–39M). Size of septa decreases at peduncle/calyx connection (Figs. 39N–39Q), where interradial longitudinal muscles (epitheliomuscular cells) inside septa become visible (Figs. 39R and 39S). Internal organization of infundibula (Figs. 39X and 39Y) similar to H. tenuis. Gastrovascular cavity without claustrum. At base of infundibulum, interradial longitudinal muscle is compressed, and then becomes divided into two bands (Figs. 5I–5L). Internal organization of gastric radial pockets and manubrium similar to H. tenuis (Fig. 6). Four gastric radial pockets laterally separated from each other by interradial septa; gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (Figs. 10P–10U); each gastric radial pocket connected to main gastrovascular cavity. Manubrium internally defined by gastrodermis, externally by epidermis. Gastric filaments (Figs. 7A–7F) similar to those of H. tenuis. Gonads with numerous vesicles, irregularly arranged in asymmetrical erected nodules, formed by an external fold of subumbrellar tissue (Figs. 38E–38I, 40A–40E). Male specimen analyzed, with spermatocytes adjacent to gastrodermis, in peripheral position; spermatozoa adjacent to epidermis, in central position (Fig. 40H). Spermatozoa divided into different sacs, delimited by cells of gastrodermal origin, connected to gastrodermis of gastric radial pocket, forming gametoduct (Figs. 40J, 40L–40P). Large batteries of nematocysts in subumbrellar epidermis, associated with gonads, between internal vesicles (Figs. 13L, 13M, 40H and 40I). Anchors absent (Figs. 38A, 38B and 38J). Arms sharply paired at interradii (perradial notches deeper than interradial notches) (Figs. 38A, 38B, 38I and 38J), with internal organization similar to H. tenuis (Fig. 11). Eight sections of coronal muscle at calyx margin (Fig. 38D), each between adjacent arms. Organization of longitudinal and coronal muscles in arms similar to H. tenuis (as in other species examined; Figs. 5 and 11). Perradial and interradial white spots of nematocysts on subumbrella (Fig. 38G), with internal organization similar to H. tenuis (Figs. 12J–12M). Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Figs. 13H–13K). Distal end of arms with intertentacular lobules, a structure between adjacent secondary tentacles delimited by gastrodermis and a central layer of mesoglea (Fig. 41). Tip of each arm with large pad-like adhesive structures (Figs. 38K and 38L), with thick epidermis, mesoglea, and hollow canals delimited by thin layer of gastrodermis; hollow gastrodermal canals gradually connected to gastrovascular cavity at tip of arms (Figs. 24A–24K). Continuous layer of internal unorganized nematocysts in subumbrellar epidermis (as in most of species examined; Figs. 15 and 16). Secondary hollow tentacles composed of two parts, knob and stem, with organization (Figs. 17A–17C) similar to H. tenuis. At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section as in most of species examined (Fig. 17F).

• Calvadosia cruciformis (Okubo, 1917) (Figs. 2G, 2H; 9J–9S; 10G–10L; 12G, 12H; 13D; 42; 43; 44; 45; 57)

Basal pedal disk of peduncle (Fig. 42A) with epidermal axial canal, a pronounced and delimited invagination with blind end (Figs. 42I, 43A–43D). Base of peduncle with four gastric perradial chambers (delimited by gastrodermis) (Figs. 43A–43G). Gradual connection of four perradial chambers, defining one central gastric chamber, and delimiting four interradial gastric septa toward apical region (Figs. 43H–43L). Peduncle without interradial longitudinal muscles (Figs. 43A–43M). Size of septa decreases at peduncle/calyx connection, and interradial longitudinal muscles (epitheliomuscular cells) inside septa become visible (Figs. 43N–43P). Internal organization of infundibula (Figs. 43N–43P) similar to H. tenuis. Gastrovascular cavity without claustrum. At base of infundibulum, interradial longitudinal muscle is compressed, and then becomes divided into two bands as in C. corbini (Figs. 5I–5L). Internal organization of gastric radial pockets and manubrium similar to H. tenuis (Fig. 6). Four gastric radial pockets laterally separated from each other by interradial septa; gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (Figs. 10G–10L); each gastric radial pocket connected to main gastrovascular cavity. Manubrium internally defined by gastrodermis, externally by epidermis. Gastric filaments similar to those of other species examined (Fig. 7). Gonads with vesicles, which are serial gastrodermal evaginations at lateral regions of interradial septa, gastric radial pockets, and arms (Figs. 44A–44C); vesicles of same gastric radial pocket formed by gastrodermis of two different interradial septa (two adjacent septa). Female specimen analyzed (Fig. 44), with ovarian vesicles with two main layers: peripheral layer with immature oocytes in different developmental stages, internal layer with mature oocytes with scattered yolk granules (Figs. 44B, 44F–44L). Mature oocytes surrounded by cells of gastrodermal origin (probably follicle cells), which are connected to gastrodermis of gastric radial pocket, forming gametoduct (Figs. 44D–44L). Perradial and interradial primary tentacles present, presenting curved knob with nematocysts, and a small white disk at stem (Figs. 9J–9S, 10G–10L, 42D and 42E). Interradial ostia connecting interradial primary tentacles with gastrovascular cavity (Figs. 10G–10L). Arms sharply paired at interradii (Figs. 42A and 42B), with internal organization similar to H. tenuis (Fig. 11). Eight sections of coronal muscle at calyx margin, each between adjacent arms. Organization of longitudinal and coronal muscles in arms similar to H. tenuis (as in other species examined; Figs. 5 and 11). Perradial and interradial white spots of nematocysts on subumbrella (Figs. 12G, 12H, 42B and 42C), with internal organization similar to H. tenuis. Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Fig. 13D). Distal end of arms with intertentacular lobules, a structure between adjacent secondary tentacles delimited by gastrodermis and a central layer of mesoglea (Fig. 45). Outermost secondary tentacles with pad-like adhesive structures (epidermal thickening) (as in M. uchidai; Figs. 24L–24N and 42H). Clearly recognizable continuous layer of internal unorganized nematocysts in subumbrellar epidermis, from base of infundibula to tips of secondary tentacles, as in most of species examined (Figs. 15 and 16). Secondary hollow tentacles composed of two parts, knob and stem, with organization similar to H. tenuis (as in other species examined; Figs. 17 and 45).

• Calvadosia vanhoeffeni (Browne, 1910) (Figs. 5E–5H, 5R, 5S; 7H; 10M–10O; 11I–11L; 12F; 13E–13G; 15A–15H; 16A–16C; 46; 47; 48; 49; 50; 57)

Basal pedal disk of peduncle with increased surface area due to invaginations (Figs. 46E and 46F). Base of peduncle with four gastric radial chambers (delimited by gastrodermis) (Figs. 47D and 47E). Gradual connection of four perradial chambers, defining one central gastric chamber, and delimiting four interradial gastric septa toward apical region (Figs. 47F–47K). Peduncle without interradial longitudinal muscles, which are visible inside septa only at peduncle/calyx connection (Figs. 47L and 47M). Internal organization of infundibula (Figs. 47O and 47P) similar to H. tenuis. Gastrovascular cavity without claustrum. At base of infundibulum, interradial longitudinal muscle is compressed, and then becomes divided into two bands (Figs. 5E–5H). Internal organization of gastric radial pockets and manubrium similar to H. tenuis (Fig. 6). Four gastric radial pockets laterally separated from each other by interradial septa; gastric radial pockets directly connected only by means of small interradial ostia at margin of calyx (Figs. 10M–10O); each gastric radial pocket connected to main gastrovascular cavity. Manubrium internally defined by gastrodermis, externally by epidermis. Gastric filaments (Fig. 7H) similar to those of H. tenuis. Vesicles of gonads with irregular shape, but clearly defined, with prominent gastrodermis (Figs. 48 and 49). Two sets of vesicles in each gastric radial pocket; each vesicle formed by gastrodermis of two different interradial septa (two adjacent septa). Male specimen analyzed (Figs. 48 and 49), with spermatocytes adjacent to gastrodermis, in peripheral position; spermatozoa adjacent to epidermis, in central position (Figs. 48B and 49L). Spermatozoa divided into different sacs, delimited by cells of gastrodermal origin, connected to gastrodermis of gastric radial pockets, forming gametoduct (Figs. 48F–48X and 49). Anchors absent (Figs. 46A and 46B). Arms with internal organization similar to H. tenuis (Figs. 11I–11L). Eight sections of coronal muscle at calyx margin, each between adjacent arms. Organization of longitudinal and coronal muscles in arms (Figs. 5R, 5S and 11L) similar to H. tenuis. Perradial and interradial white spots of nematocysts on subumbrella (Fig. 46H), with internal organization (Fig. 12F) similar to H. tenuis. Batteries of nematocysts sparsely distributed in exumbrellar epidermis (Figs. 13E–13G). Distal end of arms with intertentacular lobules (Fig. 50), a structure between adjacent secondary tentacles delimited by gastrodermis and a central layer of mesoglea. Outermost secondary tentacles with pad-like adhesive structures (epidermal thickening) as in M. uchidai (Figs. 24L–24N, 46J–46L). Clearly recognizable continuous layer of internal unorganized nematocysts in subumbrellar epidermis, from base of infundibula to tips of secondary tentacles (as in most of species examined; Figs. 15A–15H, 16A–16C). Secondary hollow tentacles (Figs. 46B, 46J and 46L) composed of two parts, knob and stem, with organization similar to H. tenuis (Figs. 17N–17Q and 50). At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section as in most of species examined (Fig. 17).

• Family Craterolophidae Uchida, 1929

• Genus Craterolophus Clark, 1863

• Craterolophus convolvulus (Johnston, 1835) (Figs. 7J; 17R–17U; 32A–32M; 51; 52; 53; 54; 55; 56; 57)

Basal pedal disk of peduncle with increased surface area due to invaginations and a central pit (Figs. 51B–51C). Peduncle with four perradial chambers (delimited by gastrodermis), and without interradial longitudinal muscles (Figs. 52A–52K). Size of four chambers gradually increases to merge as a single chamber at peduncle/calyx connection (Figs. 52M, 52N and 52P), delimiting four interradial septa (Figs. 52O and 52P). At peduncle/calyx connection, lateral projections of adjacent interradial septa, composed of a central layer of mesoglea surrounded by gastrodermis, progressively merges, defining the claustra, tissues that divide gastrovascular cavity (Figs. 52Q–52S, 53A–53F). Four accessory radial pockets delimited (separated from main gastrovascular cavity) by claustra (Figs. 53B–53D and 53F). Four infundibula funnel-shaped with blind end, delimited by epidermis, deeply developed down to base of calyx, widening apically, with broad apertures on subumbrella (Figs. 53C and 53F). Interradial longitudinal muscle (epitheliomuscular cells) divided into two bands, visible only after complete formation of claustra (Figs. 53G and 53H). Septa with complex lateral folds, delimiting auxiliary radial pockets (Figs. 53G, 53H, 53J and 54). Below manubrium delimitation, lateral folds of septa produce auxiliary radial pockets inside infundibula (Figs. 53G and 53H), composed of external epidermis, a layer of gonadal content, and internal gastrodermis (Fig. 54B). As adjacent septal gastrodermis and epidermis merge, dividing once more the gastrovascular cavity and delimiting four principal radial pockets and manubrium (similarly to M. uchidai, Fig. 20), these auxiliary radial pockets are externalized, still connected to subumbrella (Figs. 53J, 54A and 54B). Above manubrium and principal radial pocket delimitation, new auxiliary radial pockets formed inside principal radial pocket (Fig. 54C), also as a result of irregular folds of lateral tissue of principal radial pocket, always containing gonads (Figs. 54E–54H). Auxiliary radial pocket inside principal radial pocket composed of external gastrodermis, a layer of gonadal content, and internal epidermis (Fig. 54D). Gastrodermis and epidermis of auxiliary radial pockets progressively merge with lateral gastrodermis and epidermis of principal radial pocket, externalizing auxiliary radial pockets (Figs. 54I–54L). Therefore, gastrovascular system in C. convolvulus divided into numerous radial pockets (Figs. 53J, 54A and 54L): four accessory radial pockets, directly associated with perradial chambers in peduncle, arms and secondary tentacles; four principal radial pockets, associated with manubrium and gonads; and numerous auxiliary radial pockets (external and internal), associated with gonads (Figs. 53 and 54). Accessory and principal radial pockets separated by claustra (Figs. 53J, 54A and 54L). Accessory radial pockets laterally separated from each other by interradial septa (Fig. 53F); directly connected only by means of small interradial ostia at margin of calyx as in M. uchidai, and in gastric radial pockets of species without claustrum, such as C. vanhoeffeni and C. corbini (Figs. 10M–10U). Manubrium (Figs. 51I and 54C) internally defined by gastrodermis, externally by epidermis. Gastric filaments similar to those of H. tenuis in internal organization, associated with principal radial pocket (Fig. 7J). Gonadal content restricted to one layer between gastrodermis and epidermis of septa, principal radial pockets, and auxiliary radial pockets, not organized in vesicles (Figs. 53–55). Gonadal layers of same principal radial pocket formed by two different adjacent interradial septa. Female specimen analyzed (Figs. 53–55), with immature oocytes adjacent to gastrodermis and peripheral; mature oocytes adjacent to epidermis and central (Figs. 55A–55C). Mature oocytes surrounded by a layer of cells with gastrodermal origin (probably follicle cells), which merges with gastrodermis of gastrovascular cavity (including gastrodermis of principal radial pocket and gastrodermis of auxiliary radial pocket), forming gametoduct (Figs. 55D–55Q). Cilia often associated with gametoduct (Fig. 55D). Anchors absent (Fig. 51A). Each accessory radial pocket extending throughout calyx, apically continuing into two adradial arms and respective tentacular clusters. Internal organization of arms similar to H. tenuis (Fig. 11). Eight sections of coronal muscle at calyx margin, each between adjacent arms (Fig. 51A). Organization of longitudinal and coronal muscles in arms similar to H. tenuis (as in other species examined; Figs. 5 and 11). Perradial and interradial white spots of nematocysts on subumbrella, with internal organization (Figs. 32A–32M) similar to H. tenuis. Aperture of white spots at subumbrellar epidermis clearly recognizable in its central thicker region (Figs. 32G–32M): a pore divides subumbrellar epidermis and adjacent layer of mesoglea into two regions in a longitudinal section, allowing an outflow to central mature nematocysts (Fig. 32M). Batteries of nematocysts sparsely distributed in exumbrellar epidermis (as in other species examined; Fig. 13). Distal end of arms with intertentacular lobules, a structure between adjacent secondary tentacles delimited by gastrodermis and a central layer of mesoglea (Fig. 56). Outermost secondary tentacles with pad-like adhesive structures (epidermal thickening) as in M. uchidai (Figs. 24L–24N, 51E–51G). Continuous layer of internal unorganized nematocysts in subumbrellar epidermis as in most of other species examined (Figs. 15 and 16). Secondary hollow tentacles composed of two parts, knob and stem (Figs. 17R–17U and 56), with organization similar to H. tenuis. At stem base, secondary tentacles tightly joined, separated only by thin layer of mesoglea, with beehive appearance in cross section as in most of species examined (Fig. 17).

Longitudinal and coronal muscles

The musculature of stalked jellyfishes is organized into two muscular arrangements: circular (coronal) and longitudinal (radial) muscles (Uchida, 1929; Berrill, 1963; Miranda, Collins & Marques, 2013). The main muscular arrangement in stauromedusae is longitudinal, and not circular as generally observed in active and planktonic scyphomedusae and cubomedusae (Gwilliam, 1960; Arai, 1997; Satterlie, Thomas & Gray, 2005), consistent with the benthic and sessile habit of staurozoans (Gwilliam, 1960; Miranda, Collins & Marques, 2015).

The recently proposed phylogenetic hypothesis for Staurozoa (Fig. 1) suggests that interradial longitudinal muscles in the peduncle played a fundamental role in the evolution of the class (Miranda et al., 2016). The new phylogeny-based classification scheme separates the group into two suborders, Myostaurida and Amyostaurida, animals with and without interradial longitudinal muscle in the peduncle at the stauromedusa stage, respectively (Figs. 1, 5, 57 and 59; Table 3; Miranda et al., 2016). Because the last common ancestor of Staurozoa is inferred to have had peduncular muscles (Table 3), a loss of these muscles can be inferred to have happened in the lineage leading to Amyostaurida (Fig. 59). As expected, our new observations of internal anatomy corroborate the absence of interradial longitudinal muscles in the peduncle in representatives of Amyostaurida, Craterolophus and Calvadosia, differing from the condition in Myostaurida, as represented by species of Haliclystus, Lucernaria, and Manania (Fig. 57).

In Lucernaria species, the interradial longitudinal muscles are associated with the interradial septa (occupying an internal position in the septa) from the base of the peduncle (Figs. 26, 30, 35 and 57; Collins & Daly, 2005), whereas in H. tenuis and M. uchidai the interradial longitudinal muscles in the peduncle are intramesogleal because the septa are only present in the region where the calyx connects to the peduncle (Figs. 4, 19 and 57; Uchida, 1929; Ling, 1939; Miranda, Collins & Marques, 2013). In species of Haliclystus, the gastrodermis of the central chamber envelops the interradial longitudinal muscles in the region of the peduncle/calyx connection (Fig. 4H; Uchida, 1929; Uchida & Hanaoka, 1934; Ling, 1939; Miranda, Collins & Marques, 2013), delimiting the four interradial septa with internal interradial longitudinal muscles, similar to Lucernaria (Fig. 57). On the other hand, M. uchidai has a slightly different organization (Figs. 19 and 20): the interradial septa are gradually formed as the size of the four perradial chambers increases in the region of the peduncle/calyx connection. The interradial longitudinal muscles do not occupy an internal position in the septa, remaining external in relation to the septal constriction (Fig. 21), a condition that changes only at the base of the manubrium (Fig. 20I). Manania uchidai has a central tissue (mesoglea and gastrodermis) (Figs. 20A and 20B) connecting the four interradial septa that disappears only after the formation of claustra (Figs. 20D–20G). However, our observations contrast with those of Uchida & Hanaoka (1933), who reported that the septa are completely separated below the formation of the claustra in M. uchidai (identified therein as Manania distincta). Either the central connection of septa varies intraspecifically or one report is an artefact.

Species of Amyostaurida do not have interradial longitudinal muscle in the peduncle (Figs. 5, 57 and 59; Table 3). The examined species of Calvadosia have four perradial chambers at the base of the peduncle, which progressively merge, producing four interradial septa and one central gastric chamber (Figs. 39, 43, 47 and 57). The interradial longitudinal muscles only appear at the peduncle/calyx connection, internal to the septa (Figs. 39, 43, 47 and 57). Craterolophus convolvulus has four perradial chambers separated by mesoglea throughout the peduncle (Fig. 52). At the peduncle/calyx connection, the perradial chambers of C. convolvulus increase in size and merge centripetally, delimiting the interradial septa (Figs. 52J–52N). A central mesoglea delimited by gastrodermis is still visible (Figs. 52N–52P), but progressively disappears apically (Figs. 52R and 52S). Interradial muscles associated with septa are clearly visible only after claustra formation (Fig. 53G), differing from other species of Amyostaurida, in which the interradial longitudinal muscles become visible at the base of the infundibula (Figs. 39, 43 and 47).

Interradial longitudinal muscles are divided into two bands in the calyx of all species (Figs. 5D, 5H and 5L). These bands gradually assume an adradial position toward the calyx margin (Figs. 5M and 11B). In the amyostaurid C. convolvulus, when the interradial longitudinal muscles are visible in the cross sections of the calyx, they are already divided into two bands (Figs. 53G and 53H) that progressively assume a more adradial position. Each longitudinal muscle band is associated apically with one arm and the correspondent stems of secondary tentacles (Figs. 5Q–5V and 11). Accordingly, there is a continuum of the longitudinal muscles found in the peduncle (in Myostaurida) or at the base of the calyx (Amyostaurida), with the muscles in the stem of secondary tentacles. In addition, other regions of the body, such as the manubrium, are provided with thin muscle fibers (Gwilliam, 1960; Miranda, Collins & Marques, 2015).

The coronal muscle can be entire, as in M. uchidai (Figs. 18C and 18D), running as an external (exumbrellar) and non-interrupted band in the arms; or discontinuous, divided into perradial and interradial sections by the arms, as in H. tenuis (Fig. 59; Table 3; Clark, 1863; Carlgren, 1935; Gwilliam, 1956; Kramp, 1961). Cross sections in the arms clearly show two lateral bands of coronal muscle, one from interradii and the other from perradii (Figs. 5Q, 5R, 11H and 11L). The presence of coronal muscle (vestigial in Stylocoronella; Kikinger & Salvini-Plawen, 1995) and its relative position with respect to the anchors/primary tentacles have also been used to differentiate genera (Clark, 1863; Mayer, 1910; Uchida, 1929; Carlgren, 1935; Gwilliam, 1956; Kramp, 1961; Miranda et al., 2016).

The contraction of the coronal musculature, along with contraction of the longitudinal muscles, considerably reduces the total volume of the animal, probably making its adherence to substratum more efficient in wave-exposed habitats (Hyman, 1940; Miranda, Collins & Marques, 2013; see also Kier, 2012, for discussion about the arrangement of muscular fibers and their role in support and movement in hydrostatic skeletons). In addition, longitudinal muscles in arms and secondary tentacles are likely related to feeding behaviors (Hyman, 1940; Larson, 1980; Miranda, Collins & Marques, 2013). Spontaneous neuromuscular activity in Haliclystus is arrhythmic, and stauromedusae lack the ability to perform pulsating swimming motions, unlike free-swimming jellyfishes (Gwilliam, 1960).

Chambers in the peduncle

Most of the staurozoan genera have four chambers in the peduncle and this state was suggested as a potential synapomorphy of Staurozoa (Fig. 59; Table 3; Collins & Daly, 2005; Miranda et al., 2016). However, the number of chambers in the peduncle has been considered to be a useful character to distinguish staurozoan genera (Clark, 1863; Mayer, 1910; Uchida, 1929; Kramp, 1961; Larson & Fautin, 1989). The stalked jellyfishes can have either (a) one chamber in the peduncle (e.g., Lucernaria, Lipkea; Kramp, 1961); (b) four perradial chambers (e.g., Haliclystus, Craterolophus; Kramp, 1961); (c) four perradial chambers at the base, merging into one chamber medially (e.g., Calvadosia; Mayer, 1910; Miranda et al., 2016); (d) one chamber at the base splitting into four chambers medially (e.g., some Manania, Larson & Fautin, 1989).

In addition, the numbers of chambers in the peduncle were also proposed as the “most useful” character for differentiating species of Manania (Larson & Fautin, 1989: 1547). The genus Manania includes species with one chamber throughout the peduncle, four chambers throughout the peduncle, or one chamber basally with four chambers at the median region (Larson & Fautin, 1989). However, the number of chambers varies during staurozoan development (Mayer, 1910; Uchida, 1929; Hirano, 1986), as reported in Haliclystus and Manania (Wietrzykowski, 1911; Wietrzykowski, 1912; Uchida, 1929; Hirano, 1986), and this character must be cautiously employed in taxonomic studies (Miranda et al., 2016).

According to the classification proposed by Miranda et al. (2016; Fig. 1) based mainly on molecular phylogenetic analyses, Lucernariopsis, Sasakiella, and Kishinouyea are synonyms of Calvadosia. At least in principle, species of former Lucernariopsis would have one chamber throughout the peduncle, whereas former Kishinouyea and Sasakiella would have four chambers at the base of the peduncle (sometimes separated by an axial canal resulting from an invagination of epidermis), which gradually merge apically to form a single chamber (Kramp, 1961). However, the presence of four chambers only at the base of the peduncle is not easy to observe, demanding serial transversal sections of a well-preserved and straight peduncle (the bodies of stauromedusae frequently contract during preservation). Indeed, cross sections at the bases of peduncles are rare in the descriptions of species, and most of them include only information about the median region of the peduncle (Edmondson, 1930; Carlgren, 1938; Miranda et al., 2012). Consequently, it is difficult to be sure that this distinction was correctly applied in former determinations. We have sectioned and analyzed the base of the peduncle of Calvadosia vanhoeffeni, formerly Lucernariopsis vanhoeffeni (Fig. 47), and although the species has been described with a one-chambered peduncle (Browne, 1910), it has four chambers at the peduncle base (Figs. 47D–47E). Therefore, the peduncle should be re-examined in detail for other species of the genus Calvadosia, in order to verify the universality and/or variability of number of chambers in the group.

A possible function for the chambers in the peduncle of stauromedusae has never been discussed, but they probably work as a hydrostatic skeleton (fluid filled cavities; see review in Kier, 2012), in addition to being an important part of the gastrovascular system, increasing surface area (Hand, 1966). In hydrostatic skeletons the force of muscle contraction is transmitted by internal pressure (Kier, 2012). The association of antagonist muscles (longitudinal and circular) with the hydrostatic skeleton can change the shape, size, and orientation of the body (Kier, 2012), as well as possibly improving temporary attachment—all actions particularly important to these animals.

The four chambers are formed during the metamorphosis of the stauropolyp to the stauromedusa, and their organization is complete only late in development (Wietrzykowski, 1912), so they should not be considered typical “polypoid” structures. For instance, the peduncle of developing Haliclystus octoradiatus has one chamber until the 32-tentacle stage, and then, progressively, four independent chambers are formed upwards (Wietrzykowski, 1911; Wietrzykowski, 1912). Similar observations were also described for other species of Haliclystus, whose juveniles have a single-chambered peduncle, later divided into four chambers that extend from the base to the top of the peduncle (Hirano, 1986). Accordingly, the general understanding that the peduncle region retains polypoid characters (Stangl, Salvini-Plawen & Holstein, 2002) is not completely correct for stauromedusae.

Therefore, the internal anatomy of the peduncle of many stauromedusae is different and more complex than the condition found in stauropolyps (Wietrzykowski, 1912) and in other medusozoan polyps, indicating that metamorphosis in Staurozoa (stauropolyp to stauromedusa) occurs both at the apical region (calyx) and basal region (peduncle) (Uchida, 1929; Hirano, 1986). A simple comparison between states of the stauromedusa peduncle and those of polyps of medusozoans is an oversimplification. Consequently, a detailed investigation of the metamorphosis of stauropolyps into stauromedusae would help evaluate hypotheses concerning the evolution of morphological characters in Staurozoa and in cnidarians in general.

Claustrum

The gastrovascular system of some stauromedusae has an additional level of complexity because of the presence of the claustrum (Fig. 2D; Clark, 1863; Gross, 1900; Berrill, 1963; Collins & Daly, 2005). The claustra are structures formed by the fusion of lateral evaginations of adjacent interradial septa, and each claustrum is composed of a central layer of mesoglea surrounded by gastrodermis (Figs. 2D, 20 and 53). We documented the claustra in M. uchidai (Fig. 20) and C. convolvulus (Fig. 53). Besides Manania and Craterolophus, claustra have also been recorded in Depastrum, Depastromorpha, and Halimocyathus (Table 3; Clark, 1863; Kramp, 1961; Miranda et al., 2016).

In M. uchidai and in C. convolvulus, the claustra delimit the accessory radial pockets (Figs. 20 and 53), and the principal radial pockets are formed in association with the manubrium (Figs. 20I and 20J). The accessory and principal radial pockets are separated by claustra (Figs. 2D, 20L, 20M, 53J and 54A). Consequently, there are eight gastric pockets in the calyx instead of four in the animals without the claustrum (Fig. 6; Gross, 1900; Berrill, 1963). The four external pockets, known as accessory pockets (or exogon; Thiel, 1966), extend into the anchors, arms, and secondary tentacles, continuing into the peduncle as perradial chambers (Fig. 57; Berrill, 1963). Although the accessory radial pockets occupy the same position as gastric radial pockets (of the animals without the claustrum), the four internal pockets, also known as principal (main) radial pockets (or mesogon, Thiel, 1966), are apparently the true gastric radial pockets of these stauromedusae because they contain the gonads and the gastric filaments (Figs. 20, 53, 54 and 57; Clark, 1863; Gross, 1900; Berrill, 1963).

Because of the internal organization, the principal radial pockets of the animals with claustra occupy a perradial position. Consequently, their associated gonads are also perradial (Figs. 57 and 58). The accessory radial pockets, which do not contain gonads, are associated with the arms that, likewise, do not contain gonads (Figs. 57 and 58). On the contrary, animals without the claustrum have gonads associated with the gastric radial pockets, therefore in an adradial position extending into the arms (Fig. 58). Animals with interradial paired arms (such as C. cruciformis, C. corbini, L. quadricornis) have the gonads still associated with the gastric radial pockets in the arms, but then positioned at the interradii/adradii.

Therefore, as a consequence of the internal architecture, the position of gonads is a useful external character for distinguishing animals with and without the claustrum (Fig. 58). Although the claustrum is a valuable character for distinguishing genera in Staurozoa, making histological sections to ascertain the presence or absence of this structure is not always possible. Externally, animals with claustra have exclusively perradial gonads located from the base of the manubrium to the perradial margin of calyx; whereas animals without the claustrum have perradial gonads only at the base of the manubrium, assuming an adradial position at calyx margin and arms, or they are associated with the interradial paired arms (Fig. 58). This can be observed in species of Manania, Craterolophus, and Depastromorpha, but needs to be checked for species of Depastrum and Halimocyathus (the other staurozoan genera with claustra).

According to Berrill (1963: 749), species with and without claustrum indicate “two biological engineering solutions to nutritional problems but does not imply a superiority of one type over the other.” The claustra compartmentalize the body of stalked jellyfishes, and this could be a response to biotic or abiotic pressures, but does not seem to be directly related to other internal structures, such as the number of gastric chambers or the presence of interradial longitudinal muscles in peduncle. However, the claustra create an exclusive compartment for the gonads, separating them from the gastric chambers, anchors, arms, and secondary tentacles (differently from the animals without the claustrum), although basal communication exists before claustra delimitation is complete (Figs. 53A–53C). The gonads in C. convolvulus and M. uchidai, species with claustra, are similar, organized as a simple layer, between the gastrodermis and epidermis, and not as numerous vesicles (Figs. 22 and 54). In addition, they are constrained to the interior of the calyx, never associated with arms, and therefore they would be more protected from predators that graze part of a stauromedusa (Prell, 1910; Uchida & Hanaoka, 1933; Davenport, 1998; Mills & Hirano, 2007).

Clark (1863) was apparently the first author to describe the claustrum, and used the feature to divide the stalked jellyfishes into two main groups: Cleistocarpida (claustrum present) and Eleutherocarpida (claustrum absent). Since then, the claustrum has played a central role in the taxonomy of Stauromedusae (Collins & Daly, 2005), and most of the main proposals of classification have been based on that feature (see review in Miranda et al., 2016). Molecular phylogenies make it clear that neither Cleistocarpida nor Eleutherocarpida are monophyletic, dismissing the importance of the claustrum for diagnosing the primary subgroups of Staurozoa (Collins & Daly, 2005; Miranda et al., 2016). A question derived from the homoplastic appearance of the claustrum is whether it is comprised of the same structures among the different and non-closely related staurozoan genera. We analyzed the distantly related C. convolvulus and M. uchidai, and the structures of claustra in these species are morphologically similar, i.e., lateral projections of the septa composed only of mesoglea and gastrodermis (Figs. 20 and 53). In C. convolvulus, besides the accessory and principal radial pockets, there are also numerous auxiliary radial pockets containing gonads, as a result of folds of septa and principal radial pockets (Figs. 53 and 54). Collins & Daly (2005) questioned whether a claustrum might be an ancestral character for Staurozoa (Fig. 59; Table 3). If so, the claustrum was lost several times in staurozoan evolution (Fig. 59; Table 3; Miranda et al., 2016). Indeed, the claustrum has also been described in the medusa stage of Cubozoa, but the gonads are associated with the exogon in box-jellyfishes (Thiel, 1966), and the existence of a typical staurozoan-type claustrum in Cubozoa needs to be assessed (Fig. 59; Table 3; Miranda et al., 2016).

Internal layer of nematocysts, white spots of nematocysts, and exumbrellar/subumbrellar batteries of nematocysts

A continuous internal layer of nematocysts at different stages of development has been recently described for H. antarcticus in the subumbrellar epidermis of arms, radial pockets, and infundibula (Miranda, Collins & Marques, 2013). It has been hypothesized that these nematocysts are produced in the epidermis of the infundibula and migrate through the subumbrellar epidermis to the tips of the secondary tentacles in the knob, where mature nematocysts are organized in one external row and can perform their function of defense from predators, capture of prey, and adherence to substrate (Figs. 15–17; Miranda, Collins & Marques, 2013; see also Gross, 1900; Kassianow, 1901; Wietrzykowski, 1912; Weill, 1925; Weill, 1935; Leuschel, 1932; Uchida & Hanaoka, 1933; Uchida & Hanaoka, 1934). Wietrzykowski (1912) observed this layer in the epidermis of the infundibula, adjacent to the septa, beginning with the 4-tentacle stage of the stauropolyps of H. octoradiatus. According to him, most of the cells in these regions become nematoblasts and, as development proceeds, the epidermis contains nematocysts at different stages of development without direct contact with the external environment (Wietrzykowski, 1912).

We observed this internal layer of nematocysts from the epidermis at the bases of the infundibula to the knobs of the secondary tentacles (Figs. 15–17) in all species (with the possible exception of M. uchidai, in which this layer was not clearly discernible, but has been previously described by Uchida & Hanaoka, 1933: 145, identified as M. distincta), indicating its likely universality in Staurozoa. Additionally, this layer seems to be associated with the accumulation of unorganized nematocysts at the base of the secondary tentacles (Fig. 16L), internal to the epidermis and just opposite to the intertentacular lobules (Miranda, Collins & Marques, 2013). This region of accumulation was observed in all species analyzed in this study (Fig. 16), and could be important for providing the animal with a constant and dynamic supply of nematocysts.

Although Weill (1925) has suggested a similar scenario for the migration of nematocysts, he associated the internal layer of nematocysts with another set of structures, the white spots of nematocysts (Figs. 12 and 32). The white spots are located on the subumbrella at the perradii and/or interradii (depending on the species), frequently associated with the calyx margin, gonads, and arms. In a cross section, they look like “bubbles” in the mesoglea, between layers of subumbrellar gastrodermis and epidermis, containing abundant nematocysts (Fig. 12). The process responsible for the formation of these structures has been proposed as invagination (Weill, 1925) or delamination (Leuschel, 1932) of the subumbrellar epidermis (Uchida & Hanaoka, 1933). The presence of white spots of nematocysts is widespread in Staurozoa, occurring in all genera, but they were never reported as taxonomically important until Corbin (1978) and, more broadly, Hirano (1997) and Kahn et al. (2010) used their presence and position (perradial versus perradial and interradial) to diagnose and differentiate staurozoan species. As far as we know, white nematocyst spots are only known for Staurozoa, and their presence is a probable synapomorphy for the class (Fig. 59; Table 3).

Three hypothetical functions of the white spots can be raised. First, they could be structures for defense and attack, with the nematocysts able to escape outside the sacs by pressure through a narrow slit between ectodermal cells (Kling, 1879; Kassianow, 1901; Uchida & Hanaoka, 1933). Alternatively, the sacs could simply be a place for the production of nematocysts (Uchida & Hanaoka, 1933), an idea offered due to the inferred lack of an aperture in the white spots (Leuschel, 1932). The third hypothesis is that the white spots could be selective reservoirs of nematocysts produced at the base of the infundibula (Weill, 1925; Weill, 1935) and that after a period of maturation, they would migrate out to the secondary tentacles (Weill, 1925; Weill, 1935).

The white spots of the species studied herein all have the same morphology, in accordance with that described by Uchida & Hanaoka (1933: 146) for M. uchidai (as M. distincta), with a basal peripheral layer of nematoblasts adjacent to the gastrodermis, and a central group of loosely aggregated mature nematocysts (Fig. 12). The nematoblasts have opaque and darker capsules, whereas in the mature nematocysts, internal structures such as the shaft and tubule are visible. An epidermal thickening at the central region of white spots (Figs. 12 and 32) was also consistently observed.

Importantly, serial longitudinal sections of the white spots of nematocysts of C. convolvulus and L. bathyphila revealed a small opening in these sacs (Fig. 32), located in the region with the thickest epidermis, in which a well-delimited pore passing through the epidermis and adjacent mesoglea connects mature nematocysts with the outside (Fig. 32). This strengthens the hypothesis that the white spots are structures for defense and attack because there is indeed an opening through which the nematocysts could be released. Furthermore, the basal and peripheral layer of nematoblasts suggests that the nematocysts from the white spots are produced locally, contrary to the hypothesis that they would be related to maturation of nematocysts produced at the subumbrellar epidermis (Weill, 1925; Weill, 1935). Besides, there are species without white spots, such as H. antarcticus (cf. Miranda, Morandini & Marques, 2009), suggesting that the nematocysts produced in the internal subumbrellar epidermis would not need a “selective reservoir” (Weill, 1925; Weill, 1935) in order to become mature and migrate to the secondary tentacles.

Although the function of the white spots is still not known with certainty, it has also been hypothesized that they protect the stauromedusan gonads from predators (Kling, 1879; Kassianow, 1901; Weill, 1925). Indeed, we observed that they are often associated with the gonads (Fig. 20M). In addition, white spots appear concomitantly with the gonads during the development of Haliclystus octoradiatus (32-tentacle stage; Wietrzykowski, 1912, Fig. XXI). The striking color pattern provided by the presence of these white spots could also be a visual signal to either attract prey or deter visual predators, as their bright reflecting white color diverges from the usually camouflaged body of stauromedusae (Mills & Hirano, 2007).

There is no information about whether the white spots are permanent or transitory structures, i.e., whether or not these structures are retained after (and if) nematocysts are released. However, apparently their perradial/interradial position is constant, making them useful in species differentiation (Corbin, 1978; Hirano, 1997; Kahn et al., 2010). During development of different species of Staurozoa, the perradial white spots generally appear first (e.g., Haliclystus borealis, H. tenuis), but in some species (e.g., Haliclystus stejnegeri, H. octoradiatus) eight adradial white spots appear at the same time (Wietrzykowski, 1912; Uchida, 1929; Hirano, 1986). This pattern of appearance of the white spots was used to support a close relationship between H. tenuis and H. borealis (Hirano, 1986), corroborated in the recent molecular phylogeny (Miranda et al., 2016).

In summary, based on our results and on information in the literature, there are at least two regions of nematocyst formation in stauromedusae: 1) in the epidermis of the infundibula, supplying nematocysts to the secondary tentacles (Figs. 15 and 16); and 2) at the peripheral region of white spots, supplying nematocysts exclusively to the white spots (Figs. 12 and 32). However, there is not enough information to suggest whether the nematocysts of the secondary tentacles are being formed exclusively in the basal epidermis of the infundibula or throughout the whole subumbrellar epidermis, i.e., throughout the internal layer of nematocysts.

Many species of Staurozoa also have exumbrellar batteries (warts) of nematocysts (Fig. 13), comparable to those of other cnidarians. These batteries are simple epidermal clusters of nematocysts that probably play a role in the defense of stalked jellyfishes. These batteries of nematocysts are also common on the subumbrella of C. corbini, and are associated with the nodular gonads, between the areas internally corresponding to two adjacent vesicles (Figs. 13L, 13M, 40H and 40I). These subumbrellar batteries could provide additional protection to the gonads, which are probably more susceptible in these animals because they are projected to the exterior (Fig. 38). The origin of the nematocysts in these batteries, as well as the origin of the nematocysts found elsewhere, such as the gastric filaments (Miranda, Collins & Marques, 2013), is uncertain.

Intertentacular lobules and “U-shaped” space

The base of the tentacular cluster at the internal tip of the arms can have either several intertentacular lobules, or a deep “U-shaped” space (Table 3; Hirano, 1997). The intertentacular lobules are internal projections between adjacent secondary tentacles composed of a central layer of mesoglea surrounded by a layer of gastrodermis (the gastrodermis belonging to adjacent secondary tentacles; see examples in Figs. 33C and 50C). In a cross section, it is possible to see a circular gastrodermis surrounded by mesoglea (Figs. 17E and 17M). However, some species, such as H. tenuis, do not have intertentacular lobules, but have a “U-shaped” space at the base of the tentacular cluster (Figs. 14 and 59; Table 3). In this region, there is a platform composed of mesoglea and gastrodermis, similar to a large lobule in a longitudinal section (Fig. 14K), which connects the exumbrellar side of the arms to the base of secondary tentacles.

These structures were rarely mentioned hitherto. Apparently, Clark (1878) was the first author to describe the intertentacular lobules, in the stauromedusa Haliclystus auricula. More than a century later, the internal anatomy of the base of secondary tentacles was used to differentiate species of Haliclystus (Hirano, 1997): Haliclystus californiensis, H. tenuis, and H. borealis do not have intertentacular lobules but have a “U-shaped” space, whereas Haliclystus “sanjuanensis” (nomen nudum), H. auricula, H. octoradiatus, and H. stejnegeri have intertentacular lobules (also present in H. antarcticus, Miranda, Morandini & Marques, 2009). The possible function of these structures and consequences of the different shapes of the internal base of the tentacular clusters have never been discussed, but they increase the gastrovascular area (see Hand, 1966) and also likely function as a point of anchorage and communication between secondary tentacles, possibly related to their movement/contraction (Hand, 1966; Kier, 2012).

Although these structures are relatively well known for Haliclystus, their general occurrence in Staurozoa has never been studied before. We observed intertentacular lobules of different sizes in Craterolophus (C. convolvulus), Calvadosia (C. corbini, C. vanhoeffeni, and C. cruciformis), Lucernaria (L. bathyphila, L. quadricornis, and L. sainthilairei), and Manania (M. uchidai). Therefore, intertentacular lobules seem to be widespread in Staurozoa and their presence is a probable synapomorphy for the class (Fig. 59; Table 3), whereas the “U-shaped” space may be restricted to some Haliclystus species (Table 3).

Gonads and gametoduct

The gonads of Staurozoa have been considered an important character to understand the evolution of the group, and the complex ovaries with follicle cells (Eckelbarger & Larson, 1993) have been suggested as a potential synapomorphy for the class (Marques & Collins, 2004; Collins & Daly, 2005; Collins et al., 2006; Van Iten et al., 2006), although this was recently questioned (Tiemann & Jarms, 2010).

The gonads are organized inside the gastric radial pockets in species lacking claustra, from the base of the calyx to the tips of the arms (Fig. 57). In C. corbini, evaginations of the gastric radial pockets produce nodular gonads as external projections on the wide-open subumbrella (Figs. 38E–38I and 40). In animals with claustra (e.g., M. uchidai, C. convolvulus), the gonads are associated with the principal radial pockets (mesogon), at perradii, and do not extend into the arms. Craterolophus convolvulus is unique in our observations in possessing auxiliary radial pockets, which also contain gonadal tissue (Figs. 53G–53J and 54).

Gonads are generally organized in vesicles as evaginations of gastrodermis, more (i.e., H. tenuis; Fig. 8) or less (i.e., species of Lucernaria; Figs. 27, 31 and 36) delimited. Gonadal content can also be restricted to a simple layer between gastrodermis and epidermis (i.e., M. uchidai; Fig. 22), without the organization of vesicles.

Male and female gonads can only be recognized with histological sections, and their internal structures have been described in a few different studies (Uchida, 1929; Berrill, 1963; Eckelbarger & Larson, 1993; Eckelbarger, 1994; Miranda, Collins & Marques, 2013). The gonadal content in males is organized in one peripheral layer of spermatocytes adjacent to gastrodermis and a central and internal layer of spermatozoa generally adjacent to epidermis. In females, a peripheral immature layer of oocytes is adjacent to gastrodermis and the mature oocytes occupy a central position, generally adjacent to epidermis. Each gonadal male and female structure contains numerous developing and mature spermatozoa and many immature and mature oocytes, respectively. Additionally, follicle-like accessory cells surround individual oocytes in Haliclystus “sanjuanensis” (Eckelbarger & Larson, 1993).

In this study, clear regionalization was recognized in all species other than species of Lucernaria. In L. quadricornis, the oocytes, surrounded by follicle cells, are randomly arranged in the mesoglea between gastrodermis and epidermis (Fig. 27). Oocyte organization in this genus is somewhat similar to the pattern of Scyphozoa and Cubozoa (Eckelbarger, 1994). The specimens of L. bathyphila and L. sainthilairei examined are apparently males, as they have structures similar to spermatocytes, but probably immature because spermatozoa could not be recognized (Figs. 31 and 36).

Gametoducts are a direct connection between the gonadal content and the gastrodermis of the vesicles (or correspondent gonadal structures), which likely carry spermatozoa or mature oocytes to the gastrovascular cavity. In females, they are associated with cells, probably the follicle-like cells of Eckelbarger & Larson (1993), that surround and isolate mature oocytes at the central region of vesicles (or correspondent gonadal structures). The organization is similar in males; gametoducts are associated with cells surrounding the spermatozoa, sometimes producing different sacs, and directly connected with spermatocytes (Figs. 48 and 49) most likely of gastrodermal origin. Moreover, cilia (Figs. 27G–27I) were observed in the connection of gametoducts with the gastrodermis of vesicles (or correspondent gonadal structures), probably contributing to a more efficient transport of gametes. It is noteworthy that gametoducts were observed in all but one analyzed species. The only exception was L. sainthilairei, for which no gametes were observed in the specimen, raising questions about whether the permanence or transitory presence of gametoducts is related to gonadal maturity.

The presence of gametoducts in stalked jellyfishes has been described for H. auricula as “mouth of genital sac,” “entrance of the sac,” “aperture of genital sac” (Clark, 1878: 67, Figs. 54, 62, 74, 75 and 77), for L. bathyphila as “oviduct” or “oviductulus” (Haeckel, 1882: 61, Plate XVII, Figs. 17 and 19), and for H. octoradiatus as “étroit canal” (Wietrzykowski, 1912: 77, Fig. 30), but has been completely ignored for over 100 years. This is the first study to indicate the universality of gametoducts in Staurozoa, present in all analyzed genera, both in males and females, and even in species without delimited vesicles (viz., M. uchidai, C. convolvulus), and their presence is reconstructed as a putative synapomorphy for the class (Fig. 59; Table 3). Nevertheless, more detailed studies in other cnidarian classes are necessary to better understand the evolution of these structures within Cnidaria (see also discussion below about gonopores in Periphylla periphylla, Tiemann & Jarms, 2010).

Berrill (1963: 742, 745) proposed that the “expulsion of gametes” in Haliclystus salpinx and in L. quadricornis “results from the rupture of the endodermis enclosing the gonads.” Our histological studies, however, suggest that gametes are released through specialized structures, indicating a complex functional mechanism. Gametoducts allow the selective release of mature gametes, preventing damage or destruction of the gonads, and not wasting immature oocytes (peripheral) in female vesicles and spermatocytes in male vesicles (or correspondent gonadal structures).

A similar structure, the gamete-releasing pore, was described for the coronate scyphomedusa Periphylla periphylla (Tiemann & Jarms, 2010). These special pores would work as gonopores, therefore the structure of the gonad would not be destroyed, allowing a continuous gamete release (Tiemann & Jarms, 2010). Spermatozoa were seen associated with these pores, as well as oocytes, which were expelled through this narrow pore, becoming deformed, but then regaining their spherical shape (Tiemann & Jarms, 2010). In addition, there is a strong maturity gradient in gonads of both sexes, simultaneously presenting all developmental stages of gametes (Tiemann & Jarms, 2010), a regionalization also observed in Staurozoa. Tiemann & Jarms (2010) concluded that complex and specialized structures, including gonopores, strongly suggest that true sexual organs exist in Scyphozoa (interpreted broadly to include Cubozoa and Staurozoa), even though they presented observations on only a single species of coronate scyphozoan. The homology between gametoducts in Staurozoa and gonopores in Periphylla periphylla remains to be tested. Indeed, recent research is beginning to uncover more complex reproductive systems in different cnidarians (Cartwright & Nawrocki, 2010; Schiariti et al., 2012), especially some species belonging to the class Cubozoa (Garm, Lebouvier & Tolunay, 2015; Marques, García & Lewis Ames, 2015). Although plesiomorphic external fertilization would be expected in adult medusae (Marques & Collins, 2004), certain cubomedusae can present courtship behavior, specialized internal fertilization, and sperm storage (Lewis & Long, 2005; Garm, Lebouvier & Tolunay, 2015; Marques, García & Lewis Ames, 2015), which demonstrate the importance to highlight different cnidarian reproductive strategies in order to study their evolution.

However, a recent review concluded that Cnidaria has no specialization in the reproductive system, such as genital ducts, generally used for storing, transporting, or extruding gametic products (Extavour, 2007). Our results and data in Tiemann & Jarms (2010) refute this interpretation. That said, gonoducts in Bilateria generally take gonadal elements directly to the outside of the animal (Dewel, 2000; Extavour, 2007), in contrast to the structures of Staurozoa and P. periphylla (Tiemann & Jarms, 2010). Staurozoan gametoducts release gametes into the gastrovascular cavity, after which the gametes are then expelled to the outside through the mouth (Otto, 1976). In addition, staurozoan gametoduct structures are perhaps unlikely to be homologous with bilaterian gonoducts because of their different origins, the latter generally mesodermal (Extavour, 2007; Nielsen, 2008).

Convergent evolution may have produced different morphological solutions to the problems posed by sexual reproduction (Extavour, 2007), although it is possible that similarities in the underlying molecular pathways may exist. Consequently, further investigation of the occurrence of gonopores or gametoducts in other cnidarians, as well as additional developmental data, particularly on gene expression, could uncover possible homologies of these specialized structures. Such knowledge will likely have important implications for understanding the evolution of reproductive systems across Eumetazoa.

Anchors and primary tentacles

During the early metamorphosis of a stauropolyp, eight primary tentacles develop, four perradial and four interradial (Wietrzykowski, 1912; Hirano, 1986; Kikinger & Salvini-Plawen, 1995), which may have four different fates in the stauromedusa stage: 1) they disappear by resorption (in L. quadricornis, Berrill, 1963); 2) they metamorphose into adhesive structures called anchors (in species of Haliclystus, Hirano, 1986); 3) they remain as primary tentacles, although with a modified shape (in Calvadosia tsingtaoensis and C. cruciformis; Ling, 1937); 4) they are originally rather filiform, but transform into capitate tentacles and cluster together with secondary tentacles (in species of Stylocoronella, Kikinger & Salvini-Plawen, 1995).

Anchors are apparently restricted to Haliclystidae (Fig. 59; Table 3; Miranda et al., 2016), and their shapes have been broadly used to distinguish species of Haliclystus (Miranda, Morandini & Marques, 2009; Kahn et al., 2010). However, the use of this character for differentiating species can cause taxonomic problems because of ontogenetic variation (Miranda, Morandini & Marques, 2009).

Primary tentacles and anchors are hollow structures, composed of an internal layer of gastrodermis and an external layer of epidermis, with mesoglea between them (Figs. 9 and 10). Perradial primary tentacles/anchors are directly connected to the gastrodermis of the gastric radial pockets (or accessory radial pockets in animals with claustra) (Fig. 9). Interradial primary tentacles/anchors are connected to the gastrovascular cavity by means of small interradial ostia (Fig. 10) that connect adjacent gastric radial pockets (or accessory radial pockets in the animals with claustra).

We agree that anchors (or rhopalioids) and rhopalia, present in Staurozoa, Cubozoa, and Scyphozoa, are probably homologous marginal structures because they all result from metamorphosis of primary tentacles (Uchida, 1929; Thiel, 1966). However, whereas the rhopalia in Cubozoa and Scyphozoa have a sensorial function (Nakanishi et al., 2010), the rhopalioids in Staurozoa likely have a role in substrate attachment (Larson, 1988). The epidermis of the anchor is formed by tall supporting cells and glandular cells, providing evidence for an adhesive function that contributes to small movements of the animals, mainly when the peduncle is detached from the substrate (Miranda, Collins & Marques, 2013). Nematocysts are generally not observed in the anchors, but they are visible in species with a knobbed remnant of primary tentacles, such as in M. uchidai, corroborating their tentacular nature. The primary tentacles of C. cruciformis also have an external layer of nematocysts (Fig. 9L), and they probably also help in the locomotion (adhesive function) of the stauromedusa.

Ostia

The presence of an ostium between two adjacent gastric radial pockets has been recorded before for a small number of staurozoan species, such as H. salpinx, Manania atlantica (Berrill, 1963), and H. antarcticus (Miranda, Collins & Marques, 2013). The ostium connects the gastrodermis of the interradial anchor with that of the calyx (Fig. 10). In species with claustra this connection occurs in the accessory radial pockets. The main hypothesis to explain the presence of these ostia is that they might permit a nutritional extension into the four interradial anchors (Berrill, 1963: 742). However, they are small that they might not allow for effective circulation of fluids between two adjacent radial pockets (Berrill, 1963). Based on this hypothesis, species without anchors would not need an ostium connecting adjacent gastric radial pockets if ostia really only serve to allow for the connection of the anchor/primary tentacle to the calyx (Miranda, Collins & Marques, 2013).

However, all species analyzed, both with and without anchors/primary tentacles, have an ostium connecting two adjacent gastric radial pockets (Fig. 10). Therefore, two main hypotheses emerge: (1) the ostia, that probably provided gastrodermal extension to the primary interradial tentacles in the stauropolyp stage, were simply retained after the disappearance of primary tentacles in the stauromedusa stage (species without anchors/primary tentacles); (2) ostia are important in the communication of radial pockets, and probably have other function(s) besides providing nutritional extension to the four interradial anchors/primary tentacles.

Comparable structures, such as the septal ostium or connecting canal, have been observed in Scyphozoa (Korschelt & Heider, 1895; Chapman, 1966) and Cubozoa (Conant, 1898: 18, 19), but further detailed studies are necessary to assess their homology across these taxa (see also Fig. 59; Table 3).

Pad-like adhesive structures

Pad-like structures can be present individually in the outermost secondary tentacles of the tentacular cluster (Larson & Fautin, 1989), or as a broad structure on the tip of each arm (Fig. 24; Larson, 1980; Miranda et al., 2012). We found individual pad-like adhesive structures in the outermost secondary tentacles in C. convolvulus, M. uchidai, C. cruciformis, and C. vanhoeffeni. These structures are a simple lateral projection in the tentacular stem, with an epidermal thickening (Figs. 24L–24N). The only examined species with pads at the tips of arms was C. corbini (Figs. 24A–24K). This more complex structure is an exumbrellar lateral projection with thick epidermis, mesoglea, and internal hollow gastrodermal canals, connected to the gastrovascular cavity of arms.

Individual adhesive glandular pads at the outermost secondary tentacles are widespread in the class (Miranda et al., 2016), a putative synapomorphy for the class (Fig. 59; Table 3), whereas the broad pad-like adhesive structure on the tip of each arms is probably a synapomorphy of a clade within Calvadosia (Miranda et al., 2016, Fig. 16). The pad-like adhesive structures of the outermost secondary tentacles and at the tips of the arms were hypothesized to be homologous by Corbin (1978), and the broad adhesive pad-like structure on the tip of each arm was suggested to be the result of fusion of several outermost secondary tentacles (Larson, 1980). Indeed, cross sections of the pads on the tips of arms show many hollow gastrodermal canals, which could be interpreted as the “merging” of several stem canals of the secondary tentacles. However, the thin gastrodermis of the pads in the arms contrasts with the thick and glandular gastrodermis of the secondary tentacles (Figs. 17, 24A–24K), but this could be a secondary differentiation related to a different function assumed by the pads.

Adhesive pads help the animals to adhere to its substrate (Larson, 1980). They have been hypothesized to serve to temporarily reattach the stauromedusae when the peduncle is detached (Larson, 1988). Their internal anatomy, with epidermis composed of tall glandular and supporting cells, like all other adhesive regions (viz. secondary tentacles, anchors, peduncle and pedal disk) of a stauromedusa, corroborates this hypothesis (Miranda, Collins & Marques, 2013).