Revision of the genus Placospongia (Porifera, Demospongiae, Hadromerida, Placospongiidae) in the Indo-West Pacific

Abstract Species of the genus Placospongia are common within the tropical Indo-West Pacific, demonstrating a wide variety of colors and either branching or encrusting growth forms. A revision of Indo-West Pacific Placospongia was undertaken based on a redescription of the holotypes of species of Placospongia from the Indian Ocean and Western Pacific and an examination of an additional 103 specimens of Placospongia ssp. collected from Indonesia (including Vosmaer and Vernhout 1902 material), Seychelles, India, Singapore and Micronesia. One mitochondrial (COI) and one nuclear (ITS) marker were subsequently used to differentiate species. All Placospongia species are characterized by selenasters and tylostyles in two size classes. The combination of microsclere diversity and morphology as well as megasclere size were shown to be informative morphometric characters, supported by molecular evidence. Live coloration and growth form is shown to be unreliable for diagnoses. The study of holotypes found that Placospongia mixta is a valid species and that two genus transfers are necessary: Geodinella anthosigma is a Placospongia and Placospongia labyrinthica is a Geodia. A new species is also described from an anchialine pool in Indonesia, Placospongia santodomingoae sp. n.; bringing the total fauna of Placospongia species in the Indo-West Pacific to five: Placospongia anthosigma, Placospongia carinata, Placospongia mixta, Placospongia melobesioides, and Placospongia santodomingoae sp. n. An identification key is given. Two additional species, possibly morphologically cryptic, have been identified by molecular markers.


Introduction
Species of the genus Placospongia in the tropical Indo-West Pacific occur in a wide variety of habitats such as marine lakes, coral reefs and mangroves. They may display a variety of colors and growth forms, from encrusting to branching (Figs 1,2). Generally only two species have been recorded in species checklists within the Indo-West Pacific (e.g. Burton 1959, Hooper and Wiedenmeyer 1994, Hooper et al. 2000, Becking et al. 2006, de Voogd et al. 2008, de Voogd et al. 2009): Placospongia melobesioides Gray 1867, and Placospongia carinata (Bowerbank 1858). A recent collection of over 100 Placospongia specimens during fieldtrips by the author to Indonesia in 2006(Sulawesi), 2007(Papua), 2008, 2009, and to Micronesia in 2010 (Yap) revealed, however, that there were more than two species present in these faunas.
The taxonomic literature records six valid species of Placospongia worldwide, of which there are three from the Indian Ocean and Western Pacific: P. carinata (type locality "South Sea", presumably in the Pacific), Placospongia labyrinthica Kirkpatrick 1903 (type locality East London, South Africa, Indian Ocean), P. melobesioides (type locality Borneo, Pacific). In 1900 Thiele described the species Placospongia mixta from Ternate (Indonesia), which was later synonymized with P. carinata by Vosmaer andVernhout in 1902. Vosmaer andVernhout (1902) based their conclusions on a review of 26 specimens collected during the Siboga expedition to Indonesia, and this collection is presently housed at the Naturalis Biodiversity Center (Leiden, The Netherlands). Subsequently, according to the World Porifera Database (van Soest et al. 2011) Geodinella anthosigma Tanita and Hoshino 1989 (type locality Sagami Bay, Japan) should be transferred to the genus Placospongia, and P. labyrinthica should in fact be transferred to the genus Geodia. These suggested genus transfers have, however, not yet been published in the peer-reviewed literature. A molecular phylogeny constructed using the internal transcribed spacer region (ITS) indicated that there were nine evolutionary lineages worldwide within the genus Placospongia of which there were five distinct clades in the Indo-Pacific (clades C3, C4, C5, C6 & C9) that may represent five species (Nichols & Barnes 2005). The authors did not investigate the spicule morphology of the specimens in their study, therefore it is unclear which species name can be assigned to the different clades. The objectives of the present study were to revise the genus Placospongia in the Indo-West Pacific by examining the holotypes of P. melobesioides, P. carinata, P. mixta, as well as 103 specimens of Placospongia spp. that were collected from Indonesia (including the Vosmaer & Vernhout material), Singapore, Seychelles, Madagascar, and Micronesia. In order to obtain a full view of the species from the Western Pacific and Indian Ocean the holotypes of the temperate species G. anthosigma, and P. labyrinthica were also examined. Subsequently it was determined if growth form and color can be used as diagnostic characteristics to identify different species of Placospongia in the field. Finally, the aim was to provide species names to the five clades of Indo-Pacific Placospongia as published by Nichols and Barnes (2005) by combining their published ITS sequences from GenBank with ITS sequences from identified species of Indo-Pacific Placospongia.

Material and methods
Specimens from Indonesia were collected via snorkeling in marine lakes and mangroves, and scuba diving in reefs. For a detailed description of marine lakes in Indonesia see Becking et al. (2011). Where possible material was preserved in 96% ethanol for DNA analysis, and voucher specimens were preserved in 70% ethanol and deposited in the collections of the Naturalis Biodiversity Center, Leiden, The Netherlands (RMNH POR.). Records were made on the external morphology, skeletal architecture and spicules of all material. Spicule dimensions were measured of a subset of specimens indicated in Table 1, based on 25 measurements (unless noted otherwise) and given in the text as minimum-average-maximum. The following dimensions were measured: tylostyles length × shaft width × head width; selenasters length × width; streptasters total length × ray length; spherasters diameter; rhabds length × width. Only fully developed spicules were measured. To study the skeletal architecture handcut perpendicular sections of the choanosome were made. The sections were air-dried, mounted in Durcupan ® ACM on a microscope slide, and studied under a Leica high power microscope. Spicule preparations were made by dissolving the organic tissue of a small fragment of the specimen in commercial bleach, after which the spicules were washed >10 times with distilled water and once with 96% ethanol. The spicules were air-dried on microscope slides and mounted with Durcupan ® ACM. The spicules were also mounted on aluminium stubs, coated with gold-palladium and studied with a Jeol Scanning Electron Microscope.
DNA extractions were made with Qiagen DNEasy animal blood and tissue extraction kit following the manufacturer's protocol. The polymerase chain reaction (PCR) reaction volume was 25 μl and contained 5 μl Phire ® Hot Start reaction buffer, 1 unit Hotstart Phire® Hot Start DNA polymerase (Finnzymes), 2 μl 1 mM dNTPs (Gibco), 1 μl DNA template (5-20 ng) and 0.625 μl of 10mM each primer. The standard DNA-barcoding fragment of the mitochondrial cytochrome oxidase subunit I (COI) fragment was amplified by using a specific forward primer designed by the author for  Nichols & Barnes (2005), for GenBank accession numbers see Figure 11. The best-fit DNA substitution model was selected by the Akaike Information Criterion deployed in jMODELTEST v. 0.1.1 (Posada 2008)  . Encrusting to branching growth forms. Small encrustations of 3 cm 2 to large surfaces of >2m 2 to branching individual with total size of up to 45cm in length and branch diameter between 0.25-1.5cm. Total size of specimens is hard to establish as parts of the body may be encrusting within cracks. Dried material is hard, alcohol preserved and live specimens remain compressible as the choanosome is of more pliant material than the cortex. The surface is made up of smooth cortical plates separated by contractible grooves which form a kind of network on the surface while these are firmly closed in preserved specimens. See Vosmaer & Vernhout (1902) and Rützler (2002) for an extensive description of the genus. In live specimens grooves are open and oscules are visible inside contractile ridges, running between plates. Live color white, cream, orange, reddish brown to dark black-brown ( Fig. 1, 2) and come color is usually retained after alcohol preservation. The contact lines between the plates ridge up slightly and are generally a different shade of the color of the plates.
Skeleton. the cortical plates consist of densely packed selenasters and can also contain auxiliary microscleres. Developmental stages of selenasters occur throughout the choanosome. Tylostyle tracts support the margins of the cortical plates. In branching specimens radial tylostyle tracts run from the centre core (consisting of densely packed selenaster) to the cortical plates, in encrusting specimens tracts run in direction from substrate to cortex. The sharp ends of the smaller tylostyles are projected beyond the cortex surface. Microscleres occur in the cortex and scattered in choanosomal skeleton. For a detailed description of external morphology and anatomy see Vosmaer and Vernhout (1902).
Distribution. Type locality Sagami Bay, Eastern Japan, presently not recorded from any other locality.
Ecology. On rock substrate in deep temperate waters. Remarks. Originally described by Tanita and Hoshino (1989) as Geodinella anthosigma. Geodinella is no longer a valid genus. Geodinella anthosigma should be transferred to the genus Placospongia based on the external morphology with the characteristic cortical plates and the presence of selenasters, tylostyles and spherasters. Placospongia anthosigma is distinguished from the other Indo-Pacific Placospongia spp. by the presence of contorted, spirally ornamented spirasters referred to by Tanita and Hoshino (1989) as 'anthosigma' and the small class of tylostyles with blunt points.  Tanita & Hoshino (1989 Table 2 for full details per specimen) Description. Reviewed material is encrusting and/or branching. External morphology follows the description of the genus. Color of live specimens can be purple brown, chocolate brown, milk coffee brown, orange brown, orange, cream, or white ( Fig. 1, 2). Color of choanosome is pale beige. After preservation in ethanol specimens retain some color of the live coloration.
Skeleton. As description of genus with addition that microrhabds form a layer over and amidst the selenaster cortex and are also prevalent in choanosomal tissue. Spirasters scattered in choanosome.
Distribution. East African coast to eastern Indonesia (Fig. 9 , Table 2). Originally described from the 'South Sea', presumably the South Pacific Ocean. This has been interpreted by some (Rützler 2002, van Soest et al. 2011 to be Palau or Vanuatu, but this remains speculative. Based on the reviewed material and literature the minimal distribution is from Madagascar (Lévi 1956), to the Seychelles, and across Indonesia to the Aru Islands (Table 2). Distribution may extend further East.
Ecology. Depth 0-45m. In Indonesia rarely found in reef environment, but high abundance in marine lakes. Possibly higher prevalence in reefs in Eastern Africa, based on the ZMA Por. collection from the Seychelles and the publication from Madagascar (Lévi 1956).
Remarks. The Bowerbank description from 1858 should be considered as the original description of 'Geodia carinata', now accepted as P. carinata, with plates XXV Fig. 19 and XXVI Fig. 10 representing the streptasters ("arborescent elongo-subsphero-stella"). Subsequently in 1874 Bowerbank published a more extensive description of "Geodia carinata" including a drawing of the streptasters (Fig.  3, p.299) and spined microrhabds ("minute multiangulated cylindrical retentive spicula", fig.2, p.299) that he described as characteristic of the species. In neither publication registration numbers were provided, however. The habitus drawing in Fig. 5, p299 of Bowerbank publication in 1874 is identical to the specimen BMNH95.6.7.1 that I received from the BMNH after requesting the holotype for P. carinata. In addition, I received the slides of spicules (codes: R1228, 86g, Bk.1390) and of the thick cut (codes: R1275, PE01, Bk1390) that were labeled to belong to the holotype (Fig. 5). Upon inspection I discovered that the specimen BMNH 95.6.7.1 is in fact a P. melobesioides, while the two slides do indeed represent P. carinata containing the characteristic streptasters with bifurcating endings and the microrhabds as indicated in the Bowerbank images and in the images taken from these slides in Fig. 5. The slides clearly do not come from the specimen BMNH 95.6.7.1. In the 16 years between Bowerbank's 1858 and 1874 publications, I fear that there has been some exchange or misinterpretation of the labels of the specimens resulting in the incorrect assignment of specimen BMNH 95.6.7.1 to the slides and as the holotype of P. carinata. This specimen BMNH 95.6.7.1, furthermore, has two labels attached to it: one with "Geodia carinata", and one with "Placospongia melobesioides". According to Bowerbank (1874) three specimens had been reviewed for his manuscript: one received from his friend Mr. Thos. Ingall in 1854, one placed by Dr. Baird from the coral to the sponge collection in the BMNH, and one specimen purchased by Bowerbank in 1864. The first mentioned specimen is presumably the holotype, but as this specimen has not been located, I propose to designate the slides R1228-86g-Bk.1390 and R1275-PE01-Bk1390 as representing the holotype of P. carinata.  Placospongia melobesioides Gray (1867) Table 3 for full details per specimen) Description. Holotype BMNH 52.4.1.14 dry, chalky white angular branches, hard. Other examined material encrusting to branching, hard, thicker specimens slightly compressible. External morphology follows the description of the genus. Size ranging between 5-50 cm, though encrusting specimens may be larger growing within crevices. Ectosome color in life ranging from purple, dark black brown, chocolate brown, orange brown to light beige (Fig. 1, 2). Choanosome pale beige. After preservation color of ectosome is similar to live color.
Skeleton. As description of genus with addition of sporadic spherasters lodged amidst selenasters in cortex and high abundance of spherules in choanosome and cortex.
Distribution. Type locality: Borneo. Distribution from Seychelles to Micronesia (Fig. 9, Table 3). Possibly further east to Central Pacific.
Remarks. In the original description by Gray (1867) there is no mention of two size classes of tylostyles. I reexamined the original slide and conclude that the holotype does contain two size classes of tylostyles. The Systema Porifera indicates that the holotype has two size classes, the large 720-963-1200 × 13-14.1-19 μm and the small 350-438.8-560 × 8-9.1-10.5 μm, based on 10 measurements per spicule type (Rützler 2002). These measurements deviate from the holotype measurements in the present study that were based on 25 measurements per spicule type (670-880-1010 × 10-13-18 μm and 205-293-420 × 5-10-13 μm respectively), and also deviate from the range of sizes within the examined material of this study (Table 1). There is great variation in tylostyle length and spherasters are only sporadically present, often absent.

Placospongia mixta Thiele 1900
http://species-id.net/wiki/Placospongia_mixta Figure 7 Placospongia mixta Thiele, 1900: Plate III, Fig. 25 Table 4 for full details per specimen) Description. Holotype ZMB 3204 encrusting, size 5 × 2.5 cm and thickness 1-5 mm (as described by Thiele, now very small fragment), white after preservation in alcohol. The majority of the reviewed material is encrusting with a thickness of 4-10mm, but branching specimens also occur. External morphology follows the description of the genus. Color of the ectosome can be red, orange, brown orange, dark brown, chocolate brown, milk coffee brown, cream, or white (Fig. 1, 2). Color of choanosome is pale beige. After preservation in ethanol color is similar to live specimens, but lighter shade.
Distribution. East African coast to eastern Indonesia (Fig. 9, Table 4). Possibly further east to Central Pacific. Pulitzer-Finali (1993) identified a 'P. carinata' from East Africa (Mombasa) that fits the description of P. mixta based on the length of the tylostyles (up to 1200 μm) and the presence of spherasters, but no P. mixta specimens were observed in the Seychelles material deposited at ZMA.
Ecology. Depth 0-45m. Common in reefs, also occurs in marine lakes. Remarks. In 1900 Thiele described a new species named P. mixta, which was originally identified as P. melobesioides by Kieschnick (1896). The specific epithet mixta was given because the specimen contained a mixture of spicules: both spirasters like P. carinata as well as large spherasters like P. intermedia and P. melobesioides, which are absent in P. carinata. In 1902 Vosmaer & Vernhout decided that P. mixta was a junior synonym of P. carinata, because they saw no distinction between the different shapes of streptasters and stated that spherasters are never very abundant -in some 'exceedingly rare and in some we failed to find them at all' -and could therefore not be seen as a distinguishing character. The specimens that were studied by Vosmaer and Vernhout (1902) were collected in Indonesia during the Siboga Expedition (1899Expedition ( -1900 and are housed in the collection of the Naturalis Biodiversity Center (Leiden, The Netherlands). In the present study these specimens were reexamined. After inspection, the specimens labeled 'P. carinata' could be clearly and consistently divided into two species: P. carinata without spherasters, with streptasters displaying bifurcating tips, and tylostyles up to 980 μm, and P. mixta with abundant spherasters, with streptasters displaying hastate tips, and tylostyles up to 1250 μm. In none of the specimens of Vosmaer & Vernhout (1902), nor of the other specimens reviewed for this study was there a mixture of the two types of streptasters. These two species also show molecular distinction in both mitochondrial and nuclear markers (Fig. 10, 11, Table 6, 7). Placospongia santodomingoae sp. n. urn:lsid:zoobank.org:act:3C4F2599-15C0-4075-BD3B-8C6439C8F821 http://species-id.net/wiki/Placospongia_santodomingoae Figure 8 Holotype. RMNH POR. 4486, Indonesia, East Kalimantan province, Maratua island, Buli Halo anchialine pool, 02°11'16.4"N, 118°37'06.4"E, 0-1m. depth, xi.2008, coll Holotype and paratypes are branching and encrusting, size 8cm in length. Total size of specimens in situ is hard to establish as parts of the body may be encrusting within cracks. Alcohol preserved and live specimens are hard but slightly compressible. The surface is made up with typical Placospongia cortical plates separated by contractible grooves which form a network on the surface. Oscules are present in the grooves. Live color of holotype was dark brown, the paratypes were orange, and these colors were mostly retained after alcohol preservation (Fig. 8A).
Etymology. Named in honor of Nadiezhda K. Santodomingo, the collector of the types, for her years of tireless work in marine science including anchialine research.
Remarks. Placospongia santodomingoae sp. n. is similar to P. carinata, yet lacks streptasters and has shorter tylostyles. Placospongia santodomingoae sp. n. likewise differs from P. mixta by the absence of streptasters as well as the absence of spherasters. Placospongia santodomingoae sp. n. differs from P. anthosigma by the absence of anthosigma, and by having hastate endings of the smaller tylostyles. (Kirkpatrick, 1903) http://species-id.net/wiki/Geodia_labyrinthica Placospongia labyrinthica Kirkpatrick 1903: Plate V Fig. 1a Spicules. Megascleres styles, oxea; microscleres sterrasters, chiasters Remarks. This species was originally described as 'Placospongia labyrinthica', but does not have the characteristic cortical plates of Placospongia. The specimen furthermore has sieve pores, sterrasters with star-like plates, euasters, styles and oxea characteristic of the Geodiidae. In the original description, Kirkpatrick (1903) stated "the presence of chiasters is so exceptional that I thought at first that I had to deal with a geodine sponges, but there were no triaenes to be found" and as a result placed this species in the Placospongia rather than Geodia. Genus transfer to Geodia is, however, required as suggested on the World Porifera Database (van Soest et al. 2011).

Genetic data analysis
All sequences were submitted to GenBank with accession numbers KC848421 -41 (Table 5). Final alignments (excluding primers) were obtained for the sponge Placospongia of 581 bp for COI with three genetic variants (28 individuals) and 13 polymorphic sites. The three genetic variants correspond to the three species P. melobesioides, P. mixta, and P. carinata that represent monophyletic groups which are strongly supported by both Bayesian and maximum likelihood inference methods (Fig. 10).
There was no intra-specific variation within each species, regardless of geographic locality. The inter-specific p-distances ranged between 0.5-2.1% (Table 6). There were 11 substitutions between P. melobesioides and P. carinata, 12 substitutions between P. melobesioides and P. mixta, and three substitutions between P. mixta and P. carinata. The specimens of P. carinata and of P. santodomingoae sp. n. had identical genotypes for COI. No molecular work could be done on the dried holotype of Placospongia anthosigma and fresh material was not available. Final alignments (excluding primers) of 720 bp were obtained for ITS with 18 genetic variants from the present study (22 individuals). An additional 27 genetic variants from GenBank (for GenBank accession numbers see Fig. 11) were included in the phylogenetic analysis. The ITS sequences represented five clades that were strongly supported by both Bayesian and maximum likelihood inference methods (Fig. 11). These five divergent clades (see Table 7 for uncorrected p-distances) correspond to the clades C3, C4, C5, C6, and C9 as presented by the study of Nichols & Barnes (2005). Clade C9 represents specimens of the species P. melobesioides, clade C5 P. mixta, and clade C4 P. carinata. Clades C6 is represented by one specimen from the Solomon Islands (QM317896) and clade C3 by one specimen from Bynoe Harbour, Northern Territory, Australia (QM303439); none of the samples that were sequenced in the

Different species
In the Indo-West Pacific at least five species of the genus Placospongia can be identified based on spicule morphology: P. anthosigma, P. carinata, P. mixta, P. melobesioides, and P. santodomingoae sp. n.. Placospongia melobesioides, P. carinata, and P. mixta can be distinguished with the DNA barcode marker (COI) and a nuclear marker (ITS). The species P. santodomingoae sp. n. and P. carinata have the same sequence of COI. The sequence variation of COI in sponges can be low (e.g. Wörheide 2006, Pöppe et al. 2011 and this is also the case in species of Placospongia, e.g. only 0.5% nucleotide distance between the species P. mixta and P. carinata. There is further- Figure 10. Bayesian/maximum likelihood phylograms of Cytochrome Oxidase I (COI) sequences from Indo-Pacific Placospongia spp. See Table 5 for GenBank accession numbers. Only posterior probabilities of >90 and maximum likelihood values of >70 indicated. Scale bar indicates substitutions/site. Figure 11. Bayesian/maximum likelihood phylograms of genotypes of the internal transcribed spacer region of nuclear ribosomal operons (ITS) of Indo-Pacific Placospongia spp. found in this study and related species from the same genus collected from GenBank. Clades C3, C4, C5, C6 & C9 refer to the clades presented in the study by Nichols & Barnes (2005). Taxon labels are organized as follows: Specimen -Locality -Genbank code or RMNH POR. Number. Only posterior probabilities of >90 and maximum likelihood values of >70 indicated. Scale bar indicate substitutions/site. more no intraspecific variation in COI within each of the Placospongia species, not even between populations at 1000s of km distance from each other (e.g. specimens from the Seychelles are identical with specimens from Indonesia). The phylogenetic inference based on the ITS sequences does show a supported clade of P. santodomingoae sp. n. within the clade of P. carinata (Fig. 11), though the degree of divergence between the two species is low (0.6%) ( Table 7). Placospongia santodomingoae sp. n. should, however, be designated as a new species based on the spicule morphology: the absence of a distinguishing spicule type (streptasters) and consistently shorter and thicker tylostyles (maximum 760 × 20 μm) compared to P. carinata (maximum 980 × 17.5 μm) are valid arguments to distinguish a separate species within this genus. The specimens of P. santodomingoae sp. n. were collected from an anchialine pool. This kind of isolated environment has previously been shown to contain small, rapidly evolving populations, and many rare species across a large spectrum of taxa (e.g. Holthuis 1973, Tomascik & Mah 1994, Dawson & Hamner 2005, Becking et al. 2011. The divergence of P. santodomingoae sp. n. from P. carinata is likely too recent to be expressed in the molecular markers that were used. Other, faster evolving, molecular markers might show a more distinct separation between species, but for the present significant morphometric differences in spicules are reliable characters in separating these sister species. A molecular phylogeny using the internal transcribed spacer region (ITS) showed that there were five distinct clades within the genus Placospongia in the Indo-West Pacific (clades C3, C4, C5, C6 & C9) (Nichols & Barnes, 2005). Nichols & Barnes (2005) indicated that their results presented a conundrum that "specimens collected from Indonesian marine lakes that have been isolated from the surrounding marine environment since the Pleistocene are undifferentiated from individuals collected from the Seychelles indicating that populations from these geographically disparate regions are, or have recently been, connected by gene flow despite the lack of evidence of connectivity between these lakes and nearby reefs." It is important to note here that the authors did not investigate the spicule morphology of the specimens in their study, while it is in fact the spicules that can largely explain the presented conundrum. In the present study over 30 specimens from the marine lakes Kakaban and Maratua and the adjacent reefs have been reviewed as well as the specimens from the ZMA Por. collection that were used in the Nichols & Barnes (2005) study. Clade C4 represents the material from the Seychelles (ZMA Por.11367) together with the marine lakes and can all be morphologically identified as P. carinata sensu stricto. The samples from the lakes and the Seychelles are thus conspecific, but the populations of the two locations are necessarily connected by gene flow. Subsequently clade C9 is P. melobesioides (specimens from Indonesia, Miscronesia and the Seychelles) and clade C5 is of P. mixta (specimens from Indonesia, Palau and Papua New Guinae). This explains three of the five clades from the Indo-West Pacific and leaves two undetermined: clade C3 represented by one specimen from Bynoe Harbour, Northern Territory, Australia (QM303439), and clade C6 represented by one specimen from the Solomon Islands (QM317896). The morphology of these specimens should be further studied in order to correctly identify the species and determine if they may represent morphologically cryptic species.

Natural variation
Each of the five species of the genus Placospongia in the Indo-West Pacific can be distinguished based on the composition and morphology of spicules. The external morphology, however, does not allow species distinction. The most common species from the tropical Indo-West Pacific (P. melobesioides, P. mixta, and P. carinata) can have both encrusting and branching growth forms displaying a variety of colors from white to dark brown. The only observed consistent pattern was that all the red specimens belonged to P. mixta, while all the dark black-brown specimens belonged to P. melobesioides. These two colors may be useful for field identifications, yet both species can also display the range of other colors (white, cream, beige, light brown) as well. The density of canals/ridges (or size of cortical plates) appears to be related to environment as this is higher in specimens from high sediment locations such as the marine lakes than in specimens from the reefs (Fig. 1, 2). Within each species there is also some natural variation in the range of tylostyle length and spicule morphology. The streptaster morphology varies within species and even within individuals. Within one individual the number of rays can vary from 4-10 (Figs 3, 4) and between individuals the decoration and size of spines can be diverse. For example the streptasters of P. carinata specimens from Haji Buang marine lake are micro-acanthose while the specimens from other locations are not. Spherasters are always present and abundant in P. mixta and P. anthosigma, but are in low abundances or absent in P. melobesioides, as has been indicated previously by Vosmaer & Vernhout (1902). In P. carinata and P. santodomingoae sp. n. spherasters are always absent.

Ecology and distribution
P. melobesioides and P. mixta are common in the reef environment. Most of the collected material from the reefs in Indonesia were one of these two species. P. carinata appears to be rare in the reefs, in Indonesia at least, while it is highly abundant in the marine lakes Haji Buang and Kakaban in East Kalimantan, Indonesia. Placospongia santodomingoae sp. n. is restricted to an anchialine pool. Placospongia anthosigma was not found in any of the examined collections from the tropical Western Pacific, this species is restricted to more temperate and deeper waters. Placospongia melobesioides is indicated in the Systema Porifera to have a distribution from the Indo-West Pacific to the Tropical Atlantic (Rützler 2002). Both P. melobesioides and P. carinata have been recorded from the Atlantic (e.g. de Laubenfels 1936, Hechtel 1976, Coelho and Mello-Leitão 1978, Pulitzer-Finali 1986, González-Farías 1989, which would imply that these are pantropical species. Recent molecular and more detailed morphological studies have, however, shown that many cosmopolitan sponge species are in fact species complexes either delineated by morphology or molecules (e.g. Reveilleud et al. 2010. Van Soest (2009) has indicated that there are at least five species of Placospongia in the Caribbean that are morphologically different from the holotypes of P. melobesioides and P. carinata. Rua et al. (2006) and Nichols and Barnes (2005), furthermore, show that there are distinct lineages in the Carib-bean and Western Pacific, that are not shared between the two regions and that most likely represent undescribed species in the Caribbean. Considering these results as well as the large geographic distance between the Caribbean and the type localities of P. melobesioides and P. carinata (both Indo-West Pacific), it is highly unlikely that these species occur in the Tropical Atlantic. Further revision of the Atlantic and Eastern Pacific material will shed more light on this issue.
Future biodiversity surveys and species checklists both in the Atlantic as well as in the Pacific are advised to check the spicule morphology of Placospongia specimens in order to identify species, as the external morphology and color will not give an indication to the number of species. The different Placospongia spp. can occupy the same type of habitats in the tropics. An example of such sympatry is represented in Kakaban lake where in the 4 km 2 area of the marine lake the three common tropical species of Placospongia co-exist side by side. Neglecting to review the spicule morphology would mean possibly missing the true diversity of species that are present in the location of study.