Hydrobiologia 443: 103–128, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 103 Systematic revision of the genus Petromica Topsent (Demospongiae: Halichondrida), with a new species from the southwestern Atlantic Guilherme Muricy1 , Eduardo Hajdu1,2 , José Valter Minervino1, Ana Verena Madeira3 & Solange Peixinho3 1 Departamento de Invertebrados, Museu Nacional, Universidade Federal do Rio de Janeiro. Quinta da Boa Vista, s/no., São Cristóvão. 20940-040 Rio de Janeiro, RJ, Brazil E-mail: muricy@acd.ufrj.br 2 Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião, SP, Brazil 3 Departamento de Zoologia, Instituto de Biologia, Universidade Federal da Bahia. Campus Universitário, Ondina, 40210, Salvador, Bahia, Brazil E-mail: peixinho@ufba.br Received 16 November 1999; in revised form 28 September 2000; accepted 18 October 2000 Key words: taxonomy, phylogeny, morphology, Porifera, Petromica, Petromica citrina sp. n. Abstract The status, scope and classification of the sublithistid demosponge genus Petromica Topsent are revised through morphological analysis of museum specimens of all seven species (including proposed synonyms and varieties), two of which were collected and observed in situ along the Brazilian coast (P. ciocalyptoides (Van Soest & Zea) and P. citrina sp. n.). The synonymy of Petromica and Monanthus Kirkpatrick with priority to the former is justified due to the consistent presence of monocrepid rhizoclone desmas and oxeas in an halichondroid arrangement, and to the lack of co-variance in other morphological characters among the species studied (presence and shape of papillae, surface texture, ectosomal skeleton and desma shape). The proposed synonymy of P. grimaldii Topsent and P. massalis Dendy is refuted due to differences in habit and spicule shape between the two species. Three forms described as varieties of Monanthus plumosus Kirkpatrick are raised to species level: P. plumosa (Kirkpatrick), P. tubulata (Kirkpatrick) and P. digitata (Burton). Phylogenetic analysis indicates that two possibly monophyletic clades may be recognized within Petromica, although with low bootstrap support (35–59%): (P. ciocalyptoides, P. citrina) and (P. grimaldii, P. massalis) (P. plumosa) (P. tubulata) (P. digitata). The classification of Petromica within the Halichondriidae (order Halichondrida) is supported by the confused reticulation of long oxeote spicules with ascending spicule tracts, present in all species of the genus. Introduction The classification of desma-bearing sponges is a longstanding problem in sponge taxonomy. Desmas are a special type of siliceous spicules produced by sponges known as ‘lithistids’ due to their stony consistency. These spicules have a central epirhabd with a basic tetraxial or monaxial symmetry (respectively, tetracrepid and monocrepid desmas), which is branched in several recurved, irregular and often tuberculate cladii, and often fused with neighbouring desmas through junctions called zygoses. There are, however, many sponges in which the desmas are not connected through zygoses, instead they are isolated and scattered in the choanosome, mixed with a skeleton of usual spicules such as, e.g. oxeas or triaenes. Such sponges are much softer in consistency than true lithistids, and are sometimes collectively called ‘sublithistids’ (e.g. Van Soest & Zea, 1986). The accessory skeleton of megascleres and microscleres in different species suggests affinities with several orders of Demospongiae, and both lithistids and sublithistids are considered polyphyletic (e.g. Gruber, 1993; Kelly-Borges & Pomponi, 1994). The genus Petromica Topsent, 1898 is a clear example of problems in the classification of sublithistid sponges. It was originally defined as massive Azor- 104 icidae, cone-shaped, conulose, with dispersed pores and membranous oscules, with well-developed aspiculate ectosome, and with slightly fused, lightly ornamented desmas (Topsent, 1898: 226). Its type species P. grimaldii Topsent, 1898 has monocrepid desmas and oxeas arranged in a confused reticulation with longitudinal tracts. A second species, P. massalis Dendy, 1905, has oxeote spicules in a confused reticulation with few coarse tracts and non-fused, monocrepid desmas (Dendy, 1905, 1921). The two species were synonymized under P. grimaldii by Pulitzer-Finali (1970) and Boury-Esnault et al. (1994), who found Mediterranean specimens of P. grimaldii lacking the microspined tips of desmas typical of the species. Petromica was considered a synonym of Leiodermatium Schmidt, 1870 by Von Lendenfeld (1903), but it was later reinstated as valid by Topsent (1928), due to the lack of fused ectosomal desmas. The scope of the genus was enlarged when it was synonymized with Monanthus Kirkpatrick, 1903, with priority for the older name Petromica (cf. Van Soest et al., 1990). Monanthus was originally defined as “Desmanthidae in which the skeleton is formed of monocrepid desmas of the common type, separate or joined together, and of monaxon megascleres” (Kirkpatrick, 1903: 176). The type species M. plumosus has been described under three varieties (M. plumosus var. typica Kirkpatrick, 1903; M. plumosus var. tubulatus Kirkpatrick, 1903; and M. plumosus var. digitata Burton, 1929), all from South Africa. According to Kirkpatrick (1903), the typical variety has plumose columns of oxeas and styles, intermingled with monocrepid desmas isolated or loosely articulated; all three varieties are currently considered synonyms (cf. Van Soest & Zea, 1986; Diaz et al., 1993). The only other species described under Monanthus, M. ciocalyptoides Van Soest & Zea (1986) from the Caribbean, has oxeas in an halichondroid arrangement, with desmas concentrated at the base of the sponge, and a typical fistulose habit. Petromica was originally placed in the lithistid family Azoricidae by Topsent (1898), who later made it the type of a monotypical family, Petromicidae, defined as “Anoplia with monocrepid desmas and aspiculate, membranous ectosome” (Topsent, 1928: 105). De Laubenfels (1936) transferred both Petromica and Monanthus from the Lithistida to his family Monanthidae (order Halichondrida), on the grounds of a shared skeleton of smooth monaxons in a confused reticulation with vague spicule tracts. This move was disregarded by Lévi (1973) but followed by subsequent authors (Van Soest & Zea, 1986; Van Soest et al., 1990, Pomponi et al., 1991; Diaz et al., 1991, 1993; Gruber, 1993), who considered the possession of desmas as either an ancestral (plesiomorphic) or a homoplastic character. Molecular data supported the proximity of Monanthus with the Halichondrida (Kelly-Borges & Pomponi, 1994), although not forming a monophyletic group with Hymeniacidon Bowerbank, 1864. Kelly-Borges & Pomponi (loc. cit.) disregarded the proposed synonymy of Monanthus and Petromica, tentatively classifying the former as incertae sedis within the Halichondrida and the latter in order Lithistida (family Petromicidae, suborder Rhizomorina). Apart from the discussion on its correct classification, Petromica has other problems which are common to many sponge genera: its species have often been poorly described, usually after few specimens from a single locality, and no attempt has been made so far to study either intra- or interspecific morphological variations in the genus. The goals of this study were to redescribe and review the status of all known species and varieties of Petromica; to describe a new species of Petromica, P. citrina sp. n. from the Southwestern Atlantic; and to discuss the phylogeny, biogeography, status and suprageneric classification of the genus. Materials and methods Collections of P. ciocalyptoides and the new species were made by SCUBA and free diving at depths ranging from 2 to 21 m deep, in seven sites along the Brazilian coast: Fernando de Noronha (Pernambuco state), Açú da Torre and Salvador (Bahia state), Búzios, Arraial do Cabo and Rio de Janeiro (Rio de Janeiro state), and Ilhabela (São Paulo state). Specimens were fixed in formaldehyde 10% and preserved in 70% ethanol, or put directly in 96% ethanol. Field notes and in situ photographs were taken to record surface and ecological characteristics of the specimens. Identification was made through comparison with the relevant literature (Topsent, 1898; Kirkpatrick, 1903; Dendy, 1905; Burton, 1929; Van Soest & Zea, 1986; Diaz et al., 1993; Gruber, 1993; Lerner, 1996) and with reference specimens, kindly sent on loan by R.W.M. Van Soest from the Zoological Museum of Amsterdam (ZMA), Clare Valentine from the Natural History Museum (BMNH), Claude Lévi from the Muséum National d’Histoire Naturelle, Paris (MNHN), Pedro Alcolado from the Instituto de Oceanologia de Cuba (IOC) 105 and B. Mothes from the Museu de Ciências Naturais da Fundação Zoobotânica do Rio Grande do Sul, Brazil (MCN). Specimens were deposited in the sponge collections of the Universidade Federal do Rio de Janeiro [Instituto de Biologia (UFRJPOR) and Museu Nacional (MNRJ)], and Universidade Federal da Bahia (UFBA). Other abbreviations: RMNH, Rijksmuseum Van Natuurlijke Historie, Leiden. Parsimony analyses were carried out using PAUP 3.1.1 (Swofford, 1993). In all searches, the following options were used: algorithm=branch and bound; keep minimal trees only; addition sequence furthest; collapse zerolength branches; multistate taxa=polymorphisms; optimization =ACCTRAN; rooting by outgroup (=Hymeniacidon spp. and Topsentia spp. – Halichondrida; Desmanthus spp. and Gastrophanella spp. – Lithistida); all characters unordered, with equal weights. Twenty-nine characters were included (Appendix 1). Outgroups were chosen in accordance with earlier classifications, classical (Lévi, 1973; Bergquist, 1978) or phylogenetic (Van Soest et al., 1990; Kelly-Borges & Pomponi, 1994). Three searches were run, using different outgroups: Hymeniacidon spp. (=H. heliophila (Parker, 1910); H. caerulea Pulitzer-Finali, 1986; H. sanguinea Grant, 1826), Topsentia spp. (=T. ophiraphidites (De Laubenfels, 1934); T. bahamensis Diaz et al., 1993; and T. pseudoporrecta Diaz et al., 1993), and Desmanthus spp.+Gastrophanella spp. (=D. meandroides Van Soest & Hajdu, 2000; D. levii Van Soest & Hajdu, 2000; D. incrustans (Topsent, 1889); D. topsenti Hentschel, 1912; D. rhabdophorus (Hentschel, 1912); D. macphersoni Uriz, 1988; Gastrophanella implexa Schmidt, 1879; G. mammilliformis Burton, 1929; G. primore Gómez, 1998; G. stylifera Mothes & Silva, 1999; and G. cavernicola Muricy & Minervino, 2000). All other combinations of two or more outgroups make the ingroup polyphyletic and were not used. Dependence between characters was checked by searching the data matrix for characters with identical distribution of states among taxa (Appendix 2). Bootstrap support for branches in most parsimonious trees (MPTs) or in consensus trees was calculated by 1000 replicated branch and bound searches, with the same options held constant as described above (Felsenstein, 1985; Li & Zharkikh, 1994). For each MPT, we recorded the consistency index (CI), rescaled consistency index (RC) and homoplasy index (HI – Swofford, 1993). Systematic descriptions Subclass Ceractinomorpha Order Halichondrida Diagnosis (after van Soest et al., 1990): “Demospongiae with a plumoreticulate skeletal architecture built of interchangeable styles and oxeas and intermediate spicules, of widely diverging sizes, and not functionally localized”. Family Halichondriidae Vosmaer, 1887 Diagnosis (after Van Soest et al., 1990): “Halichondrida with a choanosomal skeleton consisting of (1) a high density of spicules arranged in (2) vague, ill-defined, directionless tracts, and of (3) spicules in confusion”. Genus Petromica Topsent, 1898 Petromica Topsent, 1898: 226. Type species: Petromica grimaldii Topsent 1898: 226 (by monotypy). Monanthus Kirkpatrick, 1903: 176. Type species: Monanthus plumosus Kirkpatrick, 1903 (by monotypy). (Taxonomic decision for synonymy: Van Soest et al., 1990). Diagnosis (emended from Van Soest et al., 1990): Halichondriidae with highly fused to isolate monocrepid desmas forming a sublithistid skeleton. Definition: Massive, globular or encrusting sponges, often with opened or blind papillae. Colour usually yellow, drab or light brown. Surface smooth, hispid or conulose, sometimes with papillae-like surface projections. Consistency firm but compressible to rigid, friable. A detachable dermal membrane may be present, often with a confused tangential reticulation of oxeas. Choanosomal skeleton with oxeas forming variable ascending tracts and dispersed in confusion. Desmas always present, varying from isolated and concentrated in the basal layer to highly fused and dispersed throughtout the choanosome and ectosome. Oxeas smooth, fusiform, 260–1570 µm long. Desmas monocrepid, 180–970 µm long, varying from simple, smooth and poorly ramified to complex, highly ramified forms with microspined tips. Petromica grimaldii Topsent, 1898 (Figs 1A, 2A–C and 3) Petromica grimaldii Topsent, 1898: 226; 1904: 64; 1928: 106; ?Pulitzer-Finali, 1970: 334; ?BouryEsnault et al., 1994: 57. Specimens examined (2): Lectotype MNHN DT 850, 597 m depth, Prince de Monaco Collection, stn. 106 Figure 1. Habit of Petromica species (preserved specimens). (A) P. grimaldii, lateral view of the lectotype (MNHN DT 850). (B) P. massalis, lateral view of a fragment of the holotype (BMNH 1907.2.1.19). (C) P. plumosa, upper view of a fragment of the holotype (BMNH 1902.11.16.28). (D) P. tubulata, lateral view of the holotype (BMNH 1902.5.26.2) in a deep fissure of Pachastrella isorrhopa. (E) P. digitata, lateral view of the holotype (BMNH RN. 95B), over Pachastrella monilifera and under Craniella sp.). (F) P. ciocalyptoides, lateral view of a Brazilian specimen (UFRJPOR 4173). (G) P. citrina sp. n., lateral view of the holotype (MNRJ 580). Scale bar=1.25 cm (A–C), 3 cm (D, E), 2 cm (F) and 1.5 cm (G). 107 Figure 2. Skeletons of Petromica grimaldii and P. massalis in optical microscopy. (A–C) P. grimaldii: (A) tangential section of the ectosome. (B, C) transverse sections of the ectosome. (D–F) P. massalis: (D) tangential section of the ectosome. (E) transverse section of the ectosome. (F) zygosis between two desmas. S – surface. Scale bars=250 µm (A–E), and 50 µm (F). 587, Azores. Paralectotype: BMNH 1930.7.1.14, stn. 2214, Azores. Diagnosis: Petromica without papillae, with conical shape and conulose surface, with a partly aspiculate, detachable dermal membrane. Oxeas relatively large (470–1450 µm long). Desmas short (380–780 µm long), highly ramified, often with microspined branch tips, partly fused, abundant throughout the choanosome and ectosome. Description (Fig. 1A): Massive, conical sponges, larger at the base and gradually tapering to a thinner top. Papillae absent. Maximum size 4 cm high by 3 cm in diameter. Colour in spirit whitish to beige. Surface conulose. Conules straight, acerate, 0.5–1.0 mm 108 Figure 4. Intra- and interspecific variation in length of oxeas within the genus Petromica (in µm). Specimens: P. ciocalyptoides 1 – UFRJPOR 3168; 2 – UFRJPOR 3169; 3 – UFRJPOR 4173; 4 – UFRJPOR 4377; 5 – UFBA Br95; 6 – ZMAPOR 9408; P. citrina 1 – MNRJ 736; 2 – MNRJ 737; 3 – MNRJ 589; 4 – MNRJ 581; 5 – MCN 3395; P. tubulata 1 – BMNH 1902:5:26:2; P. plumosa 1 – BMNH 1902.5.26.2a; 2 – BMNH 1902.11.16.28; P. digitata 1 – BMNH RN 95B; 2 – BMNH 1904.12.1.87; P. massalis 1 – BMNH 1907.2.1.19; 2 – BMNH 1925.11.1.162; 3 – BMNH 1925.11.1.163; P. grimaldii 1 – BMNH 1930.7.1.14; 2 – MNHN DT 850 (Prince de Monaco Collection, stn. 587). Figure 3. Spicules of Petromica grimaldii. (A) oxeas. (B) desmas. (C) detail of a desma branch. Scale bar=100 µm (A, B) or 40 µm (C). high and 1.5–2.0 mm apart. Inhalant openings, 0.1–0.5 mm in diameter, are dispersed throughout the surface. Large exhalant superficial canals 1–2 mm wide appear at the base of the sponge and lead to the top, where they open in irregular oscules, 1–2 mm in diameter, with membranous margins. Consistency firm but compressible, relatively friable. Skeleton (Fig. 2A–C): Oxeas and desmas in a confused arrangement occupy most of the ectosome, except in some areas where the dermal membrane is aspiculate, translucent, with elliptical inhalant openings. The terminations of choanosomal ascending tracts may protrude slightly above the surface. In the choanosome, multispicular ascending tracts of oxeas, irregular and sinuous, 100–300 µm in diameter and 1000–1800 µm apart, are intermingled with a confused reticulation of relatively few oxeas and abundant desmas. Desmas are present from the base to the upper surface of the sponge, partly fused through zygoses. Spicules (Fig. 3): Oxeas, smooth, fusiform, straight or slightly curved, often with acerate ends, but also with stylote and strongylote modifications: 470– Figure 5. Intra- and interspecific variation in desma total length within the genus Petromica (in µm). Specimens as in Figure 4. 968–1450/7–35 µm. Intraspecific variation in oxea length was high (Fig. 4). Monocrepid desmas, irregular, ranging from simple ‘diactinal’ forms with few, short branches to ‘triactinal’ and ‘tetractinal’ spicules highly branched in several planes. Branches long, average 45 µm thick, smooth, producing several short, thin ramifications (15–18 µm thick). Ramification endings can be recurved, flattened (zygosial extremities) or rounded, blunt, smooth or microspined (free extremities). Total length 360–500–620 µm; epirhabd 30–87–160/25–52 µm; cladii 30–174–270 µm long (n=37). There was low intraspecific variation in desma total length (Fig. 5). Ecology: The species seems to be quite common at the Azores area, 200–914 m depth, and rarer in the Bay of Naples, 60–135 m depth. Distribution: Atlantic (Azores), (?) Mediterranean (Naples, Alboran Sea) (Fig. 6). 109 Figure 6. World distribution of all valid species of Petromica. (A) P. grimaldii; (B) P. massalis; (C) P. plumosa, P. tubulata and P. digitata; (D) P. ciocalyptoides; (E) P. citrina sp. n. Remarks: Petromica grimaldii was well described by Topsent (1898), who however overemphasized the microspined character of the branch-ends of its desmas and the lack of spicules in the dermal membrane. Mediterranean specimens without microspined desma branches have been used to justify the synonymy of P. grimaldii and P. massalis Dendy, 1905 (PulitzerFinali, 1970, Boury-Esnault et al., 1994 – see below). However, not all desmas in P. grimaldii have microspined branch endings, and these may be relatively rare in some specimens. Furthermore, its detachable dermal membrane is not completely aspiculate as suggested by Topsent (1898, 1928), but only partly so (Fig. 2A). Petromica grimaldii can be distinguished from P. massalis and all other species of Petromica by the globular to conical habit without papillae but with sharp conules, the partly aspiculate, detachable dermal membrane, the large distance between the choanosomal ascending spicule tracts and the complex ramification pattern of its desmas. Petromica massalis Dendy, 1905 (Figs 1B, 2D–F and 7) Petromica massalis Dendy, 1905: 104; 1921: 8; Burton, 1928: 110. Specimens examined (3): Holotype: BMNH 1907:2:1:19a (RN 257), Herdman coll., Ceylon (Sri Lanka). Additional material: BMNH 1925.11.1.162, Figure 7. Spicules of Petromica massalis. (A) oxeas. (B) desmas. Scale bar=100 µm. Dendy coll., Sri Lanka; BMNH 1925.11.1.163, Amirante, ‘Sealark’ Expedition. Diagnosis: Petromica with short oscular papillae, reticulated surface, ectosomal skeleton with an unorganized feltwork of oxeas, stony consistency, long oxeas often with irregular outline (640–1570 µm long), and smooth, highly ramified desmas (380– 820 µm long), which are largely fused and spread throughout the choanosome and ectosome. Description (Fig. 1B): Sponge massive, attached by a broad base, sometimes compressed vertically or horizontally. The holotype is 3.7×3.7×2.4 cm (length×width×height), but specimens may attain 110 4×4.5 cm (height×width). Colour in spirit light brown or yellowish gray. Both the “numerous short papillae scattered on the upper surface, each opened at the top by a circular oscule” and the “thin, detachable, reticulate dermal membrane evident in parts of the surface” described by Dendy (1905, 1921) were not observed in the fragments of preserved material studied. Surface uneven, variable, hispid, corrugated or conulose in places. Consistency hard, incompressible, but friable. Skeleton (Fig. 2D–F): The skeleton of the papillae was not observed. The ectosome bears an unspecialized, unorganized feltwork of oxeas, with a few dispersed desmas. Choanosomal skeleton is a confused reticulation of oxeas and desmas. The oxeas may form multispicular, coalescent, ascending tracts, 300–900 µm in diameter, but are often densely strewn in confusion without recognizable tracts. Desmas are abundant in both the choanosome and ectosome and are largely fused, giving the sponge a hard consistency. Spicules (Fig. 7): Oxeas, slightly curved, ends usually acerate, but often with strongyloid and stylote modifications. The axial canal is quite large, and spicule margins are irregular in some specimens: 640–1072–1570/10–25.2–45 µm (n=41). Intraspecific variation in spicule size was moderate (Table 1). Desmas, monocrepid, smooth, highly branched, with smooth, blunt or flattened ends: total length 380–535–820 µm; epirhabd length 50–132–230 µm; epirhabd diameter 20–51–100 µm; cladii length 80– 196–380 µm (n=30). Intraspecific variation in length of oxeas and desmas was moderate (Figs 4 and 5). Ecology: found in deep waters in Sri Lanka (precise depth and bottom type unknown), at 71 m depth at Amirante, and from 82–494 m depth in Andamans. Distribution: Indian Ocean: Gulf of Manaar, Sri Lanka, Amirante, Interview Islands, Andamans (Fig. 6). Remarks: The suggestion of Pulitzer-Finali (1970; see also Boury-Esnault et al., 1994) to synonymyze P. massalis with P. grimaldii based on the absence of microspined endings in Mediterranean populations of P. grimaldii does not hold, because the two species differ also in other characters. Among these, the most obvious are the general body shape (conical in P. grimaldii, broad base with several small papillae in P. massalis), surface (superficial exhalant canals present only in P. grimaldii), position of oscules (at the lateral part of the body in P. grimaldii, at the top of papillae in P. massalis), shape of oxeas, and both general shape and size of desmas. We consider the status of Mediterranean populations of P. grimaldii Topsent uncertain due to insufficient descriptions. Petromica massalis differs from all other Petromica species by its firm consistency, highly fused network of desmas, long oxeas with irregular outline, and complex desma shape. Petromica plumosa (Kirkpatrick, 1903) (Figs 1C, 8A–D and 9) Monanthus plumosus Kirkpatrick, 1903: 176, ‘typical variety’. Specimens examined (2): Holotype: BMNH 1902.5.26.2a, off Cone Point, Natal Coast, South Africa, encrusting Pachastrella isorrhopa Kirkpatrick, 1902. Additional material: BMNH 1902.11.16.28, East London Coast, South Africa, encrusting Placospongia labyrinthica Kirkpatrick, 1903. Diagnosis: Petromica encrusting, without papillae, with smooth surface and hard consistency. Tangential ectosomal skeleton absent. Oxeas short (310–780 µm long) and smooth. Desmas poorly ramified (310–560 µm long), loosely articulated or dispersed throughout the choanosome. Description (Fig. 1C): Sponge thinly to thickly encrusting, up to 2.5 cm in diameter by 6 mm thick, sloping down to a thin rounded margin. Papillae absent. Colour in spirit cream. Surface even, smooth or microhispid, with several, dispersed, rounded oscules 1 mm in diameter flush with the surface. Dermal membrane not easily detachable. Consistency firm but compressible in spirit, friable when dry. Skeleton (Fig. 8A–D): Ectosome without a specialized tangential skeleton. The extremities of choanosomal ascending spicule tracts arrive perpendicularly to the surface, making a pulpy appearance in tangential sections. The choanosome has large, dense, coalescent tracts (up to 1000 µm in diameter) formed of confused bundles of oxeas extending from base to surface. Monocrepid desmas, common to rare, isolated or loosely articulated, are dispersed among the oxeas in a confused arrangement. Desmas are more abundant in the choanosome, but also occur in the ectosome. Spicules (Fig. 9): Oxeas, smooth, slightly curved, with acerate ends, often stylote or strongylote: 310– 500–780/10–25–40 µm (n=31). Desmas monocrepid, with smooth epirhabd, often bifurcating at each end in flattened branches with smooth ends, which are blunt or expanded in flattened zygosial extremities. Total length 310–438–560 µm; epirhabd length 50–118–240 µm; epirhabd diameter 30–46–70 µm; cladii length 80–168–250 µm (n=15). 111 Figure 8. Skeletons of Petromica plumosa and P. tubulata in optical microscopy. (A–D) P. plumosa: (A) tangential section of the ectosome. (B) transverse section of the ectosome. (C, D) transverse sections of the choanosome. (E–F) P. tubulata: (E) tangential section of the ectosome. (F) transverse sections of the ectosome. S – surface. Scale bars=250 µm. Variation in spicule measurements in two specimens was low (Table 2; Figs 4 and 5). Ecology: Found from 62 to 155 m depth, bottom broken shells. Distribution: East London and Natal coasts, South Africa (Fig. 6). Remarks: This species was known so far only from Kirkpatrick’s (1903) short original description. It is closest to P. tubulata (Kirkpatrick, 1903), from which it differs mostly in the absence of papillae. Although this could be an ecophenotypical variation (Kirkpatrick, loc. cit.; Van Soest & Zea, 1986), P. plumosa differs from P. tubulata also in other import- 112 Table 1. Variation in spicule size (min–med–max in µm) in three specimens of Petromica massalis Specimen BMNH 1907.2.1.19 (holotype) BMNH 1925.11.1.162 BMNH 1925.11.1.163 Oxeas length Oxeas diameter Desmas length Epirhabd length Epirhabd diameter Cladii length 780–1090–1300 15–28–45 430–505–650 90–142–170 20–43–60 80–159–230 640–921–1050 10–22.1–30 380–495–620 50–118–200 30–48.5–70 80–209–380 980–1183–1570 18–23.7–30 540–672–820 100–130-230 50–69–100 190–260–370 Table 2. Variation in spicule size (min–med–max in µm) in two specimens of Petromica plumosa Specimen BMNH 1902.5.26.2a (holotype) BMNH 1902.11.16.28 Oxeas length Oxeas diameter Desmas length Epirhabd length Epirhabd diameter Cladii length 310–436–600 15–22–30 350–463–540 100–151–240 40–46–60 80–164–250 430–561–780 10–28–40 310–416–560 50–90–130 30–47–70 110–172–230 Figure 10. Spicules of Petromica tubulata. (A) oxeas. (B) desmas. Scale bar=100 µm. Figure 9. Spicules of Petromica plumosa. (A) oxeas. (B) desmas. Scale bar=100 µm. ant characters: its choanosomal skeleton has larger ascending plumose tracts, the ectosome has no tangential reticulation, and its desmas are rarer (especially in the ectosome) and generally thinner and simpler than those of P. tubulata. We therefore consider it as a valid species. Petromica tubulata (Kirkpatrick, 1903) (Figs 1D, 8E–F and 10) Monanthus plumosus var. tubulatus Kirkpatrick, 1903: 177. Specimens examined (1): Holotype: BMNH 1902:5:26:2, 61 m depth, 7.2 km NW off Cone Point, Natal Coast, South Africa, Gilchrist collection. Diagnosis: Petromica with nodular shape, cylindrical oscular papillae, and hard consistency. Oxeas smooth, 380–960 µm long, arranged in large choanosomal plumose ascending tracts. Ectosome with a confused tangential reticulation of oxeas and desmas. Desmas poorly ramified (360–660 µm long), abundant throughout the base and in the papillae. Description (Fig. 1D): Sponge with a massive, nodular base, translucent cream in spirit, irregular, completely inserted in Pachastrella isorrhopa Kirkpatrick, 1902. It measures 2.5 cm in diameter and height, with two cylindrical papillae, white, smooth, 2 cm high by 2 mm in diameter, with an apical, circular oscule 1 mm in diameter. Surface even, hispid. Consistency of the papillae soft; the base is rigid to friable. Dermal membrane non-detachable. Skeleton (Fig. 8E–F): Papillae walls are composed of two layers, the inner one with plumose spicule 113 Figure 11. Skeletons of Petromica digitata, P. ciocalyptoides and P. citrina sp. n. in optical microscopy. (A–C) P. digitata: (A) tangential section of the ectosome. (B, C) transverse sections of the ectosome. (D–F) P. ciocalyptoides: (D) tangential section of the ectosome. (E) transverse section of a papilla. (F) transverse sections of the ectosome. (G–H) P. citrina sp.n.: (G) tangential section of the ectosome. (H) transverse section of the ectosome. S – surface; ic – inhalant canal. Scale bars=250 µm. 114 Figure 13. Spicules of Petromica ciocalyptoides. (A) oxeas and raphides. (B) desmas. Scale bar=100 µm. Figure 12. Spicules of Petromica digitata. (A) oxeas. (B) desmas. Scale bar=100 µm. tracts up to 250 µm in diameter, arranged more or less spirally upwards, and the outer one with a feltwork of relatively few oxeas and abundant desmas. The ectosome of the base bears a confused tangential reticulation of oxeas and desmas. The choanosomal skeleton is a confused reticulation of few oxeas and abundant desmas with moderate zygosis. The oxeas may form vague, sinuous ascending tracts 100–300 µm in diameter. Desmas occur from the base to the surface and papillae. Spicules (Fig. 10): Oxeas, fusiform, slightly curved, thick, with acerate ends, sometimes with stylote modifications: 380–598–960/5–16.4–27 µm (n=43). Desmas, monocrepid, smooth, with short epirhabd and short, contorted, poorly ramified cladii, with flattened or blunt, smooth ends: total length 360–487– 660 µm; epirhabd length 50–97–180 µm; epirhabd diameter 40–49.7–72 µm; cladii length 140–195–292 µm (n=20). Ecology: Found in broken shells bottoms, 99 m depth. Distribution: Natal coast, South Africa (Fig. 6). Remarks: This species was originally described as a variety of Monanthus plumosus, from which it differs by a massive, nodular shape, the presence of papillae, a hard, friable consistency, thinner choanosomal ascending tracts, thinner and less contorted desmas, which are relatively rare in the ectosome, and the presence of a tangential ectosomal reticulation only in P. tubulata. These differences are here considered distinctive at the species level, and the variety is raised to specific status. Petromica digitata (Burton, 1929) (Figs 1E, 11A–C and 12) Monanthus plumosus var. digitata Burton, 1929: 9. Specimens examined (2): Holotype: BMNH RN. 95B, 97 m depth, 15.3 km N of False Is., South Africa. Additional material: BMNH 1904.12.1.87 (no. 97), 101 m depth, 14.4 km NW of O’Neil Peak, South Africa. Diagnosis: Petromica with short digitiform papillae, smooth surface and firm but compressible consistency. Dermal membrane detachable with dispersed oxeas and relatively few desmas. Oxeas with high size variation, 260–1270 µm long. Desmas large, poorly ramified (440–970 µm long), abundant in the choanosome. Description (Fig. 1E): Subspherical to thinly encrusting sponge, 5×3 cm wide by 5–10 mm thick, with 1–4 apical, hollow, digitiform papillae about 11–30 mm high by 3–7 mm in diameter, which may open at the top in circular oscules, 1–2 mm in diameter. Colour cream to drab in spirit. Surface even, smooth to microhispid, with a detachable dermal membrane covering superficial canals in a vein-like pattern. Consistency firm but compressible, friable when dried. Skeleton (Fig. 11A–C): Ectosome unspecialized. The detachable dermal membrane contains abundant, dispersed oxeas, the extremities of the choanosomal ascending tracts and relatively few desmas. The papillae have an inner layer of plumose bundles of oxeas arranged spirally upwards, and an outer tangential layer of oxeas in confusion. The choanosome is made up of loosely articulated or partly fused desmas and a confused reticulation of oxeas. Oxeas also form irregular, ill-defined ascending tracts, 150–300 µm in diameter. Desmas are abundant from the base to the surface of the sponge. 115 Table 3. Variation in spicule size (min–med–max in µm) in two specimens of Petromica digitata Specimen BMNH RN. 95B (holotype) BMNH 1904.12.1.87 Oxeas length Oxeas diameter Desmas length Epirhabd length Epirhabd diameter Cladii length 260–715–1270 8–32–55 440–631–970 80–148–240 40–52–80 110–234–370 560–700–910 20–26–42 510–663–750 90–173–250 80–83–90 180–243–300 Spicules (Fig. 12): Oxeas, smooth, fusiform, slightly curved at the middle, usually with acerate endings but occasionally stylote or strongylote: 260– 710–1270/8–30.3–55 µm (n=42). Desmas monocrepid, with smooth epirhabd, which usually bifurcates at each end and often bears several lateral branches. Articular faces of branches are flattened or concave, smooth: total length 440–642– 970 µm; epirhabd length 80–157–250 µm; epirhabd diameter 40–63.5–90 µm, cladii length 110–237–370 µm (n=35). Size variation between spicules of two specimens was moderate (Table 3). Ecology: Found from 97–101 m depth, in bottoms of sand and shells or brackish shingle. The holotype encrusts a specimen of Pachastrella monilifera Schmidt, 1868. Distribution: South Africa (Fig. 6). Remarks: This species has been originally described as a variety of Monanthus plumosa Kirkpatrick, 1903. It was known only by the short description of Burton (1929). It differs from P. plumosa in the presence of papillae and a tangential ectosomal skeleton, from P. tubulata in papillae shape, and to both species in the larger size of its spicules and general shape of its desmas. It differs from all other species of Petromica in the shape and size of its desmas. Petromica ciocalyptoides (Van Soest & Zea, 1986) (Figs 1F, 11D–F and 13) Monanthus ciocalyptoides Van Soest & Zea, 1986: 202. Petromica ciocalyptoides; Van Soest et al., 1990:41; Diaz et al., 1991: 143; Muricy et al., 1991: 1187; Diaz et al., 1993: 288; Gruber, 1993: 47; Muricy & Moraes, 1998: 215. Holotype: RMNH Por. 1309, Saba Bank Expedition station 136 (17◦ 23′ N–63◦ 33′ W). Paratypes: ZMAPOR 5837, 5838, Nenguangue Bay, Santa Marta, Colombia (11◦ 20′ N–74◦ 09′ W). Specimens Examined (9): Brazil: Rio de Janeiro State: UFRJPOR 3168, 3169, 5 m depth, coll. E. Hajdu, 22.II.1990, Prainha, Arraial do Cabo, 22◦ 57.3′ S–42◦ 01.5′ W. Bahia State: UFRJPOR 4173, 2.5 m depth, 12.X.1987; UFRJPOR 4377, 2.5 m depth, 03.X.1992, coll. E. Hajdu, Ponta de Humaitá, Salvador, 12◦ 55.6′ S–38◦ 31.1′ W; UFBA-Br 95, 2 m depth, 03.28.1986, UFBA-Br 99, 3 m depth, 23.VII.1988, coll. A. V. Madeira, Farol da Barra, Salvador, 13◦ 00.6′ S–38◦ 32.0′ W. UFBA-LN-3, 21 m depth, 03.XII.1992, coll. W. Andrade, Açú da Torre, 12◦ 44′ S–38◦ 07′ W. Pernambuco State: UFRJPOR 3910, 12 m depth, 02/03/1996, coll. G. Muricy, Ponta da Sapata, Fernando de Noronha, 03◦ 51′ S–33◦ 10′ W. Caribbean: ZMAPOR 9408 (topotypical specimen), 34 m depth, Saba Bank Expedition station 136, 17◦ 23′ N–63◦ 33′ W. Diagnosis: Petromica with long, smooth, blind papillae. Dermal membrane with a tangential, confused reticulation of oxeas. Oxeas short (300–670 µm long) and smooth. Desmas poorly ramified, 384–780 µm long, not fused and concentrated at the base of the sponge. Description (Fig. 1F): Basal mass encrusting, with irregular outlining, buried in sand and agglutinating fragments of shells, corals, and sediment. Basal mass ranges from 9×8–48×25 mm wide by 4–33 mm thick. One to several erect papillae project from the basal mass, gradually tapering from 2 to 15 mm in diameter at the base to 1 to 6 mm at the closed apex; they range from 0.4 to 17.0 cm high. Papillae walls vary from 0.5 to 3.0 mm thick, and the internal canal is 2 to 6 mm in diameter. Papillae often anastomose, with fusion occurring either along all the side or only in parts of the papillae. Colour yellow, pale yellow or whitish alive, becoming white to drab in spirit. Papillae are translucent in spirit, with longitudinal spicule tracts visible in the walls. Surface even, slightly hispid, with a distinct but hardly detachable dermal membrane, both in the papillae and basal mass. Superficial canals in a vein-like pattern may be present. Oscules flush, circular, 0.5–1.0 mm in diameter, dispersed in the surface. Consistency soft, inelastic, relatively friable. Some portions of the basal mass are more rigid due to the incorporation of foreign mineral inclusions. Skeleton (Fig. 11D–F): Papillae walls are sustained by ascending, sinuous, irregularly disposed, longitudinal spicule tracts (50–150 µm thick, 5–20 spicules 116 in a row). A confused, halichondroid reticulation of oxeote spicules extends from the external surface to the borders of the exhalant canal, where most spicules are arranged tangentially. Papillae skeleton is composed of oxeas with stylote and strongylote modifications, and eventual raphides in some specimens. Dermal membrane in both the basal mass and papillae bears a tangential, confused reticulation of oxeote spicules, which may form vague, sinuous spicule tracts. Choanosomal skeleton is a dense, confused reticulation of oxeote spicules, with little spongin. In larger specimens, sinuous ascending spicule tracts are present at the upper part of the choanosome, and may terminate in a plumose fashion at the surface of the sponge. Spicule tracts are absent in the deeper portions of the choanosome of the basal mass, where non-fused desmas are scattered within the dense reticulation of oxeote spicules. Desmas are most abundant close to the basal surface of the sponge, and rare or absent at the upper surface and papillae. Spicules (Fig. 13): Oxeas smooth, fusiform, straight or slightly curved, relatively thin, with acerate ends. Styloid and strongyloid modifications are common: 300–461–670/1.6–8.5–21.2 µm (n=180). Intraspecific variation in spicule size is small (Table 4). Raphides, thin and straight, rare or absent in most specimens: 161–286–400 µm long (n=45). Monocrepid desmas, often with a short, straight, smooth epirhabd and relatively long, scarcely ramified, contorted cladii. Extremities of cladii rounded, smooth or irregular. Developmental stages are also present, recognizable by their smooth, reduced cladii, with acerate endings. Abundance of desmas varied widely between different individuals. Total length 384–611–780 µm; epirhabd 80–210–370/21.2– 39.5–63.6 µm; cladii 70–355–726/16.3–32.4–97.8 µm (n=30). Ecology: Petromica ciocalyptoides is usually found in shallow, sheltered bays or in deeper areas, protected from direct wave impact. It inhabits horizontal or sub-horizontal rocky or sand and shells substrates, often with the base buried in sand and only the papillae exposed. Depth of collection ranged from 2 to 34 m. The surface is usually clean from epibionts, except for a few hydroids, and commensals are rare inside canals and papillae. Distribution (Figs 6 and 14): Tropical Western Atlantic: Caribbean (Saba Bank, Colombia, Venezuela), Figure 14. Distribution of Petromica ciocalyptoides ( ) and P. citrina sp. n. ( ), with location of collection sites along the Brazilian coast (numbered): 1, Ilha da Galé (SC); 2, São Sebastião (SP); 3, Rio de Janeiro (RJ); 4, Arraial do Cabo and Búzios (RJ); 5, Salvador and Açú da Torre (BA); 6, Fernando de Noronha (PE). and Brazil: Fernando de Noronha (PE), Salvador and Açú da Torre (BA), Arraial do Cabo and Búzios (RJ). Remarks: Petromica ciocalyptoides shows intraspecific variation in some morphological characters, particularly colour (white to yellow) and presence of raphides in some specimens. Morphological characters of Brazilian populations fall relatively well within the range of variation found in Caribbean specimens (van Soest & Zea, 1986; Diaz et al., 1993), and intraspecific variation in spicule measurements was very low (Table 4; Figs 4 and 5). We found in both Brazilian and Caribbean specimens a relatively well distinct category of thinner oxeas and raphides, which were considered as growth stages by Van Soest & Zea (1986). Subdermal canals in vein-like patterns were observed on the surface of Caribbean specimens, but not on Brazilian ones. Monaxons in Caribbean specimens are most frequently strongylote, although with a great deal of variation reported, whereas acerate oxeote spicules predominate in Brazilian specimens. Such subtle morphological differences could suggest that Brazilian and Caribbean populations of P. ciocalyptoides represent distinct, sibling species. This question would be better approached by cytological 117 Table 4. Intraspecific variation in spicule measurements of Petromica ciocalyptoides (min–med–max in µm) Specimens UFRJPOR 3168 UFRJPOR 3169 UFRJPOR 4173 UFRJPOR 4377 UFBA Br95 ZMAPOR 9408 Oxeas length Oxeas width Raphides length Desmas length Epirhabd length Epirhabd diameter Cladii length 300–475–670 1–7.5–18 250–306–360 380–494–570 80–132–210 13–19.2–30 120–198–330 320–468–670 2–9.1–21 250–334–400 490–595–720 80–143–200 20–28.7–35 180–279–400 300–471–630 1–9.7–20 160–216–276 430–617–800 130–210–370 21–39.2–98 70–226–450 370–527–660 4–9.9–19 240–278–320 430–560–700 50–158–240 18–35.5–80 150–234–310 392–488–614 8–11–21 221–271–307 384–510–726 80–149–213 26–34–51 130–245–375 350–431–550 3–8.8–17 210–311–380 450–553–710 120–153–220 18–24.9–30 90–260–410 or molecular methods (e.g. allozyme electrophoresis, DNA sequencing), which are outside the scope of this paper. We presently consider Brazilian and Caribbean populations of P. ciocalyptoides as conspecific on a strictly morphological basis. Petromica ciocalyptoides shows superficial similarities in habit (long, slightly conical, smooth, blind papillae) to Ciocalypta penicillus Bowerbank, 1862 and to Coelocalypta porrecta Topsent, 1928. However, Ciocalypta Bowerbank, 1862 have a distinct ectosomal tangential reticulation of spicule tracts, occurring as bundles or single spicules, and ectosomal styles together with predominantly stylote choanosomal megascleres (Van Soest et al. 1990). Coelocalypta Topsent, 1928 is a junior synonym of Topsentia Berg, 1899, which has an ectosomal crust of compact, smaller ectosomal oxeas lying paratangentially, and oxeas of a wide size range, usually in 2–3 size classes, often twisted, bent or doubly-bent (Van Soest et al., 1990; Diaz et al., 1993). Petromica ciocalyptoides shares with P. grimaldii the presence of thin superficial canals, a relatively friable consistency, multispicular tracts of oxeas in the upper choanosome, dispersed oxeas in the choanosome and ectosome, and the size range of desmas. They differ in P. ciocalyptoides having long papillae, smooth surface, a completely spiculate, non-detachable dermal membrane, desmas concentrated in the basal layer, and a simpler ramification pattern of desmas. Petromica ciocalyptoides differs from other species of Petromica by its relatively long, smooth papillae closed at the top, the thin oxeas, and the presence of raphides in some specimens. Petromica citrina sp. n. (Figs 1G, 11G–H and 15) Figure 15. Spicules of Petromica citrina sp. n. (A) oxeas. (B) desmas (scale bar=100 µm). Halichondria lutea; Lerner, 1996: 109 (non: Halichondria lutea Alcolado, 1984: 10; Diaz et al., 1993: 294). Petromica sp. n.; Hajdu et al., 1999: 23. Specimens Examined (9, all from the Brazilian coast): Holotype: MNRJ 580, 25 m depth, 18.VI.1997, coll. M. LeBlanc, Ponta do Frade, Ilha de São Sebastião, São Paulo State, 23◦ 54.9′ S–45◦ 27.5′ W. Paratypes: MNRJ 581, 3 m depth, 18.VII.1997, coll. E. Hajdu & R. Albano, Praia Vermelha, 22◦ 57.2′ S–43◦ 09.8′ W; MNRJ 1710–1713, 15–20 m depth, 12.V.1998, coll. P. S. Young and P. Paiva, Laje de Santo Antônio, off Ipanema, 22◦ 59.4′ S–43◦ 11.8′ W, Rio de Janeiro, Rio de Janeiro State. MNRJ 736, 737, 15 m depth, 09.I.1996, coll. E. Hajdu, Ponta Grossa, Ilha de São Sebastião, Ilhabela, São Paulo State, 23◦ 46.5′ S–45◦ 13.8′ W. MCN 3395, 14 m depth, 23.III.1997, coll. C. Lerner, Ilha da Galé, Bombinhas, Santa Catarina State, 27◦ 10.6′ S–48◦ 24.3′ W. Material studied for comparison: Halichondria lutea Alcolado, 1984, IOC 475 (holotype), Playa de la Concha, Habana, Cuba, 50 m depth, coll. J.M. Corpas. 118 Diagnosis: Petromica with short, conulose, conical, truncated papillae, often fused together in groups. Ectosomal skeleton with irregular tangential dermal reticulation of oxeas dispersed and forming vague, sinuous tracts. Oxeas short (320–780 µm long). Desmas short, smooth, poorly ramified (180–620 µm long), not fused, and concentrated at the base of the sponge. Description (Fig. 1G): Thickly encrusting to massive, irregular sponge, with a relatively thick base from which rise small cone-shaped or digitiform surface projections and larger papillae. Colour alive is bright orange-yellow, slightly darker in the choanosome. In spirit, it becomes whitish, drab or pale yellow. Base measures 3–9×1.6–6 cm wide and is 4–20 mm thick. The base typically encrusts hard substrate and agglomerates loose sediment, calcareous shells, etc.; it is often covered by sand, leaving only the papillae and projections exposed. Base outline irregular, roughly circular, with rounded margins well marked by a short region with smoother surface, 2–5 mm wide. Papillae vary from 1 to 9 per individual, and sometimes appear to be produced by several coalescent surface projections. They are slightly compressed laterally, truncated at the top, and may either be isolated, dispersed over the sponge body, or be partly fused and aligned forming an irregular ridge along the longest axis of the sponge. Size of papillae ranges from 4 to 25 mm high by 4 to 12 mm in diameter. They may be reduced in smaller specimens (e.g. MCN 3395). Surface projections range from small ‘bumps’ or cones 0.5 mm wide and high to thin digitiform processes 10 mm high and 2 mm in diameter. They are closely spaced (0.5– 5 mm apart) and abundant in all the surface of the sponge except at the smoother lateral margins of the base. Surface uneven and hispid, particularly at the top of surface projections. A thin, easily detachable dermal membrane covers the ectosome. Between projections the surface usually shows smoother, irregular 2–5 mm wide translucent areas with a high concentration of inhalant ostia, 25–50 µm in diameter. Oscules are round or irregular, membranous, flush with the surface, 1–5 mm in diameter, scattered in the top of papillae and surface projections. Consistency of the base is firm, hardly compressible. Papillae and surface projections are softer, compressible but inelastic. Skeleton (Fig. 11G–H): The ectosome is 50–200 µm thick. Ectosomal skeleton is composed of an irregular, tangential dermal reticulation of free oxeote spicules in a confused (‘halichondroid’) arrangement and vague, sinuous tracts of oxeas (3–20 spicules in cross section, 70–160 µm in diameter). In the upper parts of the choanosome and papillae, the skeleton is composed of sinuous ascending tracts of oxeote spicules, with low amount of spongin (3–40 spicules in cross section, 80–400 µm thick), forming a confused reticulation with abundant free spicules. The extremities of the choanosomal primary tracts sustain the surface in a plumose or plumoreticulate fashion, protruding only at the top of surface projections; free spicules protrude at the dermal surface in variable angles. The basal portion of the choanosome has a dense, halichondroid reticulation of single oxeote spicules and non-fused desmas hold together by a relatively high amount of spongin. Desmas are concentrated at the most basal layer of the sponge, and are rare in the upper parts of the body. Spicules (Fig. 15): Oxeas, smooth, fusiform, straight, slightly curved, or bent in the middle portion. Both extremities are usually acerate, but occasional stylote and strongylote modifications may occur: 320– 527–780/3.2–26 µm (n=97). Desmas, monocrepid, irregular, with a short, thin epirhabd in which the crepidial axis is often visible, and short, thin, scarcely ramified, contorted, smooth cladii. Cladii endings blunt, smooth, irregular. Total length 180–337–620 µm; epirhabd 40–87.4–190/9.8– 32.6 µm; cladii 50–126–300 µm long (n=36). Intraspecific variation in oxea and desma length was small (Table 5; Figs 4 and 5). Ecology: Petromica citrina is found in shallowwater rocky shores (3–25 m depth), exposed to direct sunlight, encrusting hard substrate. The base of the sponge is often partly buried in sand and conglomerating calcareous shells and bits of sediment. It is rather uncommon in Southwestern Brazil, with relatively dense populations in restricted areas. Distribution (Figs 6 and 14): This species appears restricted to Paulista biogeographic province in the Southeastern Brazilian coast (Coelho & Santos, 1980), from Rio de Janeiro (22◦ 53′ S–43◦ 17′ W) to Ilha da Galé, Santa Catarina State (27◦ 10′ S–48◦ 24′ W). Etymology: The latin name citrina refers to the typical bright yellow–orange colour of living specimens. Remarks: Lerner (1996) reported Halichondria lutea Alcolado, 1984 from Southern Brazil, a species which is similar to P. citrina in every respect except for its alleged absence of desmas. Examination of one of her specimens (MCN 3395) demonstrated the presence of basal monocrepid desmas formerly overlooked, indicating the conspecificity of Rio de Janeiro, São Paulo and Santa Catarina populations of P. citrina. 119 Table 5. Intraspecific variation in spicule measurements of Petromica citrina sp. n. (min–med–max in µm) Specimen MNRJ 580 (holotype) MNRJ 581 (paratype) MNRJ 736 (paratype) MNRJ 737 (paratype) MCN 3395 (paratype) Oxeas length Oxeas width Desmas length Epirhabd length Epirhabd diameter Cladii length 420–572–750 5.7–14.2–26.1 190–372–500 40–94–190 19–25–33 70–141–300 380–512–770 3.2–7.6–14.7 240–378–620 60–88–160 10–16–23 70–137–260 460–583–780 5–14.3–23 180–318–420 40–98–160 16–24–42 60–124–200 320–547–760 3.2–14.6–25 220–325–380 40–79–130 16–23–33 50–109–180 320–517–715 3.5–12.4–26 190–344–500 50–70–90 16–21–28 70–131–200 Caribbean populations of Halichondria lutea (Alcolado, 1984; Diaz et al., 1993; and examination of the holotype IOC 475) show the same general shape, colour, surface and skeletal characteristics of P. citrina, but they differ by the complete absence of desmas in H. lutea, its softer consistency, and oxea shape. Oxeas of P. citrina are fusiform, gradually tapering to more or less acerate points, and do not have telescopic ends like those of H. lutea. The new species clearly belongs to the genus Petromica as shown by the halichondroid organization of oxeote spicules together with non-fused, monocrepid desmas. Petromica citrina sp. n. shares with P. grimaldii Topsent, 1898 a firm but compressible consistency and a conulose surface. They differ however in that P. citrina has erect papillae, a bright yellow colour, a tangential ectosomal skeleton with a feltwork of oxeas, absence of superficial canals, smaller and more simple desmas with smooth ends, and smaller oxeas. Petromica plumosa (Kirkpatrick, 1903) differs from the new species by its white colour, absence of papillae, and thicker desmas. Petromica tubulata (Kirkpatrick, 1903) can be distinguished by the shape of the body (nodulose) and papillae (tubular), and by the shape and size of desmas. Petromica digitata (Burton, 1929) is similar in habit but has longer oxeas and larger, thicker, and more complex desmas than P. citrina. Petromica massalis Dendy, 1905 is similar to the new species in that both species bear a distinct, detachable dermal membrane. However, in P. massalis the surface has a network of anastomosed fibrils, which converge to the apices of small papillae or conuli (cf. Dendy, 1921 – not observed in the fragments examined here). Such fibrils are absent in P. citrina. Oxeas and desmas of P. massalis are larger, and its desmas are much more complex and ramified, than are those of P. citrina. Petromica ciocalyptoides (Van Soest & Zea, 1986) dif- Figure 16. Most parsimonious phylogenetic tree of the genus Petromica, using the lithistids Desmanthus spp. and Gastrophanella spp. as outgroups (88 steps, CI=0.78, HI=0.60, RC=0.40). Encircled numbers are synapomorphies and numbers within rectangles are homoplasies. Numbers outside circles and rectangles represent the bootstrap support for the branch. For character and states names, see Appendix 1. fers from P. citrina by an even, smooth surface, long cylindrical papillae, and larger desmas. Phylogeny Parsimony analysis using all four outgroups together, or in any combination of two or more except Desmanthus spp.+Gastrophanella spp., resulted in a polyphyletic ingroup. This indicates a high amount of homoplasy in the database, reflected in HI values between 0.55 and 0.60 and low boostrap values in all MPTs (between 1.3 and 78%; Figs 16–19), as well as in the great number of polymorphisms both within the ingroup and the outgroups (Appendix 2). Although the use of different outgroups induced different polarizations of many characters, it did not change significantly the topology of the MPTs except for the 120 Figure 17. Most parsimonious phylogenetic tree of the genus Petromica, using the halichondriid Hymeniacidon spp. as outgroup (67 steps, CI=0.79, HI=0.55, RC=0.43). Key as in Figure 16. Figure 19. The other most parsimonious phylogenetic tree the genus Petromica, using the halichondriid Topsentia spp. outgroup, in which P. ciocalyptoides and P. citrina appear non-monophyletic (70 steps, CI=0.77, HI=0.56, RC=0.37). Key in Figure 16. of as as as Figure 18. One of two most parsimonious phylogenetic trees of the genus Petromica, using the halichondriid Topsentia spp. as outgroup, in which P. ciocalyptoides and P. citrina appear as a monophyletic group (70 steps, CI=0.77, HI=0.56, RC=0.37). This tree was identical to the bootstrap majority-rule tree. Key as in Figure 16. separation of a clade (P. ciocalyptoides, P. citrina) in two of them (Figs 16–18). A main clade (P. grimaldii, P. massalis) (P. digitata) (P. plumosa) (P. tubulata) is present in all MPTs. Using the lithistids Desmanthus spp.+Gastrophanella spp. as outgroups resulted in a single MPT 88 steps long, with CI=0.78, RC=0.40, and HI=0.60. In this tree (Fig. 16), the clade (P. ciocalyptoides, P. citrina) appears monophyletic with 59% of boostrap support, and defined by the synapomorphies “sinuous ascending tracts in papillae” and “desmas restricted to the base of sponge” (chracter-states 16.1 and 28.1, respectively). This clade is absent in the single MPT resulting from the use of Hymeniacidon spp. as the outgroup, which was 67 steps long and had the low- Figure 20. Areagrams resulting from replacement of terminal taxa in Figures 16–19 by their areas of occurrence. (A) from Figures 16 and 18; (B) from Figure 17; (C) from Figure 19. est homoplasy levels (CI=0.79, RC=0.43, HI=0.552; Fig. 17). The use of Topsentia spp. as the outgroup resulted in two MPTs 70 steps long (CI=0.77, RC=0.37, HI=0.557; Figs 18 and 19). One of these had the same topology of that using Desmanthus spp.+Gastrophanella spp. as outgroups, with a distinct clade (P. ciocalyptoides, P. citrina) (Fig. 18), and the other was identical to that with Hymeniacidon spp., except for a change in the relative position of P. ciocalyptoides and P. citrina as the most basal ingroup taxon (Fig. 19). 121 The strict consensus of all these trees (not shown) includes the well resolved main clade (P. grimaldii, P. massalis) (P. plumosa) (P. digitata) (P. tubulata), and a basal trichotomy involving the relative position of P. ciocalyptoides and P. citrina. Petromica grimaldii and P. massalis appear as sister-species, a clade supported by 1.3–42% of boostrap and by character-states 9.2 (beige colour in spirit), 10.2 (conulose surface) and 25.1 (high degree of desma ramification). A conulose surface appears homoplastic (shared also with P. citrina) in all reconstructions, and high ramification degree of desmas appears plesiomorphic in the reconstruction using the lithistids as outgroups (since most species of Desmanthus and Gastrophanella have highly ramified desmas). The positions of P. digitata, P. plumosa and P. tubulata as progressively more distant sister-group of this clade are supported mainly by homoplastic traits, except in some reconstructions for synapomorphic characters 6.0 (open papillae), 16.0 (papillae skeleton with spiral plumose tracts), 22.1 (monaxon length up to more than 1000 µm), 24.1 (desmas length up to more than 500 µm), 27.1 (moderate degree of zygosis) and 28.0 (desmas distributed in whole choanosome and ectosome). Discussion Status of Petromica Most or all sponge taxonomists today would agree that Petromica is a valid genus, and the only doubt on its status refers to the validity of its synonymy with Monanthus, proposed by Van Soest et al. (1990) but not followed by Kelly-Borges & Pomponi (1994). The original diagnosis of Monanthus (“Desmanthidae in which the skeleton is formed of monocrepid desmas of the common type, separate or joined together, and of monaxon megascleres”; Kirkpatrick, 1903) does not exclude the original definition of Petromica (“massive Azoricidae, cone-shaped, conulose, with dispersed pores and membranous oscules, with well-developed aspiculate ectosome, and with slightly fused, lightly ornamented desmas”; Topsent, 1898). In this definition, Topsent (loc. cit.) overemphasized external morphological characters, while virtually ignoring skeletal traits such as the confused reticulation of oxeotes and the choanosomal ascending spicule tracts. Kirkpatrick (1903) did the opposite when defining Monanthus, concentrating on spicule composition and making no comments on skeletal organization or external characteristics. The type species of both genera (Petromica grimaldii and Monanthus plumosus) share the absence of papillae, the choanosomal skeleton composed of oxeote spicules in a confused arrangement with ascending spicule tracts and partly fused monocrepid desmas, and the absence of a specialized ectosomal skeleton. However, they differ in several other characters: specimens of M. plumosus have an even surface and a hard consistency, whereas P. grimaldii specimens show a conulose surface with thin superficial canals, and a compressible consistency. The desmas of M. plumosus are smooth, simple, poorly ramified, with flattened branch tips (Fig. 9B). In contrast, desmas of P. grimaldii are highly ramified in complex patterns, and the ends of branches are rounded and often finely tuberculate (Fig. 3B,C). Furthermore, P. grimaldii has thin, widely spaced ascending tracts and abundant, moderately fused desmas throughout the choanosome, whereas M. plumosus has dense, coalescent tracts of oxeas and relatively few desmas, sparsely fused. It could be argued that these characters would mark a generic distinction between the two species, however they are not well correlated in the other species described either as Petromica or as Monanthus, and considered together they allow no clear groupings of the seven species of the genus (c.f. phylogenetic analysis). Due to its uniqueness, the combination of an halichondroid skeleton of oxeote spicules and partly or non-fused monocrepid desmas characterizes quite well this assemblage within the Halichondriidae. The differences discussed above seem important only at the specific, and not at the generic level. We therefore consider the synonymy of Petromica and Monanthus as valid, with priority to the former. So defined, the genus Petromica is heterogeneous in several characters, of which at least one has been considered homoplastic (Van Soest et al., 1990): presence of papillae (supposed to be independently derived in some species of Ciocalypta Bowerbank, 1862, Topsentia Berg, 1899, Halichondria Fleming, 1828, Hymeniacidon Bowerbank, 1864, Collocalypta Dendy, 1905, and Petromica). Petromica includes encrusting, massive and fistulose species, with smooth or conulose surface, with or without a detachable dermal membrane. A tangential ectosomal skeleton is absent in some species, and desma shape, degree of fusion and distribution within the body vary widely among species. The only character firmly uniting the group is the presence of monocrepid desmas associated with oxeas and derivatives in an halichondroid organization. The sublithistid skeleton of Petromica is 122 usually considered either as plesiomorphic or homoplastic (Burton, 1929; Van Soest & Zea, 1986; Van Soest et al., 1990, Pomponi et al., 1991; Diaz et al., 1991, 1993; Gruber, 1993), and ideally it should not be used as a diagnostic feature at the generic level. If desmas are plesiomorphic, then Petromica would be considered paraphyletic. On the other hand, if desma production is a convergence (non-homologous) rather than a retained ancestral character, than it could be used as a synapomorphy for Petromica at this level of universality. The polarization of the production of desmas is still not completely solved, waiting for comprehensive phylogenetic or molecular analyses. However, the combination of monocrepid desmas with halichondrid traits such as vaguely plumose spicule tracts and oxeas in confusion is unique, and therefore we use it to characterize the genus Petromica within the Halichondriidae in the sake of stability of the classification. Scope of Petromica Overall, our results show that the biodiversity of Petromica has been underestimated. Diaz et al. (1991, 1993), considered only three valid species in the genus: P. grimaldii, P. plumosa and P. ciocalyptoides. The proposed synonymy of P. grimaldii and P. massalis was based on a single character (spiny vs. smooth tips of branches – Pulitzer-Finali, 1970; BouryEsnault et al., 1994), and did not take into account several other differences between the two species (body shape, surface, oscules, spicule shape and size of desmas). Similarly, the synonymy of the three varieties of Monanthus plumosus (vars. plumosa, tubulata and digitata) was based on the supposed ecophenotypical nature of the presence/absence of papillae (Kirkpatrick, 1903; Van Soest & Zea, 1986), but also disregarded other differences among the three forms (width of choanosomal ascending tracts, tangential ectosomal reticulation and desma shape, abundance and distribution). In this study, it was clear that similarity and differences among these forms were of the same magnitude of those among the other, currently considered valid species of the genus: P. citrina sp. n., P. grimaldii, P. plumosa and P. ciocalyptoides. Accordingly, P. massalis is reinstated as a valid species, and all three varieties of Monanthus plumosus are raised to specific rank. We therefore consider the genus to contain seven valid species: P. grimaldii (type species), P. massalis, P. plumosa, P. tubulata, P. digitata, P. ciocalyptoides and P. citrina sp. n. It has been recently demonstrated that production of desmas in the Mediterranean sublithistid sponge Crambe crambe (Poecilosclerida) is dependent on silicon concentration in the water (Maldonado et al., 1999). If the same is true to Petromica, then its present scope may simply reflect the silicon concentration of the environment, and the ability of different species to produce desmas in low silicon concentrations. As silicon concentration is higher in deep than in shallow waters (Nelson et al., 1995), it would be expected that deep water species have more developed desma skeletons than the shallow water species. An analysis of the bathymetrical distribution of Petromica species seems to support this, as the two shallow water species P. citrina and P. ciocalyptoides differ from the other five, deep-water species in the absence of zygozis (desma fusion), in the lower abundance of desmas, which are concentrated in the base of the sponge (Table 6), and in the simpler desma shape. It is still unclear if and how changes in silicon concentration could affect the scope of Petromica, since it has not been shown so far that desmas would be produced by other sponges with halichondroid skeleton in higher silicon concentrations. Phylogeny of Petromica Phylogenetic analysis of the species of Petromica with different outgroups (Hymeniacidon, Topsentia: Halichondriidae, and Desmanthus-Gastrophanella: Lithistida) allows two basic interpretations: in the first one the genus can be divided in two monophyletic clades (Figs 16 and 18); in the second, all species form a single monophyletic group, and Petromica cannot be divided in monophyletic, non-monotypical subgenera (Figs 17 and 19). The two possible clades within Petromica were (P. grimaldii) (P. massalis) (P. digitata) (P. plumosa) (P. tubulata) and (P. ciocalyptoides, P. citrina). The first clade is defined in different reconstructions mainly by the synapomorphic states 16.0 (spiral plumose tracts in papillae – an underlying synapomorphy), 27.1 (moderate degree of zygosis), and 28.0 (desmas distributed throughout the choanosome and ectosome), and by homoplasies 3.2 (body size smaller than 5 cm), 4.0 (papillae absent – another underlying synapomorphy), and 15.0 (consistency firm but compressible). Characters 27.1 and 28.0 appear homoplastic in two MPTs (Figs 16 and 17). The clade (P. ciocalyptoides, P. citrina) is supported by sinuous tracts in papillae skeleton, desmas concentrated in the base of the sponge (synapomorphic), more than five 123 Table 6. Degree of desma fusion, distribution of desmas within the sponge body, and bathymetrical distribution of Petromica species Species Degree of fusion Desma distribution Depth range (m) P. grimaldii P. massalis P. plumosa P. tubulata P. digitata P. ciocalyptoides P. citrina moderate high moderate moderate moderate absent absent base to surface base to surface base to surface base to surface base to surface base only base only 60–914 71–494 62–155 99 97–101 2–34 3–25 papillae per individual, and soft consistency (homoplastic). This clade appears in two of the four MPTs, with low bootstrap support (35–59%), and one of the major characters defining it (namely, desmas concentrated in the base of the sponge) might simply reflect a low concentration of silicon in shallow waters (Maldonado et al., 1999). The alternative arrangement, with a single monophyletic clade, is supported by synapomorphies 5.0 (cylindrical-digitiform papillae) and 7.0 (papillae smaller than 5 cm), and homoplasy 13.0 (oscular rim absent). Therefore, although we accept that a clade (P. ciocalyptoides, P. citrina) might be truly monophlyletic, it is unclever to propose a new subgenus for it in the absence of stronger support from other characters (secondary metabolites, DNA, etc.). In none of the MPTs two clades were formed separating species without papillae (P. grimaldii, P. plumosa) from papillate species (all the other five species); presence of papillae appeared homoplastic or plesiomorphic in all reconstructions. Also, no MPTs allowed the separation of P. grimaldii and P. plumosa in two monophyletic groups ‘Petromica’ and ‘Monanthus’; our data therefore support the synonymy of the two genera. Biogeography of Petromica Replacing the terminal taxa in Figures 16–19 by their areas of occurrence results in three possible areagrams (Fig. 20). The most consistent of these is that supported by 2 out of 4 most parsimonious trees (Fig. 20A), and we will thus concentrate our discussion on it. In this areagram, the basalmost vicariant event separates species from Atlantic South America from those of South Africa. This event could be attributed to the formation of the Atlantic Deep-Water Barrier in the Albian (middle-upper Creataceous). The next vicariance occurred between P. ciocalyptoides and P. citrina along the SE South American coastline. A possible scenario involves ecophysiological barriers due to temperature maxima and minima. P. citrina, which is known only from a narrow sector along the southern and southeastern Brazilian coastline, might have its distribution to the north limited by average temperature maxima by the Brazilian Current north of Rio de Janeiro state (e.g. Palacio, 1982). Conversely, average temperature minima could prevent P. ciocalyptoides from establishing viable populations south of Rio de Janeiro state. The southernmost population of this species has been found in the ‘Coralline Oasis’ of Arraial do Cabo (Laborel, 1969), a well known biogeographic limit to several species of marine organisms (e.g. Palacio, 1982; Yoneshigue, 1985; Muricy et al., 1991). Salinity fluctuations could also have played a role in the vicariance between these two species. The next speciations observed occurred nearly locally in South Africa, all in a similar bathymetric zone (ca. 100 m depth), and are difficult to associate to major vicariance events. Petromica digitata, P. plumosa and P. tubulata, all from South Africa, occur in an area under the influence of the Agulhas Current, which is guided by the edge of the continental shelf in the southeastern African region (Rae, 1991). This current contours the southern African continent, flowing back to the Indian Ocean when it meets the Benguela Current. Under such an apparently homogeneous current flow, speciation could be attributed to local shifts on prevailing ecological settings associated to varying sea-level changes (e.g. Pielou, 1979). Sea-levels 160 m below present levels have been estimated for the penultimate glacial (Illinoian/Riss; Kurtén, 1972). A restriction of shelf area may have increased extinction, range retractions, and/or the formation of pockets of 124 endemism in isolated bay-type areas, being a likely explanation for quasi-sympatric speciation where a continuous ancestral species once existed. The Agulhas current was prevented from reaching the south Atlantic at glacial periods, thus creating considerable fluctuations in oceanographic conditions at the southern tip of the African Continent (M. Maslin, pers. comm., 2000). Colonisation of the central Indian Ocean, the western Mediterranean and the Azores by Petromica massalis and P. grimaldii is harder to explain. This appears as a relatively recent event in the history of Petromica, and given the highly disjunct distribution of the species in the most basal clades, jump-dispersal (e.g. rafting) seems the most likely explanation. Insufficient collections or widespread extinction could also be responsible for the apparently disjunct pattern of occurrence when the P. grimaldii–P. massalis clade is compared to their South African sister clades. An alternative scenario for an older era would have a widespread ancestor for the clade (P. grimaldii, P. massalis) (P. digitata) (P. plumosa) (P. tubulata) distributed over most of the Tethys paleoocean. A north–south vicariance could then be hypothesized, such as the closure of the Tethys Ocean, followed by an east–west event possibly associated to the Mediterranean–Messinian crisis. A similar east–west vicariance was observed in other sponge genera such as Didiscus, Acarnus and Rhabderemia (Hiemstra & Van Soest, 1991; Van Soest et al., 1991; Van Soest & Hooper, 1993). Family-level classification of Petromica The genus Petromica was originally classified in the Order Lithistida, sub-order Anoplina, family Azoricidae (Topsent, 1898), and later transferred to a new, monogeneric family Petromicidae in the same suborder (Topsent, 1928; Lévi, 1973; Kelly-Borges & Pomponi, 1994). The suborder Anoplina (=Rhizomorina Zittel) is clearly polyphyletic, being defined solely by the presence of desmas in absence of microscleres, tetractinal spicules and ectosomal megascleres. Burton (1929) classified Monanthus within the Axinellidae, but such a relationship is unlikely in view of the absence of axially-condensed skeleton in all species here described. De Laubenfels (1936) created the family Monanthidae (order Halichondrida) for genera “closely related to the Axinellidae or similar families, but which are set apart by the possession of ( . . . ) desmas” (De Laubenfels, 1936: 139). This family included Monanthus, Petromica and Stylinos Topsent, 1892. This ensemble is clearly artificial, and the family Monanthidae has been abandoned by all subsequent authors (e.g. Lévi, 1973; Bergquist, 1978; Van Soest & Hajdu, 2000). Van Soest & Zea (1986) transferred Monanthus (later synonymyzed with Petromica – Van Soest et al., 1990) to the family Halichondriidae, based on the possession of the following synapomorphic traits: high spicular density, vague spicule tracts and haphazard arrangement of spicules (Van Soest et al., 1990). This classification was followed by Gruber (1993), but not by Kelly-Borges & Pomponi (1994). DNA sequences support the proximity of Monanthus with the Halichondrida (Kelly-Borges & Pomponi, 1994), although not forming a monophyletic group with Hymeniacidon. TLC patterns of secondary metabolites support a close relationship between Topsentia, Coelocalypta and Petromica (Pomponi et al., 1991). Other traits have been considered synapomorphic at lower levels within the Halichondriidae, of which two are also shared with Petromica: loss of collagenous mesohyl, and reduction of spongin (Van Soest et al., 1990). The present study has confirmed these traits in all species of Petromica. Although the validity of some of these ‘synapomorphies’ may be disputable, this large ensemble of shared morphological and biochemical characters strongly favors the inclusion of Petromica within the family Halichondriidae. Alternative classifications (Topsent, 1928; Burton, 1929; De Laubenfels, 1936; Lévi, 1973) appear less parcimonious as they imply in the parallel development of a confused skeleton of oxeotes with high size variation and straigth or sinuous ascending choanosomal tracts, as well as of specific DNA sequences and secondary metabolites, independently in Petromica and in halichondriids. Other characters which could add to this discussion such as the anatomy of the aquiferous system, cytology and more detailed secondary metabolite chemistry still await investigation in Petromica. Acknowledgements We are grateful to Clare Valentine (BMNH), Klaus Rützler (USNM), Kate Smith (USNM), Rob Van Soest (ZMA), Claude Lévi (MNHN), Pedro Alcolado (IOC), Beatriz Mothes (MCN) and Clea Lerner (MCN) for the kind loans of specimens. Mark Maslin (ECRC, University College, London) and Rob Van Soest contributed with useful biogeographical comments. Critical reading by two anonymous reviewers greatly im- 125 proved the manuscript. This work was supported by grants and fellowships from CNPq, FAPERJ, FAPESP and FUJB. References Alcolado, P. M., 1984. Nuevas espécies de esponjas encontradas en Cuba. Poeyana 271: 1–22. Berg, C., 1899. Substitución de nombres genéricos, 3. Commun. Mus. nac. Buenos Aires 1: 77–80. Bergquist, P. R., 1978. Sponges. University of California Press, Los Angeles: 268 pp. Boury-Esnault, N., M. Pansini & M. J. Uriz, 1994. Spongiaires bathyaux de la mer d’Alboran et du golfe ibéro-marocain. Mém. Mus. natn. Hist. nat. Paris, A, Zool. 160: 1–174. Bowerbank, J. 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Papillae number: 4.0 absent, 4.1 from one to five, 4.2 more than five. 5. Papillae shape: 5.0 cylindrical-digitiform, 5.1 conical-truncate. 6. Papillae termination: 6.0 open; 6.1 blind. 7. Papillae heigth: 7.0 smaller than 5 cm, 7.1 higher than 5 cm. 8. Colour in vivo: 8.0 yellow, 8.1 orange, 8.2 red, 8.3 blue, 8.4 white, 8.5 grey, 8.6 purple. 9. Colour in spirit: 9.0 whitish-drab-cream, 9.1 yellowish, 9.2 beige–tan–light brown, 9.3 grey, 9.4 blue. 10. Body surface: 10.0 smooth; 10.1 hispid; 10.2 conulose, 10.3 rough–uneven–lumpy, 10.4 with surface projections. 11. Superficial canals: 11.0 absent, 11.1 present (thin, vein-like). 12. Oscules diameter (mm): 12.0 less than 2 mm, 12.1 up to 5 mm. 13. Membranous oscular rim: 13.0 absent, 13.1 present. 14. Dermal membrane: 14.0 hardly detachable, 14.1 easily detachable. 15. Consistency: 15.0 firm but compressible, 15.1 soft, 15.2 rigid. 16. Papillae skeleton: 16.0 spiral plumose tracts; 16.1 sinuous tracts, 16.2 inner tangential layer. 17. Ectosomal skeleton: 17.0 absent, 17.1 confused tangential layer, 17.2 paratangential, 17.3 palisade. 18. Choanosomal ascending tracts: 18.0 absent, 18.1 vague, sinuous, 18.2 large, coalescent. 19. Diameter of choanosomal tracts (in µm): 19.0 less than 500, 19.1 up to more than 500. 20. Confused choanosomal reticulation: 20.0 absent, 20.1 monaxons, 20.2 monaxons and desmas, 20.3 desmas. 127 21. Monaxon type: 21.0 oxea, 21.1 style, 21.2 subtylostrongyle. 22. Monaxon length (µm): 22.0 up to 1000, 22.1 longer than 1000. 23. Curvature of monaxons: 23.0 straight-slightly curved, 23.1 abruptly bent. 24. Desma maximum length (µm): 24.0 up to 500, 24.1 longer than 500. 25. Desmas (degree of ramification): 25.0 lowmoderate, 25.1 high. 26. Desmas branch tips: 26.0 smooth, 26.1 microspined. 27. Degree of desma fusion (zygosis): 27.0 none, 27.1 parcial-moderate, 27.2 high. 28. Desmas distribution: 28.0 whole choanosome and ectosome, 28.1 base only. 29. Raphides: 29.0 absent, 29.1 present. 128 Appendix 2. Data marix used in phylogenetic analyses. For character-state names, see Appendix 1. ?, unknown state; N.A., not applicable Characters P. P. P. P. P. P. P. Hymeniacidon Topsentia Desmanthus Gastrophanella grimaldii massalis plumosa tubulata digitata ciocalyptoides citrina spp. spp. spp. spp. Body shape Body margins Maximum body size (in cm – width) Number of papillae Papillae shape Papillae termination Papillae height Colour in vivo Colour in spirit Surface Superficial canals Oscules diameter (mm) Membranous oscular rim Dermal membrane Consistency Papillae skeleton Tangential ectosomal skeleton Choanosomal ascending tracts Diameter of choanosomal tracts (µm) Confused choanosomal reticulation Monaxon type Monaxon length (µm) Monaxon curvature Desmas length (µm) Desmas degree of ramification Desmas branch tips Degree of zygosis Desmas distribution Raphides 0 0 1 0 1 1 0 0 1 0 0 0 0 1 0–1 0 0 0 1 0 0–2 0–1 2 0 N.A. N.A. N.A. ? 0–2 2 1 0 1 1 0 – 2 2 0 0 0 ? 1–2 2 0 ? 0 1 2 ? 2 0 N.A. N.A. N.A. ? 0 1 0 0 0 0 0 – 2 1 0 1 0 ? 0 1 0 0 0 0 0 0 1 1 0 0–1 0 ? 0 1 1 0 0 1 0 0 1 2 0 1 1 0–4 0 1 1 0 0 0 1 1–2 1 2 1 0–1 0 0–1 0–1 2 0 1 1 1 2 1 0–1 0–2 N.A.–1 N.A.–0–1 N.A.–0 1–2–3 0–4 1 0 0 1 0 1 1 0–1 0–1 N.A.–0-1 N.A.–0–1 N.A.–1 2–4 0–2 1 0 0 0 0 0 1 0–1 0 N.A. N.A. N.A. 0–1–3–5 3 1 0–1 0 0–1 0 2 N.A. 0–1–2 0 N.A. N.A. N.A. 0–4–6 0–2–3 1 0–1 1 0–1 0 2 N.A. 0–1 1 0 1 1 1 1 1 2 0–1 3 1 2 2 1 2 1 1 1 0 0 0–1 0 1 1 0 1 ? 0 0 N.A. N.A. 0 2 0 1 0 1 2 0 1 0 1 2 0 0 0 1 2 0 0 0 1 2 0 1 1 1 2 0 0 0 1 2 0 0 1 1 1 1 0 0 N.A. 1 0 1 0–1 N.A. 0 1 1 0 0 3 2 0 0 0 1 1 1 0 0 1 0 2 0 0 0 0 1 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 1 0 N.A. N.A. N.A. N.A. 0 N.A. N.A. N.A. N.A. 0 1 0 2 0 0 1 0 2 0 0