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Six new bisabolane-type phenolic sesquiterpenoids, including plakordiols A−D ( 1−4 ), (7R, 10R)-hydroxycurcudiol ( 5 ) and (7R, 10S)-hydroxycurcudiol ( 6 ) were isolated from the marine sponge Plakortis simplex collected from the South China Sea. Their structures were determined based on extensive analysis of spectroscopic data. Their configurations were assigned by coupling constant analysis, NOESY correlations, and the modified Mosher’s method. Furthermore, their cytotoxic and antibacterial activities were evaluated.

Marine sponges from the Plakinidae family are well known for hosting cytotoxic secondary metabolites and the Brazilian Atlantic coast and its oceanic islands have been considered as a hotspot for the discovery of new Plakinidae species. Herein, we report the chemical profile among cytotoxic extracts obtained from four species of Plakinidae, collected in Fernando de Noronha Archipelago (PE, Northeastern Brazil). Crude organic extracts of Plakinastrella microspiculifera, Plakortis angulospiculatus, Plakortis insularis, and Plakortis petrupaulensis showed strong antiproliferative effects against two different cancer cell lines (HCT-116: 86.7-100%; MCF-7: 74.9-89.5%) at 50 μg/mL, by the MTT assay. However, at a lower concentration (5 μg/mL), high variability in inhibition of cell growth was observed (HCT-116: 17.3-68.7%; MCF-7: 0.00-55.5%), even within two samples of Plakortis insularis which were collected in the west and east sides of the Archipelago. To discriminate the chemical profile, the samples were investigated by UHPLC-HRMS under positive ionization mode. The produced data was uploaded to the Global Natural Products Social Molecular Networking and organized based on spectral similarities for purposes of comparison and annotation. Compounds such as dipeptides, nucleosides and derivatives, polyketides, and thiazine alkaloids were annotated and metabolomic differences were perceived among the species. To the best of our knowledge, this is the first assessment for cytotoxic activity and chemical profiling for Plakinastrella microspiculifera, Plakortis insularis and Plakortis petrupaulensis, revealing other biotechnologically relevant members of the Plakinidae family.

Plakortis bergquistae Muricy, 2011 (Figures 7 A–G) Synonymy: Plakortis bergquistae Muricy, 2011: 311–313, fig. 7. Material examined: 2 specimens, ZSI/ANRC–20445, February 20, 2016, B. Quarry, Great Nicobar Island, Coll. Sudhanshu Dixit; 1 specimen, ZSI/ANRC–20971, July 21, 2018, Rutland Island (site II), South Andaman, Coll. Preeti Pereira. Description: Thickly encrusting sponge forming cushions, 4 cm in length and thickness up to 2 cm; colour of the specimen dark brown (Fig. 7A) when alive and light brown (Fig. 7B) in isopropyl alcohol; surface smooth and irregular; consistency soft and compressible; oscules raised when alive, become flush with the surface on preservation. Skeleton: Ectosome not clearly differentiated from the choanosome; skeleton reticulate, forming irregular/circular meshes, 80–275 µm in diameter (Fig. 7C & 7D). Spicules: Diods and triods; diods abundant, some straight or slightly curved, smooth, thickened at the centre and in two size categories (Fig. 7E & 7F). I: 61.0–104.1–169.7 × 2.1–4.2– 6.5 µm; II: 182.5–240.1–315.1 × 5.8–8.1– 10.7 µm; triods smooth, with all actines of equal length (Fig. 7G); 20.5–40.5–60.0 × 2.4–3.5– 4.8 µm. Distribution: This species is reported herein for the first time in Indian waters from the Andaman and Nicobar Islands. The type locality of this species is north Sulawesi in the West Coral Triangle province and has also been described from Western Australia, Northwest Australian Shelf province (Muricy 2011). The current report extends the distribution of this species to the Andaman province and also the West Indo-Pacific realm. Remarks: Plakortis bergquistae is the third species of the genus Plakortis to be reported from the Andaman and Nicobar Islands. It differs from its congeners, P. badabaluensis Ubare & Mohan, 2016 (originally described from Badabalu of South Andaman) and P. communis Muricy, 2011 in spicule morphology as the former has a single size category of diods (vs. two size categories of diods based on thickness) while the latter has smaller diods and triods in a size range (vs. larger diods and triods)., Published as part of Pereira, Preeti & Raghunathan, Chelladurai, 2020, New records of Indo-Pacific sponges from the Andaman and Nicobar Islands India, pp. 81-97 in Zootaxa 4894 (4) on page 93, DOI: 10.11646/zootaxa.4894.1.4, http://zenodo.org/record/4315490, {"references":["Muricy, G. (2011) Diversity of Indo-Australian Plakortis (Demospongiae: Plakinidae), with description of four new species. Journal of the Marine Biological Association of the United Kingdom, 91 (2), 303 - 319. https: // doi. org / 10.1017 / S 0025315410000743","Ubare, V. V. & Mohan, P. M. (2016) A new species of genus Plakortis Schulze, 1880 (Porifera: Homoscleromorpha) from Badabalu, Andaman and Nicobar Islands, India. Zoological Studies, 55, 2. https: // doi. org / 10.6620 / ZS. 2016.55 - 02"]}

Four new polyhydroxylated steroids plaksterols A-D (1-4), together with two known related steroids ergost-7,9(11),22-trien-3β,5α,6α-triol (5) and ergosta-6β-methoxy-7,22-diene-3β,5α-diol (6), were isolated from methanol extract of the South China Sea marine sponge Plakortis sp. Their structures were identified by spectroscopic analysis, including NMR, MS, and IR. The cytotoxicity of the polyhydroxylated steroids were evaluated, and compound 6 showed moderate inhibitory activities against K562, HL-60 and BEL-7402 cells.

Plakortis ruetzleri sp. nov. (FIg. 11, TAbLE 3) Definition. SMALL, cUSHION-SHAPEd Plakortis WITH dIOdS ANd RARE TRIOdS IN A dISORgANIZEd ARRANgEMENT IN bOTH THE EcTOSOME ANd cHOANOSOME. Type Material. HOLOTyPE: MNRJ 21833 (MQ32-GR-TP9), cOLLEcTEd IN HATUTU ISLANd, MARqUESAS ISLANdS, FRENcH POLyNESIA (7°54.387'S, 140°07.240’W), IN A LARgE OVERHANg WHIcH cONTINUES IN A dARk UNdERWATER cAVE AT 10 M dEPTH. JANUARy 27, 2012, cOLL. THIERRy PÉREZ. PARATyPE: MNRJ 18462 (MQ32-GR-TP9), SAME dATA Of HOLOTyPE. AddITIONAL MATERIAL: MNRJ 21924, MNRJ 21925, SAME dATA Of TyPE SPEcIMENS. Description. SPONgE THINLy ENcRUSTINg TO cUSHION-SHAPEd, 0.5–2.0 cM WIdE by 3–5 MM THIck, WITH SLIgHTLy ROUNdEd bORdERS. COLOUR in vivo ANd AfTER fIXATION A gRAdIENT fROM LIgHT bROWN TO A SLIgHTLy dARkER REddISH bROWN (FIgS. 11A–C). SURfAcE SMOOTH. OScULES SPARSE, cIRcULAR, WITH A TRANSLUcENT RIM, 0.7–1.1 MM IN dIAMETER. CONSISTENcy RELATIVELy SOfT. Anatomy. IN TRANSVERSE SEcTIONS, THE cHOANOSOMAL SkELETON IS dISORgANIZEd ANd RELATIVELy dENSE, WITH dIOdS ANd TRIOdS dISPERSEd THROUgHOUT THE cHOANOSOME (FIg. 11D). EcTOSOMAL SPEcIALIZATION AbSENT. TANgENTIAL EcTOSOMAL SkELETON dISORgANIZEd, WITH RARE VAgUE TRAcTS Of dIOdS fORMINg AN IRREgULAR RETIcULATION AROUNd THE OVOId PORES, WHIcH MEASURE 30–50 µM IN dIAMETER (FIg. 11E). AqUIfEROUS SySTEM LEUcONOId. BOTH EcTOSOMAL ANd bASAL cAVITIES AbSENT. Spicules (TAbLE 3). DIOdS AbUNdANT (FIg. 11F), VARIAbLE IN SIZE ANd SHAPE. THE SMALLER ARE SLENdER, STRAIgHT, SMOOTH, WITH bLUNT ENdS ANd A cONSPIcUOUS, USUALLy S-bENT cENTRAL IRREgULARITy. THE LARgER dIOdS ARE STOUT, IRREgULAR, SMOOTH, WITH AcERATE ENdS ANd cENTRAL IRREgULARITy MOdERATELy dEVELOPEd, MAINLy cENTROTyLOTE-LIkE. FULL SIZE 30– 110.7–150 / 2.5–5.6–7.5 µM. TRIOdS RARE, UNIfORM, Y- OR T-SHAPEd, SMOOTH, WITH AcERATE ENdS (FIg. 11G). AcTINES 12–40–49 / 1.8–2.5–4.0 µM. Reproduction. ROUgHLy SPHERIcAL EMbRyOS, 120–360 µM IN dIAMETER, ARE dISPERSEd IN THE cHOANOSOME (FIg. 11D). Ecology. Plakortis ruetzleri sp. nov. LIVES IN SEMI-dARk cAVES IN SyNTOPy WITH Plakinastrella pseudolopha sp. nov. ANd Plakinastrella nicoleae sp. nov. THIS SPONgE WAS cOLLEcTEd AT 10 M dEPTH ON THE ROOf Of A LARgE OVERHANg dOMINATEd by LARgE bLAck cORAL cOLONIES (PÉREZ et al. 2016). NO SIgN Of EPIbIOSIS OR PREdATION WERE ObSERVEd. Distribution. MARqUESAS ISLANdS, FRENcH POLyNESIA (FIgS. 1, 3). Etymology. THE NAME ruetzleri IS gIVEN IN HONOUR Of DR. KLAUS RüTZLER, fOR HIS PIONEERINg STUdIES Of SPONgES fROM SUbMARINE cAVES ANd fOR HIS OUTSTANdINg LONg-TERM cONTRIbUTION TO THE dEVELOPMENT Of SPONgE TAXONOMy. Taxonomic remarks. THE gENUS Plakortis HAS 29 VALId SPEcIES ANd fEW MORPHOLOgIcAL cHARAcTERS fOR SPEcIES dIScRIMINATION. THE MAIN MORPHOLOgIcAL cHARAcTERISTIcS Of 27 SPEcIES WAS REcENTLy TAbULATEd (UbARE ANd MOHAN 2016), ANd ONLy TWO SPEcIES WERE dEScRIbEd THEREAfTER (VIcENTE et al. 2016). EIgHT SPEcIES ARE dISTINgUISHEd by THE PRESENcE Of MIcRORHAbdS: P. lita dE LAUbENfELS, 1954, P. microrhabdifera MORAES ANd MURIcy, 2003, P. hooperi MURIcy, 2011, P. myrae ERESkOVSky, LAVROV ANd WILLENZ, 2014, P. petrupaulensis DOMINgOS, MORAES ANd MURIcy, 2013, P. spinalis DOMINgOS, MORAES ANd MURIcy, 2013, P. potiguarensis DOMINgOS, MORAES ANd MURIcy, 2013, ANd P. badabaluensis UbARE ANd MOHAN, 2016. ONE SPEcIES HAS qUASIAMPHIASTERS (P. quasiamphiaster DÍAZ ANd VAN SOEST, 1994). TRIOdS ARE AbSENT IN P. nigra LÉVI, 1953, P. zyggompha dE LAUbENfELS, 1934, ANd P. halichondrioides WILSON, 1902. Plakortis bergquistae MURIcy, 2011 HAS LARgER TRIOdS (30–122 vs. 12–49 µM IN THE NEW SPEcIES) ANd dIOdS (TyPE I 91 –163 µM, TyPE II 202–365 µM vs. 54–126 µM IN P. ruetzleri sp. nov.). FOUR SPEcIES ARE dISTINgUISHEd by THE LARgE SIZE Of dIOdS, UP TO 200 µM OR MORE: P. kenyensis PULITZER-FINALI, 1993; P. angulospiculatus (CARTER, 1879), P. japonica (HOSHINO, 1977), ANd P. fromontae MURIcy, 2011. THE REMAININg 12 SPEcIES HAVE dIOdS SHORTER THAN 180 µM ANd TRIOdS UP TO 65 µM (AcTINE LENgTH) LIkE THE NEW SPEcIES. THREE Of THESE HOWEVER HAVE dIOdS IN TWO SIZE cATEgORIES (P. galapagensis DESqUEyROUX-FAúNdEZ ANd VAN SOEST, 1997, P. edwardsi ERESkOVSky, LAVROV ANd WILLENZ, 2014, ANd P. dariae ERESkOVSky, LAVROV ANd WILLENZ, 2014). IN P. clarionensis ANd P. erythraena LÉVI, 1958 THE dIOdS ARE SHORTER THAN 90 µM. Plakortis albicans CRUZ-BARRAZA ANd CARbALLO, 2005 IS WHITE TO IVORy, WITH THIN SURfAcE cANALS. TWO SPEcIES, P. deweerdtaephila VIcENTE, ZEA ANd HILL, 2016 ANd P. symbiotica VIcENTE, ZEA ANd HILL, 2016 HAVE ObLIgATORy ASSOcIATIONS WITH HAPLOScLERId SPONgES. Plakortis ruetzleri sp. nov. IS MOST SIMILAR TO THE REMAININg fOUR SPEcIES Of THE simplex SPEcIES gROUP, WITH bROWN cOLOUR, dIOdS, TRIOdS, ANd WITHOUT MIcRORHAbdS. Plakortis simplex ScHULZE, 1880 cAN bE bROWN, WHITE, yELLOW OR TAN, ANd HAS bOTH EcTOSOMAL ANd bASAL cAVITIES WHIcH ARE LAckINg IN THE NEW SPEcIES. Plakortis insularis MORAES ANd MURIcy, 2003 IS cHOcOLATE TO LIgHT bROWN, RELEASES A dARk EXUdATE WITH STRONg TERPENOId SMELL IN ETHANOL, HAS A fRAgILE cONSISTENcy, EcTOSOME WITH PIgMENTEd cELLS, ANd LOW SPIcULE dENSITy. Plakortis copiosa PULITZER-FINALI, 1993 IS gREy OR bROWN OUTSIdE ANd cREAM INSIdE, WITH HARd OR fRAgILE cONSISTENcy, ANd HAVE PARTIcULARLy SyMMETRIcAL TRIOdS. Plakortis communis MURIcy, 2011 IS gREyISH-bROWN TO bLAck, HAS A TANgENTIAL EcTOSOMAL RETIcULATION WITH cIRcULAR MESHES, ANd A PARTLy ALVEOLAR cHOANOSOMAL SkELETON. THE NEW SPEcIES THEREfORE dIffERS fROM ALL OTHER Plakortis SPEcIES by A UNIqUE cOMbINATION Of cHARAcTERS: bROWN cOLOUR, dISORgANIZEd SkELETON, PRESENcE Of dIOdS ANd TRIOdS, ASyMMETRIcAL SHAPE Of TRIOdS, ANd THE AbSENcE Of MIcRORHAbdS, bASAL ANd EcTOSOMAL cAVITIES, dARk EXUdATES, ANd Of TERPENOId SMELL. ……continued on the next page ……continued on the next page ……continued on the next page GBR, Great Barrier Reef. *, type localities. **, identification probably inaccurate, records needs revision.

Genus Plakortis Schulze, 1880 Definition. PLAkINIdAE ALWAyS WITH dIOdS, ANd WITH TRIOdS IN VARIAbLE AbUNdANcE OR AbSENT. CALTHROPS ARE ALWAyS AbSENT. MIcRORHAbdS, SPHERES ANd SPINEd dIOdS (qUASIAMPHIASTERS) MAy bE PRESENT IN SOME SPEcIES (AMENdEd fROM MURIcy 2011). Type species: Plakortis simplex ScHULZE, 1880., Published as part of Lage, Ana��ra, Muricy, Guilherme, Ruiz, C��sar & P��rez, Thierry, 2018, New sciaphilic plakinids (Porifera, Homoscleromorpha) from the Central-Western Pacific, pp. 8-38 in Zootaxa 4466 (1) on page 24, DOI: 10.11646/zootaxa.4466.1.4, http://zenodo.org/record/1454345, {"references":["Schulze, F. E. (1880) Untersuchungen uber den Bau und die Entwicklung der Spongien. Neuten Mittheilung. Die Plakiniden. Zeitschrift fur wissenschaftliche Zoologie, 34, 407 - 451.","Muricy, G. (2011) Diversity of Indo-Australian Plakortis (Demospongiae: Plakinidae), with description of four new species. Journal of the Marine Biological Association of the United Kingdom, 91 (2), 303 - 319. https: // doi. org / 10.1017 / S 0025315410000743"]}

The sponge class Homoscleromorpha is a key model for the evolutionary biology of the Metazoa but its diversity remains poorly known. Here we describe six new species of the homoscleromorph family Plakinidae found in shaded habitats (submarine caves, tunnels and overhangs) of New Caledonia and Marquesas Islands, Central-Western Pacific. The new species belong to four genera: Corticium (Corticium vaceleti sp. nov.), Plakina (Plakina finispinata sp. nov.), Plakinastrella (Plakinastrella osculifera sp. nov., Plakinastrella nicoleae sp. nov. and Plakinastrella pseudolopha sp. nov.), and Plakortis (Plakortis ruetzleri sp. nov.). Plakinastrella pseudolopha sp. nov. has a novel spicule type called here ‘pseudolophose spicules’. The diversity of Homoscleromorpha is raised to 50 species in the Pacific Ocean and 120 spp. worldwide.

Plakortis ruetzleri sp. nov. (FIg. 11, TAbLE 3) Definition. SMALL, cUSHION-SHAPEd Plakortis WITH dIOdS ANd RARE TRIOdS IN A dISORgANIZEd ARRANgEMENT IN bOTH THE EcTOSOME ANd cHOANOSOME. Type Material. HOLOTyPE: MNRJ 21833 (MQ32-GR-TP9), cOLLEcTEd IN HATUTU ISLANd, MARqUESAS ISLANdS, FRENcH POLyNESIA (7��54.387'S, 140��07.240���W), IN A LARgE OVERHANg WHIcH cONTINUES IN A dARk UNdERWATER cAVE AT 10 M dEPTH. JANUARy 27, 2012, cOLL. THIERRy P��REZ. PARATyPE: MNRJ 18462 (MQ32-GR-TP9), SAME dATA Of HOLOTyPE. AddITIONAL MATERIAL: MNRJ 21924, MNRJ 21925, SAME dATA Of TyPE SPEcIMENS. Description. SPONgE THINLy ENcRUSTINg TO cUSHION-SHAPEd, 0.5���2.0 cM WIdE by 3���5 MM THIck, WITH SLIgHTLy ROUNdEd bORdERS. COLOUR in vivo ANd AfTER fIXATION A gRAdIENT fROM LIgHT bROWN TO A SLIgHTLy dARkER REddISH bROWN (FIgS. 11A���C). SURfAcE SMOOTH. OScULES SPARSE, cIRcULAR, WITH A TRANSLUcENT RIM, 0.7���1.1 MM IN dIAMETER. CONSISTENcy RELATIVELy SOfT. Anatomy. IN TRANSVERSE SEcTIONS, THE cHOANOSOMAL SkELETON IS dISORgANIZEd ANd RELATIVELy dENSE, WITH dIOdS ANd TRIOdS dISPERSEd THROUgHOUT THE cHOANOSOME (FIg. 11D). EcTOSOMAL SPEcIALIZATION AbSENT. TANgENTIAL EcTOSOMAL SkELETON dISORgANIZEd, WITH RARE VAgUE TRAcTS Of dIOdS fORMINg AN IRREgULAR RETIcULATION AROUNd THE OVOId PORES, WHIcH MEASURE 30���50 ��M IN dIAMETER (FIg. 11E). AqUIfEROUS SySTEM LEUcONOId. BOTH EcTOSOMAL ANd bASAL cAVITIES AbSENT. Spicules (TAbLE 3). DIOdS AbUNdANT (FIg. 11F), VARIAbLE IN SIZE ANd SHAPE. THE SMALLER ARE SLENdER, STRAIgHT, SMOOTH, WITH bLUNT ENdS ANd A cONSPIcUOUS, USUALLy S-bENT cENTRAL IRREgULARITy. THE LARgER dIOdS ARE STOUT, IRREgULAR, SMOOTH, WITH AcERATE ENdS ANd cENTRAL IRREgULARITy MOdERATELy dEVELOPEd, MAINLy cENTROTyLOTE-LIkE. FULL SIZE 30��� 110.7���150 / 2.5���5.6���7.5 ��M. TRIOdS RARE, UNIfORM, Y- OR T-SHAPEd, SMOOTH, WITH AcERATE ENdS (FIg. 11G). AcTINES 12���40���49 / 1.8���2.5���4.0 ��M. Reproduction. ROUgHLy SPHERIcAL EMbRyOS, 120���360 ��M IN dIAMETER, ARE dISPERSEd IN THE cHOANOSOME (FIg. 11D). Ecology. Plakortis ruetzleri sp. nov. LIVES IN SEMI-dARk cAVES IN SyNTOPy WITH Plakinastrella pseudolopha sp. nov. ANd Plakinastrella nicoleae sp. nov. THIS SPONgE WAS cOLLEcTEd AT 10 M dEPTH ON THE ROOf Of A LARgE OVERHANg dOMINATEd by LARgE bLAck cORAL cOLONIES (P��REZ et al. 2016). NO SIgN Of EPIbIOSIS OR PREdATION WERE ObSERVEd. Distribution. MARqUESAS ISLANdS, FRENcH POLyNESIA (FIgS. 1, 3). Etymology. THE NAME ruetzleri IS gIVEN IN HONOUR Of DR. KLAUS R��TZLER, fOR HIS PIONEERINg STUdIES Of SPONgES fROM SUbMARINE cAVES ANd fOR HIS OUTSTANdINg LONg-TERM cONTRIbUTION TO THE dEVELOPMENT Of SPONgE TAXONOMy. Taxonomic remarks. THE gENUS Plakortis HAS 29 VALId SPEcIES ANd fEW MORPHOLOgIcAL cHARAcTERS fOR SPEcIES dIScRIMINATION. THE MAIN MORPHOLOgIcAL cHARAcTERISTIcS Of 27 SPEcIES WAS REcENTLy TAbULATEd (UbARE ANd MOHAN 2016), ANd ONLy TWO SPEcIES WERE dEScRIbEd THEREAfTER (VIcENTE et al. 2016). EIgHT SPEcIES ARE dISTINgUISHEd by THE PRESENcE Of MIcRORHAbdS: P. lita dE LAUbENfELS, 1954, P. microrhabdifera MORAES ANd MURIcy, 2003, P. hooperi MURIcy, 2011, P. myrae ERESkOVSky, LAVROV ANd WILLENZ, 2014, P. petrupaulensis DOMINgOS, MORAES ANd MURIcy, 2013, P. spinalis DOMINgOS, MORAES ANd MURIcy, 2013, P. potiguarensis DOMINgOS, MORAES ANd MURIcy, 2013, ANd P. badabaluensis UbARE ANd MOHAN, 2016. ONE SPEcIES HAS qUASIAMPHIASTERS (P. quasiamphiaster D��AZ ANd VAN SOEST, 1994). TRIOdS ARE AbSENT IN P. nigra L��VI, 1953, P. zyggompha dE LAUbENfELS, 1934, ANd P. halichondrioides WILSON, 1902. Plakortis bergquistae MURIcy, 2011 HAS LARgER TRIOdS (30���122 vs. 12���49 ��M IN THE NEW SPEcIES) ANd dIOdS (TyPE I 91 ���163 ��M, TyPE II 202���365 ��M vs. 54���126 ��M IN P. ruetzleri sp. nov.). FOUR SPEcIES ARE dISTINgUISHEd by THE LARgE SIZE Of dIOdS, UP TO 200 ��M OR MORE: P. kenyensis PULITZER-FINALI, 1993; P. angulospiculatus (CARTER, 1879), P. japonica (HOSHINO, 1977), ANd P. fromontae MURIcy, 2011. THE REMAININg 12 SPEcIES HAVE dIOdS SHORTER THAN 180 ��M ANd TRIOdS UP TO 65 ��M (AcTINE LENgTH) LIkE THE NEW SPEcIES. THREE Of THESE HOWEVER HAVE dIOdS IN TWO SIZE cATEgORIES (P. galapagensis DESqUEyROUX-FA��NdEZ ANd VAN SOEST, 1997, P. edwardsi ERESkOVSky, LAVROV ANd WILLENZ, 2014, ANd P. dariae ERESkOVSky, LAVROV ANd WILLENZ, 2014). IN P. clarionensis ANd P. erythraena L��VI, 1958 THE dIOdS ARE SHORTER THAN 90 ��M. Plakortis albicans CRUZ-BARRAZA ANd CARbALLO, 2005 IS WHITE TO IVORy, WITH THIN SURfAcE cANALS. TWO SPEcIES, P. deweerdtaephila VIcENTE, ZEA ANd HILL, 2016 ANd P. symbiotica VIcENTE, ZEA ANd HILL, 2016 HAVE ObLIgATORy ASSOcIATIONS WITH HAPLOScLERId SPONgES. Plakortis ruetzleri sp. nov. IS MOST SIMILAR TO THE REMAININg fOUR SPEcIES Of THE simplex SPEcIES gROUP, WITH bROWN cOLOUR, dIOdS, TRIOdS, ANd WITHOUT MIcRORHAbdS. Plakortis simplex ScHULZE, 1880 cAN bE bROWN, WHITE, yELLOW OR TAN, ANd HAS bOTH EcTOSOMAL ANd bASAL cAVITIES WHIcH ARE LAckINg IN THE NEW SPEcIES. Plakortis insularis MORAES ANd MURIcy, 2003 IS cHOcOLATE TO LIgHT bROWN, RELEASES A dARk EXUdATE WITH STRONg TERPENOId SMELL IN ETHANOL, HAS A fRAgILE cONSISTENcy, EcTOSOME WITH PIgMENTEd cELLS, ANd LOW SPIcULE dENSITy. Plakortis copiosa PULITZER-FINALI, 1993 IS gREy OR bROWN OUTSIdE ANd cREAM INSIdE, WITH HARd OR fRAgILE cONSISTENcy, ANd HAVE PARTIcULARLy SyMMETRIcAL TRIOdS. Plakortis communis MURIcy, 2011 IS gREyISH-bROWN TO bLAck, HAS A TANgENTIAL EcTOSOMAL RETIcULATION WITH cIRcULAR MESHES, ANd A PARTLy ALVEOLAR cHOANOSOMAL SkELETON. THE NEW SPEcIES THEREfORE dIffERS fROM ALL OTHER Plakortis SPEcIES by A UNIqUE cOMbINATION Of cHARAcTERS: bROWN cOLOUR, dISORgANIZEd SkELETON, PRESENcE Of dIOdS ANd TRIOdS, ASyMMETRIcAL SHAPE Of TRIOdS, ANd THE AbSENcE Of MIcRORHAbdS, bASAL ANd EcTOSOMAL cAVITIES, dARk EXUdATES, ANd Of TERPENOId SMELL. ������continued on the next page ������continued on the next page ������continued on the next page GBR, Great Barrier Reef. *, type localities. **, identification probably inaccurate, records needs revision., Published as part of Lage, Ana��ra, Muricy, Guilherme, Ruiz, C��sar & P��rez, Thierry, 2018, New sciaphilic plakinids (Porifera, Homoscleromorpha) from the Central-Western Pacific, pp. 8-38 in Zootaxa 4466 (1) on pages 25-30, DOI: 10.11646/zootaxa.4466.1.4, http://zenodo.org/record/1454345, {"references":["Ubare, V. V. & Mohan, P. M. (2016) A new species of genus Plakortis Schulze 1880 (Porifera: Homoscleromorpha) from Badabalu, Andaman and Nicobar Islands, India. Zoological Studies, 55 (2), 1 - 9.","Moraes, F. C. & Muricy, G. (2003) Taxonomy of Plakortis and Plakinastrella (Demospongiae: Plakinidae) from oceanic islands off north-eastern Brazil, with description of three new species. Journal of the Marine Biological Association of the United Kingdom, 83 (2), 385 - 397. https: // doi. org / 10.1017 / S 0025315403007239 h","Muricy, G. (2011) Diversity of Indo-Australian Plakortis (Demospongiae: Plakinidae), with description of four new species. Journal of the Marine Biological Association of the United Kingdom, 91 (2), 303 - 319. https: // doi. org / 10.1017 / S 0025315410000743","Ereskovsky, A. V., Lavrov, D. V. & Willenz, P. (2014) Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis. Journal of the Marine Biological Association of the United Kingdom, 94 (02), 285 - 307. https: // doi. org / 10.1017 / S 0025315413000295","Domingos, C., Moraes, F. & Muricy, G. (2013) Four new species of Plakinidae (Porifera: Homoscleromorpha) from Brazil. Zootaxa, 3718 (6), 530 - 544. https: // doi. org / 10.11646 / zootaxa. 3718.6.2","Diaz, M. C. & Soest, R. W. M. van (1994) The Plakinidae: A systematic review. In: Soest, R. W. M. van, Kempen, Th. M. G. van & Braekman, J. - C. (Eds.), Sponges in Time and Space. Balkema, Rotterdam, pp. 93 - 109.","Levi, C. (1953) Description de Plakortis nigra nov. sp. et remarques sur les Plakinidae (Demosponges). Bulletin du Museum national d'Histoire naturelle, Series 2, 25 (3), 320 - 328.","Laubenfels, M. W. de (1934) New sponges from the Puerto Rican deep. Smithsonian Miscellaneous Collections, 91 (17), 1 - 28.","Wilson, H. V. (1902 [1900]) The sponges collected in Porto Rico in 1899 by the U. S. Fish Commission Steamer Fish Hawk. Bulletin of the United States Fish Commission, 2, 375 - 411.","Pulitzer-Finali, G. (1993) A collection of marine sponges from East Africa. Annali del Museo Civico di Storia Naturale \" Giacomo Doria \", 89, 247 - 350.","Carter, H. J. (1879) Contributions to our knowledge of the Spongida. Annals and Magazine of Natural History, Series 5, 3, 284 - 304, 343 - 360. https: // doi. org / 10.1080 / 00222937908694101","Hoshino, T. (1977) Demosponges from the Kii channel and its environs, western Japan. Proceedings of the Japanese Society of Systematic Zoology, 13, 5 - 15.","Desqueyroux-Faundez, R. & Soest, R. W. M. van (1997) Shallow water Demosponges of the Galapagos Islands. Revue Suisse de Zoologie, 104 (2), 379 - 467. https: // doi. org / 10.5962 / bhl. part. 80003","Levi, C. (1958) Resultats scientifiques des campagnes de la ' Calypso'. Campagne 1951 - 1952 en Mer Rouge (suite). 11. Spongiaires de Mer Rouge recueillis par la ' Calypso' (1951 - 1952). Annales de l'Institut oceanographique, 34 (3), 3 - 46.","Cruz-Barraza, J. A. & Carballo, J. L. (2005) First record of Plakortis Schulze (Porifera: Homosclerophorida) from the Northeast Pacific coast, with the description of Plakortis albicans sp. nov. Zootaxa, 868 (1), 1 - 12. https: // doi. org / 10.11646 / zootaxa. 868.1.1","Vicente, J., Zea, S. & Hill, R. T. (2016) Sponge epizoism in the Caribbean and the discovery of new Plakortis and Haliclona species, and polymorphism of Xestospongia deweerdtae (Porifera). Zootaxa, 4178 (2), 209 - 233. https: // doi. org / 10.11646 / zootaxa. 4178.2.3","Schulze, F. E. (1880) Untersuchungen uber den Bau und die Entwicklung der Spongien. Neuten Mittheilung. Die Plakiniden. Zeitschrift fur wissenschaftliche Zoologie, 34, 407 - 451."]}

Cryptococcus gattii is a human pathogen and causative agent of a pernicious, sometimes fatal, disseminated fungal disease. Investigation of antifungal extracts of the marine sponge association Plakortis halichondrioides-Xestospongia deweerdtae and the sponge Plakortis zyggompha from the Bahamas led to the discovery and isolation of 6-epi-7,8-dihydroplakortide K (1), plakortide AA (2), and three new plakinic acids, N-P (4-6; unstable 1,2-dioxolanes bearing benzyl-substituted conjugated dienes), along with known plakinic acids L, K, and M.5 Chiroptical comparisons and DFT calculations of (13)C NMR chemical shifts were used to assign the absolute stereostructure of 4. The stereospecific base-promoted rearrangement-saponification of 1 to 10 was briefly investigated and showed tight kinetic control and stereospecific formation of the new C-2 stereocenter with inversion at C-3. Plakinic acid M and plakortides 9 and 11 exhibited antifungal activity against C. gattii (MIC90 = 2.4 to 36 μM), but plakinic acids N-P were inactive under the same conditions.

Three new plakortides, 7,8-dihydroplakortide E (1), 2, and 10, along with known natural products 3, 4, spongosoritin A (5), 6-8, and plakortide P (9), were isolated from Brazilian specimens of Plakortis angulospiculatus. Compounds 2, 3, 5, and 7-9 displayed cytotoxic activities with IC50 values ranging from 0.2 to 10 μM. Compounds that contained a dihydrofuran ring were generally less active and displayed time dependence in their activity. The activities of compounds 2 and 7-9, carboxylic acids bearing a common six-membered endoperoxide, were higher overall than for compounds 3 and 5. The modes underlying the cytotoxic actions of plakortides 2, 3, 5, 7, and 9 were further investigated using HCT-116 cells. While dihydrofurans 3 and 5 induce a G0/G1 arrest, six-membered peroxides 2, 7, and 9 delivered a G2/M arrest and an accumulation of mitotic figures, indicating a distinctly different antimitotic response. Confocal analysis indicated that microtubules were not altered after treatment with 2, 7, or 9, therein suggesting that the mitotic arrest may be unrelated to cytoskeletal targets. Overall, we find that two related classes of natural products obtained from the same extract offer cytostatic activity, yet they do so through discrete pathways.

Five new polyketide endoperoxides, manadodioxans A−E, were isolated from the marine sponge Plakortis bergquistae. Manadodioxan E showed antimicrobial activity against Escherichia coli at 10 μg/disk, while its oxo congener, manadodioxan D, was inactive.

Plakortis insularis Moraes & Muricy, 2003 Figures 122 a–c Plakortis insularis Moraes & Muricy, 2003: 389, fig. 5; Moraes 2011: 68, several unnumbered figs. Material examined. RMNH Por. 9852, 9917, Suriname, ‘ Luymes O.C.P.S. II’ Guyana Shelf Expedition, station M97, 7.3083°N 54.1667°W, depth 130 m, bottom coarse sand, 16 April 1969; RMNH Por. 9870, Suriname, ‘ Snellius O.C.P.S. ’ Guyana Shelf Expedition, station F45, 6.4417°N 56.5467°W, depth 34 m, Van Veen grab, 7 May 1966. Description. (Fig. 122 a) Lobate masses with smooth surface. Size of largest specimen 7 x 3 x 3 cm. Color (in alcohol) orange-brown. Consistency firm, corky. Skeleton. At the surface there are rounded openings of 100–150 µm surrounded and subdivided into smaller openings by groups of perpendicular diods. Choanosome alveolar, with meshes of 30–40 µm diameter. Spicules. (Figs 122 b–c) Diods, triods. Diods (Figs 122 b), in a large size range, many are curved; they are possibly divisible in (1) larger 81–129 µm, and (2) smaller 11–28 µm spicules; overall sizes 11– 55 –129 x 0.5– 2.4 –6.5 µm. Triods (Figs 122 c), mostly equiactinal, actines 10– 31.9 – 38 x 1.5– 3.4 –4.5 µm (‘cladomes’ 22–61 µm). Distribution and ecology. Guyana Shelf, NE Brazil, 1–130 m depth (previously 1–12 m). Remarks. Of all the Plakortis simplex -like Central West Atlantic species, the specimens match closest with P. insularis. Using the key in Ereskovsky et al. (2014), likewise P. insularis comes out at the most likely match. The ectosome of my specimens is distinctly reticulate, which distinguishes it from P. edwardsi Ereskovsky, Lavrov & Willenz, 2014. The diods are very variable in size, but do not seem to be readily divisible in two distinct size categories such as found in P. dariae Ereskovsky, Lavrov & Willenz, 2014. From P. zyggompha (De Laubenfels, 1936) the present specimens differ in having larger diods (these are only up to 59 µm in the type of P. zyggompha). All these species are very similar and further research is necessary to establish whether they are all valid species., Published as part of Van, Rob W. M., 2017, Sponges of the Guyana Shelf, pp. 1-225 in Zootaxa 1 on pages 193-194, DOI: 10.5281/zenodo.272951, {"references":["Moraes, F. C. & Muricy, G. (2003) Taxonomy of Plakortis and Plakinastrella (Demospongiae: Plakinidae) from oceanic islands off north-eastern Brazil, with description of three new species. Journal of the Marine Biological Association of the United Kingdom, 83 (2), 385 - 397.","Ereskovsky, A. V., Lavrov, D. V. & Willenz, P. (2014) Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis. Journal of the Marine Biological Association of the United Kingdom, 94 (2), 285 - 307.","De Laubenfels, M. W. (1936) A discussion of the sponge fauna of the Dry Tortugas, in particular, and the West Indies in general, with material for a revision of the families and orders of the Porifera. Carnegie Institution of Washington Publication, 467 (Tortugas Laboratory Paper, 30, 1 - 225."]}

Plakortis angulospiculatus (Carter, 1879) Figures 121 a–d Suberites angulospiculatus Carter, 1879a: 346, pl. XXVIII figs 8a–b. Plakortis angulospiculatus; Van Soest 1981: 3; Zea 1987: 222, fig. 81, Pl. 13 figs 6–7; Moraes & Muricy 2003: 386, fig. 2; Rützler et al. 2014: 16. Material examined. RMNH Por. 9918, Suriname, ‘ Luymes O.C.P.S. II’ Guyana Shelf Expedition, station M97, 7.3083°N 54.1667°W, depth 130 m, bottom coarse sand, 16 April 1969. Description. (Fig. 121 a) Pale beige (in alcohol), massive fragments, size of largest 5 x 3 x 2 cm. The fragments are overgrown by a crust with digitations of Haliclona (Gellius) aff. flagellifera, which likely affected color and surface characteristics. No apparent oscules. Consistency firm. Skeleton. Surface skeleton consists of a tangential crust pierced by regularly distributed pores, 80–250 µm in diameter. Spicules. (Figs 121 b–d) Diods, triods. Diods robust, in a large size variation (Figs 121 b–c), but no clear size categories, 57– 134 –202 x 2 – 5.1 – 8 m. Triods (Fig. 121 d), both equiangular and Y-shaped forms, actines 21– 44.1 – 72 x 1 – 3.4 –5 µm (‘cladomes’ 39– 69.5 –123 µm). Distribution and ecology. Guyana Shelf, Jamaica, Curaçao, Colombia, Belize, from shallow water down to 130 m. Remarks. In the key to the Central West Atlantic Plakortis species provided by Ereskovsky et al. 2014 (pp. 303–304) the present specimen keys out as Plakortis angulospiculatus (Carter, 1879a). Few other species of Plakortis have diods larger than 200 µm as found in my specimen. Several more species have been described from NE Brazil (Domingos et al. 2013), but all these have distinctly smaller diods. Brazilian records of Plakortis angulospiculatus (cf. Muricy et al. 2011) may belong to one of these new species, at least it is likely that not all belong to P. angulospiculatus, so their presence is here left undecided. We noticed a few small spheres (7–10 µm diameter), the significance of which is unclear. Muricy (2011) was the first to report the presence of these spicules in Australian Plakortis species, and subsequently also found them in Brazilian species (Domingos et al. 2013). It remains to be decided if the presence of these, usually uncommon, spheres has taxonomic significance, as there is a distinct possibility that they are a product of high environmental silica content, or more simple spicular reduction (in this case reduced diods)., Published as part of Van, Rob W. M., 2017, Sponges of the Guyana Shelf, pp. 1-225 in Zootaxa 1 on pages 192-193, DOI: 10.5281/zenodo.272951, {"references":["Carter, H. J. (1879 a) Contributions to our knowledge of the Spongida. Annals and Magazine of Natural History, (5) 3, 284 - 304, 343 - 360.","Zea, S. (1987) Esponjas del Caribe Colombiano. Catalogo Cientific, Bogot, Colombia, 286 pp.","Moraes, F. C. & Muricy, G. (2003) Taxonomy of Plakortis and Plakinastrella (Demospongiae: Plakinidae) from oceanic islands off north-eastern Brazil, with description of three new species. Journal of the Marine Biological Association of the United Kingdom, 83 (2), 385 - 397.","Rutzler, K., Piantoni, C., Van Soest, R. W. M. & Diaz, M. C. (2014) Diversity of sponges (Porifera) from cryptic habitats on the Belize barrier reef near Carrie Bow Cay. Zootaxa, 3805 (1), 1 - 129. http: // dx. doi. org / 10.11646 / zootaxa. 3805.1.1","Ereskovsky, A. V., Lavrov, D. V. & Willenz, P. (2014) Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis. Journal of the Marine Biological Association of the United Kingdom, 94 (2), 285 - 307."]}

Six new cyclic peroxides (1–6) were isolated from the Korean sponge Plakortis simplex, along with two new alkylpyridinium alkaloids (7 and 8). The structures of these compounds were completely determined by a combination of NMR analysis and chemical reactions. Compounds 1–6 exhibited cytotoxic/antifungal activities against RAW264.7 cells and Candida albicans.

Five new species of Homoscleromorpha (Porifera) of four genera,Oscarella,Plakortis,PlakinaandCorticium, are described from vertical walls of reef caves at depths ranging from 23 to 28 m in the Caribbean Sea.Oscarella nathaliaesp. nov. has a leaf-like thinly encrusting, flat body, loosely attached to the substrate and a perforated, not lobate surface.Oscarella nathaliaesp. nov. contains two bacterial morphotypes and is characterized by two mesohylar cell types with inclusions.Plakortis myraesp. nov. has diods of two categories: abundant large ones (83–119 μm long) and rare small ones (67–71 μm long) with sinuous, S-bent centres; triods Y- or T-shaped (18–5 μm long), and abundant microrhabds (5–12 μm long).Plakortis edwardsisp. nov. has diods of one category with thick, sinuous, S-bent centres (110 to 128 μm long); triods T-shaped (actines 28–59 μm long). It is the only species of this genus showing small diods (22–31 μm long).Plakortis dariaesp. nov. has diods of two categories: large ones (67–112 μm long) and small, rare, irregular ones, slightly curved, often deformed with one end blunt (30–59 μm long); triods rare and regular (actines 20–44 μm long).Corticium diamantensesp. nov. has oscula situated near its border, regular non-lophose calthrops of one size-class, very rare tetralophose calthrops and candelabra with the fourth actine ramified basally in 4–5 microspined rays. In addition, a re-description ofPlakina jamaicensisis based on newly collected material and the type specimen.Plakortis jamaicensishas a convoluted brain-like surface; well developed sub-ectosomal cavities; irregular sinuous diods, triods, calthrops, rare monolophose calthrops, rare dilophose calthrops, rare trilophose calthrops and common tetralophose calthrops. Molecular ‘barcoding’ sequences for mitochondrial cob are given forPlakortis edwardsisp. nov.,P. dariaesp. nov.,Plakina jamaicensisandCorticium diamantensesp. nov. An identification key for all western Atlantic Homoscleromorpha is provided.

Plakortinic acids A (2) and B (3), two polyketide endoperoxides with a bicyclo[4.2.0]octene unit, were isolated as minor constituents from the sponge–sponge symbiotic association Plakortis halichondrioides–Xestospongia deweerdtae, along with known epiplakinic acid F (1). The structures of the mixture of two inseparable compounds were determined by spectroscopic analysis. Screening for cytotoxic activity of the mixture against two human tumor cell lines revealed that these compounds are very active at sub-micro molar concentration.

There is an urgent need for novel strategies to fight drug resistance and multi-drug resistance. As an alternative to the classic antibiotic therapy, attenuation of the bacteria virulence affecting their Quorum sensing (QS) system is a promising approach. Quorum sensing (QS) is a genetic regulation system that allows bacteria to communicate with each other and coordinate group behaviors. A new γ-lactone that is capable of inhibiting the LasI/R QS system, plakofuranolactone (1), was discovered in the extract of the marine sponge Plakortis cf. lita, and its structure, including absolute configuration, was determined by NMR spectroscopy, MS spectrometry, and quantum-mechanical prediction of optical rotation. The quorum quenching activity of plakofuranolactone was evaluated using reporter gene assays for long- and short-chain signals (E. coli pSB1075, E. coli pSB401, and C. violeaceum CV026) and was confirmed by measuring the total protease activity (a virulence factor which is under control of the LasI/R system) of the wild-type P. aeruginosa PAO1. Further research will be pursued to assess the potential of plakofuranolactone as a new antivirulence lead compound and a chemical tool to increase the knowledge in this field.

The recently described epizoic sponge-sponge symbioses between Xestospongia deweerdtae and two species of Plakortis present an unusual series of sponge interactions. Sponges from the genus Plakortis are fierce allelopathic competitors, rich in cytotoxic secondary metabolites, and yet X. deweerdtae flourishes as an epizoic encrustation on Plakortis deweerdtaephila and Plakortis symbiotica. Our objective in this study was to evaluate the hypothesis that X. deweerdtae grows epizoic to these two species of Plakortis due to a shared chemical defense against predators. We collected free-living individuals of X. deweerdtae and symbiotic pairs from a wide geographical range to generate crude organic extracts and a series of polarity fractions from sponge extract. We tested the deterrency of these extracts against three common coral reef predators: the bluehead wrasse, Thalassoma bifasciatum, the Caribbean sharpnose puffer, Canthigaster rostrata, and the white spotwrist hermit crab, Pagurus criniticornis. While the chemical defenses of P. deweerdtaephila and P. symbiotica are more potent than those of X. deweerdtae, all of the sponge species we tested significantly deterred feeding in all three generalist predators. The free-living form of X. deweerdtae is mostly defended across the region, with a few exceptions. The associated form of X. deweerdtae is always defended, and both species of Plakortis are very strongly defended, with puffers refusing to consume extract-treated pellets until the extract was diluted to 1/256× concentration. Using diode-array high performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (LC-MS/IT-TOF), we found two secondary metabolites from P. deweerdtaephila, probably the cyclic endoperoxides plakinic acid I and plakinic acid K, in low concentrations in the associated-but not the free-living-form of X. deweerdtae, suggesting a possible translocation of defensive chemicals from the basibiont to the epibiont. Comparing the immense deterrency of Plakortis spp. extracts to the extracts of X. deweerdtae gives the impression that there may be some sharing of chemical defenses: one partner in the symbiosis is clearly more defended than the other and a small amount of its defensive chemistry may translocate to the partner. However, X. deweerdtae effectively deters predators with its own defensive chemistry. Multiple lines of evidence provide no support for the shared chemical defense hypothesis. Given the diversity of other potential food resources available to predators on coral reefs, it is improbable that the evolution of these specialized sponge-sponge symbioses has been driven by predation pressure.

Plakortis deweerdtaephila sp. nov. (Fig. 2; Table 1) Plakortis halichondrioides; Zea et al. 2009 (photographic guide). Non: Plakortis halichondrioides (Wilson 1902), a valid species. Plakortis sp. 1; Vicente et al. 2014 (ecology and symbiosis). Plakortis sp. 1-���under Xestospongia deweerdtae associated���; Zea et al. 2014 (photographic guide). Type material. Holotype and type locality: USNM 12 54645, Dolphin Rock, Bocas del Toro, Panama (9.35076�� N, - 82.1863�� W), 14 m depth, coll. Jan Vicente, May 20, 2015. Paratype: USNM 1254647, San Salvador, Bahamas (24.0406 �� N, - 74.5314�� W), 32 m depth, coll. Jan Vicente, July 19, 2011. Specimens examined for comparison (other than those described here). Plakortis halichondrioides: PHBH, San Salvador (24.0406 �� N, - 74.5314�� W), Bahamas, 32 m coll. Jan Vicente, July 19, 2011; PHPR, La Parguera, Puerto Rico (17.8883�� N, - 66.9981�� W), 32 m depth coll. Jan Vicente, June 12, 2012. Diagnosis. Thinly and thickly encrusting to massive cushions with a soft surface and compressible body. Found always associated as basibiont of X. deweerdtae. The latter may partially cover (Bahamas) or may completely overgrow P. deweerdtaephila sp. nov. (Panama). Oscules can be large and slightly elevated (Bahamas) or can be small and even with the surface when growing underneath X. deweerdtae (Panama). Color is dark brown with occasional olive green patches in vivo and exudes a light brown pigment when preserved in ethanol. Reticulated tangential ectosomal skeleton and a vaguely reticulated choanosomal skeleton with lacunae. Spicules are triods, diods and very small diods. Description. External morphology is influenced by the growth progression of X. deweerdtae on the body of Plakortis deweerdtaephila sp. nov. For example, in Panama it can be thinly encrusting (Fig. 2 A) growing underneath a thick (1 cm) mat of X. deweerdtae; very small oscules (1���3 mm). In the Bahamas, sponge pairs form 3 �� 30 cm by 1���8 cm thick compressible cushions. X. deweerdtae may overgrow the entire P. deweerdtaephila individual except around the elevated oscules, which are 0.2���0.9 cm in diameter (Fig. 2 B). Oscules in preserved specimens are contracted. External color is dark brown and internal color is light brown. Surface is smooth, soft and irregular. Consistency is compressible, and easily torn. Skeleton. Ectosome is composed of a disorganized tangential reticulation of diods and triods. Multispicular tracts are not well defined but form circular meshes, 114��� 205 ���329 ��m diameter (n=20; Fig. 2 C). Spicules never break the surface of the ectosome. When X. deweerdtae forms inner channels within the choanosome of P. deweerdtaephila sp. nov. the ectosome forms a barrier between the two sponge species, as observed in Vicente et al. (2014, their Fig. 7 B). The ectosome (30���50 ��m thick) can be easily distinguished from the choanosome with an abundance of subectosomal lacunae and by having a denser aggregation of pigmented cells (Fig. 2 D). The choanosome is dense with a confused reticulation of diods and triods that form circular meshes of varying diameters (Fig. 2 E). Spicules. Diods can be very small to large. Large diods are slightly bent but mostly straight, slightly sinuous, with a thick center. Ends of large diods are sharp and sometimes bent (Fig. 2 F���G): Small diods are rare, also thicker in the center and mostly straight. Small diods in Panama (Fig. 2 F) can be as small as 50 ��m and in the Bahamas (Fig. 2 H) can be as small as 24 ��m. Size (length �� width) for Panama, 50��� 173.2 (��37.1)���234 ��m �� 4.3��� 7.9 (��1.7)��� 11.0 ��m; Bahamas, 24��� 107.6 (��43.4)���172 ��m x 2.4��� 3.7 (��0.7)���4.8 ��m (Table 1). Triods are not abundant, being Yshaped, smooth, and with sharp endings that are sometimes bent (Fig. 2 I) Size for Panama, 40��� 64.2 (��15.8)���103 ��m �� 1.8��� 5.9 (��2.2)���10.9 ��m; Bahamas, 26��� 45.1 (��11.3)���67 ��m �� 2.4��� 3.3 (��0.5)���4.8 (Table 1). Microrhabds, quasiamphiasters and spheres are absent. Spicule Specimen Location* Length (��m) Width (��m) Diod Plakortis deweerdtaephila USNM 1254645 (h) BDT, Panama 50��� 173.2 (��37.1)���234 4��� 7.9 (��1.7)���11 Plakortis deweerdtaephila USNM 1254647 (p) SS, Bahamas 24��� 107.6 (��43.4)���172 2��� 3.7 (��0.7)���5 Plakortis symbiotica USNM 1254650 (h) LP, Puerto Rico 72��� 113.1 (��16.7)���142 2��� 3.6 (��0.8)���5 Triods Plakortis deweerdtaephila USNM 1254645 (h) BDT, Panama 40��� 64.2 (��15.8)���103 1��� 5.9 (��2.2)���11 (actine) Plakortis deweerdtaephila USNM 1254647 (p) SS, Bahamas 26��� 45.1 (��11.3)���68 2��� 3.3 (��0.5)���5 Plakortis symbiotica USNM 1254650 (h) LP, Puerto Rico 20��� 40.4 (��12.8)���71 2��� 3.3 (��0.7)���5 *Location BDT refers to Bocas del Toro, SS refers to San Salvador and LP to La Parguera. Habitat and ecology. Extensive surveys performed in the Caribbean suggest that this sponge is obligately associated with X. deweerdtae as free-living forms of P. deweerdtaephila sp. nov. have not been observed in more than 25 surveys that spanned four countries in the Caribbean: Mexico, Bahamas, Puerto Rico (Vicente et al. 2014) and Panama in this study. P. deweerdtaephila / X.deweerdtae sponge pairs have been documented from small sponge recruits to massive adults (Fig. 3 A���C in Vicente et al. 2014). These sponge pairs are found on the upper level (30��� 36 m) of mesophotic reef habitats, on vertical walls, shaded sides of pinnacles, as well as cryptic habitats (roof of overhangs and reef caves) of the Caribbean. In Panama the new species was found in a depth of 14 m, on the shaded sides of spur and groove hard bottom habitats exposed to high wave energy; individuals are completely overgrown by X. deweerdtae to the point where the new species is not visible unless the sponge pair is broken. Distribution. Bahamas (Little San Salvador, San Salvador, Acklins, Mayaguana, Mira Por Voz, Plana Keys, Hogsty Reef; see also Vicente et al. 2014, Zea et al. 2014) and Panama (Dolphin Rock, Bocas del Toro) (Fig. 1 B, E). Etymology. The name deweerdtaephila denotes the close association with Xestospongia deweerdtae, from phila meaning ���living or growing by preference���. Taxonomic remarks. There are currently 12 Plakortis species known for the TWA of which seven occur in the Caribbean: P. angulospiculatus (Carter 1883), P. zyggompha (De Laubenfels 1934), P. halichondrioides (Wilson 1902), P. myrae Ereskovsky et al., 2014, P. e d w a rd s i Ereskovsky et al., 2014, and P. da r i a e Ereskovsky et al., 2014. P. deweerdtaephila sp. nov. does not have any microrhabds or quasiamphiasters which places it among the P. simplex species group according to Muricy (2011). This species complex in the TWA includes, P. insularis (Moraes & Muricy, 2003), P. zyggompha, P. edwardsi, and P. dariae. By having two size classes of diods the new species is more similar to P. e dw ard s i and P. dariae that inhabit vertical shaded sides of reef boulders in Martinique and were collected along depths of 22��� 26 m. The external morphology of the new species differs from P. e d w a rds i by having an irregular surface with oscules that can be elevated with large openings. In P. e d w a rds i oscules are flush with the surface (Ereskovsky et al. 2014). The ectosome of the new species has more organized circular meshes than P. edwardsi, without spicules cluttering open circular spaces. There is also an abundance of subectosomal lacunae that separates the ectosome from the choanosome in the new species that is not mentioned in the description of P. e d w a rd s i. The choanosome also has more abundant circular meshes than in P. e d w a rds i. Spicules of specimens from the Bahamas, however, are similar in size and shape to P. e dw ard s i. Despite having similar spicule sizes to P. dariae, the new species differs in external morphology and in the skeleton arrangement of the ectosome and choanosome. Also, the color of P. dariae is green, contrasting with the brown of the new species. Sponge individuals of the new species are larger and thicker than P. dariae. The new species also has larger oscules and an irregular surface. The ectosome of P. dariae is poorly differentiated without subectosomal lacunae. Spicules of P. d ar i ae cross the surface of the ectosome while spicules in the ectosome of the new species never cross the surface. The shape of the small diods of P. d ar i ae is also irregular with one end blunt which does not coincide with the shape of small diods in the new species that are always symmetrical. The specimen also shows morphological differences to P. insularis, in that it has an irregular surface with large oscules. The skeleton of P. insularis consists of a loose and confused arrangement of diods in low density and in P. deweerdtaephila diods are present in high density forming organized circular meshes. The dense arrangement of diods also forms subectosomal lacunae which are not present in P. insularis. Small diods in P. insularis are also absent. The thick massive shape, brown color and dark color exudate, plus having some spicules reaching sizes>150 ��m, led Zea et al. (2009) to erroneously identify P. deweerdtaephila as P. halichondrioides. This fact was later corrected (Zea et al. 2014) after the molecular and spicular comparisons of Vicente et al. (2014) demonstrated their distinctiveness. To understand their differences, we made direct comparisons of specimens of P. deweerdtaephila with specimens of P. halichondrioides from the Bahamas and Puerto Rico. Morphologically, the ectosomal skeleton of P. halichondrioides can be distinguished from P. deweerdtaephila in that spicules break the surface of the ectosome. Spicules also protrude inside circular meshes of the ectosome of P. halichondrioides and in P. deweerdtaephila circular meshes are free of spicules. Spicule sizes are also significantly smaller in P. deweerdtaephila than in P. halichondrioides (Vicente et al. 2014). Differences between P. deweerdtaephila and P. symbiotica are given below in the taxonomic remarks of the latter species. Recently, P. angulospiculatus was reported from Carrie Bow Cay, Belize, completely overgrown by X. deweerdtae (R��tzler et al. 2014). Spicule size and shape of the P. angulospiculatus identified by R��tzler et al. (2014) are in agreement with P. deweerdtaephila. Thus, its identity needs to be confirmed through molecular analysis, as the sequences of P. angulospiculatus we used herein (see below) turned out to be distantly related to those of P. deweerdtaephila or P. symbiotica. P. angulospiculatus is a widespread Caribbean species, and its many reports may encompass several of the species newly described in the last decade (e.g., Ereskovski et al. 2013; Domingos et al. 2013); it needs to be reassessed, preferably from holotype material. The cob and cox1 gene sequences we used from P. angulospiculatus individuals were determined by Ereskovski et al. (2014) and Erpenbeck et al. (2008), respectively. The only character that apparently may distinguish P. angulospiculatus from our two new species of Plakortis is its lack of dark brown exudate (Ereskovski et al. 2013), but even this needs to be confirmed. The most important morphological character that differentiates P. deweerdtaephila from any Plakortis species is that individuals have only been found associated with X. deweerdtae. No other sponge associations between Plakortis and a Xestospongia have been reported other than the P. symbiotica / X. deweerdtae sponge pair (described below). This is the first time that an obligate symbiosis with a heterospecific sponge species is a taxonomic character of any sponge species. The larger size of spicules in specimens from Panama in comparison to those from the Bahamas (Table 1), reflect a Caribbean wide geographical pattern present in many groups of sponges, attributed to the apparent enrichment of silicon that continental locations experience, in comparison to oceanic ones (Zea 1987)., Published as part of Vicente, Jan, Zea, Sven & Hill, Russell T., 2016, Sponge epizoism in the Caribbean and the discovery of new Plakortis and Haliclona species, and polymorphism of Xestospongia deweerdtae (Porifera), pp. 209-233 in Zootaxa 4178 (2) on pages 212-215, DOI: 10.11646/zootaxa.4178.2.3, http://zenodo.org/record/255301, {"references":["Zea, S., Henkel, T. & Pawlik, J. (2009) The Sponge Guide: a picture guide to Caribbean sponges. 2 nd Edition. Available from: www. spongeguide. org / 2 ndEdition (Accessed 3 Mar. 2016)","Wilson, H. (1902) The sponges collected in Porto Rico in 1899 by the US Fish Commission steamer Fish Hawk. Bulletin of the United States Fish Commission, 2, 375 - 411.","Vicente, J., Zea, S., Powell, R. J., Pawlik, J. R. & Hill, R. T. (2014) New epizooic symbioses between sponges of the genera Plakortis and Xestospongia in cryptic habitats of the Caribbean. Marine Biology, 161, 2803 - 2818. http: // dx. doi. org / 10.1007 / s 00227 - 014 - 2546 - z","Carter, H. (1883) Contributions to our knowledge of the Spongida. Annals and Magazine of Natural History, 12, 308 - 329. http: // dx. doi. org / 10.1080 / 00222938309459636","De Laubenfels, M. W. (1934) New sponges from the Puerto Rican deep. Smithsonian Miscellanous Collections, 91, 1 - 28 ..","Ereskovsky, A. V., Lavrov, D. V. & Willenz, P. (2014) Five new species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis. Journal of the Marine Biological Association of the United Kingdom, 94, 285 - 307. http: // dx. doi. org / 10.1017 / S 0025315413000295","Muricy, G. (2011) Diversity of Indo-Australian Plakortis (Demospongiae: Plakinidae), with description of four new species. Journal of the Marine Biological Association of the United Kingdom, 91, 303 - 319. http: // dx. doi. org / 10.1017 / S 0025315410000743","Moraes, F. C. & Muricy, G. (2003) Taxonomy of Plakortis and Plakinastrella (Demospongiae: Plakinidae) from oceanic islands off north-eastern Brazil, with description of three new species. Journal of the Marine Biological Association of the UK, 83, 385 - 397. http: // dx. doi. org / 10.1017 / S 0025315403007239 h","Rutzler, K., Piantoni, C., van Soest, R. W. & Diaz, M. C. (2014) Diversity of sponges (Porifera) from cryptic habitats on the Belize barrier reef near Carrie Bow Cay. Zootaxa, 3805 (1), 1 - 129. http: // dx. doi. org / 10.11646 / zootaxa. 3805.1.1","Domingos, C., Moraes, F. & Muricy, G. (2013) Four new species of Plakinidae (Porifera: Homoscleromorpha) from Brazil. Zootaxa, 3718 (6), 530 - 544. http: // dx. doi. org / 10.11646 / zootaxa. 3718.6.2","Zea, S. (1987) Esponjas del Caribe colombiano: Catalogo cientifico, Colombia"]}

Genus Plakortis Schulze, 1880 Definition. Plakinidae with the skeleton formed by diods, with a variable abundance or absence of triods. Diactine derived microscleres (microrhabds), quasiamphiasters, spined diods and spheres may be present in some species; calthrops are absent (Muricy 2011)., Published as part of Vicente, Jan, Zea, Sven & Hill, Russell T., 2016, Sponge epizoism in the Caribbean and the discovery of new Plakortis and Haliclona species, and polymorphism of Xestospongia deweerdtae (Porifera), pp. 209-233 in Zootaxa 4178 (2) on page 212, DOI: 10.11646/zootaxa.4178.2.3, http://zenodo.org/record/255301, {"references":["Schulze, F. E. (1880) Untersuchungen u ¨ ber den Bau und die Entwicklung der Spongien. Die Plakiniden. Zeitschrift fur Wissenchaftliche Zoologie, 34, 407 - 451.","Muricy, G. (2011) Diversity of Indo-Australian Plakortis (Demospongiae: Plakinidae), with description of four new species. Journal of the Marine Biological Association of the United Kingdom, 91, 303 - 319. http: // dx. doi. org / 10.1017 / S 0025315410000743"]}

Plakortis symbiotica sp. nov. (Fig. 3; Table 2) Plakortis sp. 2; Vicente et al. 2014 (ecology and symbiosis). Type material. Holotype and type locality: USNM 1254650, basibiont of Haliclona plakophila sp. nov., Old Buoy, La Parguera, Puerto Rico (17.9552�� N, - 67.0532�� W), 32 m depth, coll. Jaaziel Garc��a Hern��ndez, October 15, 2015. Paratype USNM 1254649, basibiont of X. deweerdtae, Old Buoy, La Parguera, Puerto Rico, (17.9552�� N, - 67.0532�� W), 32 m depth, coll. Jan Vicente, August 13, 2012. Specimens examined for comparison (other than those described here). Plakortis halichondrioides: PHBH, Little San Salvador, Bahamas, PHPR, La Parguera, Puerto Rico. Diagnosis. Thickly encrusting cushions with a soft irregular surface and compressible body. Found as basibiont of X. deweerdtae but also of Haliclona plakophila sp. nov. In Puerto Rico, both X. deweerdtae and H. plakophila can be found on the same individual of P. symbiotica sp. nov. Both sponge epibionts grow as patches on the P. symbiotica body, never fully covering the basibiont as observed in P. deweerdtaephila, but can penetrate the basibiont body forming inner channels. Oscules are slightly elevated from the surface. Color can be dark green and dark brown in vivo and exudes a brown pigment when preserved in ethanol. Reticulated tangential ectosomal skeleton and reticulated choanosomal skeleton with lacunae present. Spicules consist of triods and diods of one size class. Description. Thick cushions 3�� 30 cm by 1���3 cm in thickness (Fig. 3 A). Oscules are slightly elevated measuring 0.3���1.7 cm in diameter. Oscules in preserved specimens are contracted. External and internal color is dark brown; a dark brown pigment is exuded when preserved. Surface is smooth, soft and irregular where X. deweerdtae or H. plakophila sp. nov. grows. Consistency is compressible, and easily torn. Skeleton. Ectosome is a disorganized tangential reticulation of diods and triods. Multispicular tracts are not well defined but form circular meshes, 43��� 73 ���121 ��m diameter (N=20; Fig. 3 B). Spicules barely break the surface of the ectosome. When X. deweerdtae or H. plakophila sp. nov. form inner channels within the choanosome of P. symbiotica sp. nov., the ectosome forms a barrier between the two sponge species as observed by Vicente et al. (2014, their Fig. 7 E and F). The ectosome (60���70 ��m thick) is dense and sometimes hard to distinguish from the choanosome. Subectosomal lacunae are present (Fig. 3 C). The choanosomal skeleton is dense but has an abundance of irregular circular meshes of varying diameters formed by a confused reticulation of diods and triods (Fig. 3 D). Spicules. One size class of diods and triods. Diods are significantly sinuously bent, with a thick center. Ends are sharp and significantly bent (Fig. 2 E���F). Size (length x width): 72��� 113.1 (��16.7)���142 ��m x 2.2��� 3.6 (��0.8)���5.0 ��m (Table 1). Triods are rare, Y-shaped, smooth, with sharp endings that are sometimes bent (Fig. 2 I): 20��� 40.4 (��12.8)���71 ��m long by 2.0��� 3.3 (��0.7)���4.7 ��m in width (Table 1). Microrhabds, quasiamphiasters and spheres are absent. Habitat and ecology. Sponge pairs are found on vertical walls (> 30 m), shaded side of pinnacles, and in reef cave habitats. Like Plakortis deweerdtaephila sp. nov., the new species has only been found either associated with X. deweerdtae or H. plakophila, never free-living (Vicente et al. 2014). Sponge pairs have been documented from small recruits (Fig. 3 D in Vicente et al. 2014) and growth morphologies of sponge pairs remain stable for long periods of time (Supplementary Fig. 4 in Vicente et al. 2014). Distribution. Bahamas (Little Inagua) and Puerto Rico (Mona Island, La Parguera, Desecheo) (Figure 1 B, D). Taxonomic remarks. As in Plakortis deweerdtaephila sp. nov., the lack of microrhabds or quasiamphiasters in P. symbiotica sp. nov., places it within the P. simplex species group (see above, cf. Muricy 2011). P. symbiotica can be distinguished from all species of this complex by its association status with either haplosclerid, as well as by color, shape, size consistency and spicule composition. For example, P. symbiotica has an irregular surface with large oscules which sets it apart from other Plakortis spp. in this group. Unlike P. edwarsii, only large diods are present in P. symbiotica that form organized ectosomal meshes without cluttering opened circular spaces. Diods are also densely packed in both the ectosome and choanosome. P. symbiotica occasionally forms lacunae which are not present in P. da r i a e or P. insularis. Other than P. deweerdtaephila, the only other species to associate with X. deweerdtae is P. symbiotica (but see remarks above regarding P. angulospiculatus as basibiont of X. deweerdtae from Belize, cf. R��tzler et al. 2014). In Puerto Rico, H. plakophila is also an epibiont of P. s y m b i o t i c a. There are several morphological differences between P. deweerdtaephila and P. symbiotica. For example, P. s y m b i o t i c a has only large diods with small diods absent. The large diods are significantly more bent than in P. deweerdtaephila (Fig. 2 E, F). The ectosome has circular meshes with smaller diameters (43��� 73 ���121 ��m; Fig. 3 B) than the ectosomal circular meshes of P. deweerdtaephila ( 114��� 205 ���329 ��m; Fig. 2 C). The ectosome is denser and harder to differentiate in cross-sections of P. symbiotica than of P. deweerdtaephila. Subectosomal lacunae are present but much fewer than in P. symbiotica; the choanosome also appears to have more circular meshes than P. symbiotica. As for P. deweerdtaephila, P. symbiotica spicules were compared with those of P. halichondrioides by Vicente et al. (2014), and shown to be larger in the latter. Our direct comparisons with a specimen of P. halichondrioides from Puerto Rico show that P. s y m b i o t i c a can be distinguished by having smaller spicules and like P. deweerdtaephila spicules never cross the surface of the ectosome or the open spaces of the circular meshes. Circular meshes are therefore better defined in P. deweerdtaephila than in P. halichondrioides. Phylogenetic analysis. Our phylogenetic analysis used maximum likelihood to compare partial sequences of the cytochrome b (cob) and c (cox1) genes of our new Plakortis spp. to other homoscleromorph sponge sequences deposited in GenBank. Our analysis confirmed that P. deweerdtaephila sp. nov. and P. symbiotica sp. nov. are more closely related to one another than to any other homoscleromorph or Plakortis species (Fig. 4). Sequence homology between both species was 93% for cob and 94% for cox1 and formed a clade that was supported with bootstrap values (85 and 78% respectively). On the other hand, these differences are enough to support their status as separate species, which is further supported by the morphological differences outlined above. The closest relatives of the new species were P. simplex and P. dariae with a 92% sequence homology for both genes. Despite having similar morphological features compared to P. e dw ard s i, this species turned out to be more closely related to P. halichondrioides and P. angulospiculatus in the phylogenetic analysis of both genes (Ereskovsky et al. 2013). Support values in the phylogenetic analysis for the P. symbiotica and P. deweerdtaephila clades suggests that the symbiotic association of these sponges with other sponges is perhaps an ancestral, and even a synapomorphic character. Etymology. The name symbiotica denotes the tendency of the new species to associate with X. deweerdtae and H. plakophila sp. nov., Published as part of Vicente, Jan, Zea, Sven & Hill, Russell T., 2016, Sponge epizoism in the Caribbean and the discovery of new Plakortis and Haliclona species, and polymorphism of Xestospongia deweerdtae (Porifera), pp. 209-233 in Zootaxa 4178 (2) on pages 215-218, DOI: 10.11646/zootaxa.4178.2.3, http://zenodo.org/record/255301, {"references":["Vicente, J., Zea, S., Powell, R. J., Pawlik, J. R. & Hill, R. T. (2014) New epizooic symbioses between sponges of the genera Plakortis and Xestospongia in cryptic habitats of the Caribbean. Marine Biology, 161, 2803 - 2818. http: // dx. doi. org / 10.1007 / s 00227 - 014 - 2546 - z","Muricy, G. (2011) Diversity of Indo-Australian Plakortis (Demospongiae: Plakinidae), with description of four new species. Journal of the Marine Biological Association of the United Kingdom, 91, 303 - 319. http: // dx. doi. org / 10.1017 / S 0025315410000743","Rutzler, K., Piantoni, C., van Soest, R. W. & Diaz, M. C. (2014) Diversity of sponges (Porifera) from cryptic habitats on the Belize barrier reef near Carrie Bow Cay. Zootaxa, 3805 (1), 1 - 129. http: // dx. doi. org / 10.11646 / zootaxa. 3805.1.1"]}

The 6-epimer of the plakortide H acid (1), along with the endoperoxides plakortide E (2), plakortin (3), and dihydroplakortin (4) have been isolated from a sample of the Caribbean sponge Plakortis halichondrioides. To perform a comparative study on the cytotoxicity towards the drug-sensitive leukemia CCRF-CEM cell line and its multi-drug resistant subline CEM/ADR5000, the acid of plakortin, namely plakortic acid (5), as well as the esters plakortide E methyl ester (6) and 6-epi-plakortide H (7) were synthesized by hydrolysis and Steglich esterification, respectively. The data obtained showed that the acids (1, 2, 5) exhibited potent cytotoxicity towards both cell lines, whereas the esters showed no activity (6, 7) or weaker activity (3, 4) compared to their corresponding acids. Plakortic acid (5) was the most promising derivative with half maximal inhibitory concentration (IC50) values of ca. 0.20 µM for both cell lines.

The new discovery by Vicente et al. (2014) of specialized epizoic symbioses between sponges of the genera Plakortis and Xestospongia revealed the obligate interaction of two new Plakortis spp. associating with Xestospongia deweerdtae and a new Xestospongia sp. In this study we formally describe the two new Plakortis spp. as Plakortis deweerdtaephila sp. nov. (previously reported as Plakortis sp. 1), Plakortis symbiotica sp. nov. (previously reported as Plakortis sp. 2) and describe the new Xestospongia sp. epibiont as Haliclona ( Halichoclona ) plakophila sp. nov. Plakortis deweerdtaephila associates only with X. deweerdtae , and has very small to large straight diods (24.2–233.7 μm long) and triods (26.4–102.6 μm long) that form large ectosomal circular meshes (114–329 μm diameter). P. symbiotica associates with both X. deweerdtae and H. plakophila, has larger curved diods (71.9–141.8 μm long) and triods (20.4–70.6 μm long) that form smaller ectosomal circular meshes (43–121 μm diameter) than P. deweerdtaephila . Phylogenetic analysis of cox1 and cob gene fragments revealed a strongly supported clade that grouped both Plakortis spp. nov. distantly from any other known Plakortis spp. H. plakophila is described as a thin encrusting veneer of tissue with occasional papillae, so far only found associated with P. symbiotica in La Parguera, Puerto Rico. Phylogenetic analysis of 18S rRNA and cox1 gene fragments place it distantly from any known clade of Haplosclerida. We found a new associated morphotype of X. deweerdtae from Bocas del Toro Panama, which completely overgrew P. deweerdtaephila . In addition, free-living morphotypes from Panama produce larger S-shaped and round bracket shaped strongyles never before observed for this species, leading us to redescribe X. deweerdtae . All X. deweerdtae morphotypes shared >99% sequence homology of cox1 , 18S rRNA and 28S rRNA genes with the holotype of X. deweerdtae . This study highlights the highly variable morphological characters of X. deweerdtae influenced by lifestyle and environmental factors. This is also the first time that an obligate symbiosis with a heterospecific sponge is used as a key taxonomic character.

Three new polyketides, woodylides A-C (1-3), were isolated from the ethanol extract of the South China Sea sponge Plakortis simplex. The structures were elucidated by spectroscopic data (IR, 1D and 2D NMR, and HRESIMS). The absolute configurations at C-3 of 1 and 3 were determined by the modified Mosher's method. Antifungal, cytotoxic, and PTP1B inhibitory activities of these polyketides were evaluated. Compounds 1 and 3 showed antifungal activity against fungi Cryptococcus neoformans with IC₅₀ values of 3.67 and 10.85 µg/mL, respectively. In the cytotoxicity test, compound 1 exhibited a moderate effect against the HeLa cell line with an IC₅₀ value of 11.2 µg/mL, and compound 3 showed cytotoxic activity against the HCT-116 human colon tumor cell line and PTP1B inhibitory activity with IC₅₀ values of 9.4 and 4.7 µg/mL, respectively.

Two novel polyketides, simplextones A (1) and B (2), were isolated from the sponge Plakortis simplex. Their structures were established by spectroscopic methods. The absolute configurations were assigned by modified Mosher's method, X-ray crystallographic analysis, and quantum mechanical calculation of the electronic circular dichroism (ECD) spectrum. Compounds 1 and 2 featured an unprecedented polyketide skeleton via the connection of a single carbon-carbon bond to form a cyclopentane. These compounds also exhibited moderate cytotoxicity.

Spectroscopy-guided chemical analysis of a marine sponge from the genus Plakortis, collected in Tonga, yielded seven new metabolites of polyketide origin, lehualides E-K (5-11), four of which incorporate various sulfur functionalities. The structures of compounds 5-11 were elucidated by interpretation of spectroscopic data and spectral comparison with model compounds. The biological activities of compounds 6-9 were investigated against human promyeloid leukemic HL-60 cells and two yeast strains, wild-type and a drug-sensitive mutant.

Two new five-membered-ring polyketide endoperoxides, epiplakinic acid F methyl ester (1) and epiplakinidioic acid (3), and a peroxide-lactone, plakortolide J (2), were isolated from the Puerto Rican sponge Plakortis halichondrioides, along with two previously reported cyclic peroxides, 4 and 5. The structures of the new metabolites were determined by spectroscopic and chemical analyses. The absolute stereostructures of 1, 2, and 5 were determined by degradation reactions followed by application of Kishi's method for the assignment of absolute configuration of alcohols. Biological screening of cycloperoxides 1-5 and semisynthetic analogues 7-12 for cytotoxic activity against various human tumor cell lines revealed that compounds 3, 4, and 11 are very active. Upon assaying for antimalarial and antitubercular activity, some of the compounds tested showed strong activity against the pathogenic microbes Plasmodium falciparum and Mycobacterium tuberculosis.

A multidisciplinary approach, based on molecular dynamics/mechanics, ab initio calculations, dynamic docking studies, and chemical reactions, has been employed to gain insight into the mechanism of the antimalarial action of plakortin and dihydroplakortin, simple 1,2-dioxanes isolated from the sponge Plakortis simplex. Our results show that these molecules, after interaction of the endoperoxide bond with Fe(ii), likely coming from the heme molecule, give rise to the formation of an oxygen radical, followed by rearrangement to give a carbon radical centered on the "western" alkyl side-chain. The carbon radicals generated on the side-chain, amenable for intermolecular reactions, should represent the toxic intermediates responsible for subsequent reactions leading to plasmodium death. The minimal structural requirements necessary for the activity of this class of antimalarial agents have been identified and discussed throughout the paper.

Circular dichroism (CD) is a powerful tool for assignment of configuration in natural products,[1] however, its use in acyclic molecules is limited by motional averaging that may reduce or eliminate Cotton effects (CE's). Recently, we reported application of liposomal exciton coupled CD (LECCD) for determination of both relative and absolute configuration of 1,n-acyclic diols (n >5)[2] which exploited two properties: dual chromophores with very large electronic charge-transition dipole moments and ordering of the long-chain carbon backbones within uniform unilamellar liposomes. This report now describes a sensitive technique – liposomal circular dichroism (LCD) – for assignment of configurations at remote methyl-branched stereocenters in long-chain natural products at sub-µmole levels by exploiting a single chromophore appended to the chain terminus. LCD reveals a general principle: simple Cotton effects arising from perturbation of single chromophores may be amplified by constraining molecules within lipid bilayers. Application of LCD is applied here to an outstanding problem: configurational assignment of the remote stereocenters in methyl-branched in polyketide peroxides (e.g. 1 and 2) from marine sponges of the genera Plakortis and Plakinastrella.

Investigation of the bioactive crude extract from the sponge Plakortis angulospiculatus from Brazil led to the isolation of plakortenone ( 1) as a new polyketide, along with five known polyketides ( 2- 6) previously isolated from other Plakortis sponges. The known polyketides were tested in antileishmanial, antitrypanosomal, antineuroinflammatory, and cytotoxicity assays. The results show that plakortide P ( 3) is a potent antiparasitic compound, against both Leishmania chagasi and Trypanosona cruzi, and exhibited antineuroinflammatory activity. The known polyketides 2- 6 were tested for cytotoxicity against four human cancer cell lines, but displayed only moderate cytotoxic activity.

Total synthesis of (±)-gracilioether F was achieved via a pivotal reductive cleavage of 1,2-dioxane from allenic ester in 11 steps. The key 1,2-dioxane species, derived from singlet oxygen and a diene, could be used as a common precursor for a stereocontrolled formation of the crucial 1,4-diol through a reductive cleavage.

The Gram-positive, alkali- and salt-tolerant marine bacterium strain P203T is described together with its closest phylogenetic neighbour, terrestrial isolate LMG 21005T. Strain P203T was isolated from material from the sponge Plakortis simplex that was obtained from the Sula-Ridge, Norwegian Sea. Strain LMG 21005T was an undescribed strain that was isolated from a church wall mural in Germany. Strains P203T and LMG 21005T were identified as novel alkalitolerant members of the Bacillus rRNA group 6 with a 16S rRNA gene sequence similarity of 99.5 %. The closest described neighbour, Bacillus gibsonii DSM 8722T, showed 99.0 % gene sequence similarity with P203T and 98.8 % similarity with strain LMG 21005T. Despite the high 16S rRNA gene sequence similarity, DNA–DNA cross-hybridization revealed only 25.8–34.1 % similarity amongst the three strains. The DNA G+C contents were 41.1 mol% for strain P203T and 39.6 mol% for strain LMG 21005T. Both strains grew well between pH 7 and pH 11. Strain P203T showed growth at moderate temperatures (from 4 to 30 °C) and in the presence of up to 12 % (w/v) NaCl at pH 9.7, whereas strain LMG 21005T was not salt tolerant (up to 4 % NaCl) and no growth was observed at 4 °C. The major fatty acids of strains P203T, LMG 21005T and the type strain of B. gibsonii were the saturated terminally methyl-branched compounds iso-C15 : 0 (19.8, 15.6 and 28.0 %, respectively) and anteiso-C15 : 0 (57.1, 48.6 and 45.2 %, respectively). Physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains P203T and LMG 21005T from the six related Bacillus species with validly published names and supported the proposal of two novel species, Bacillus plakortidis [type strain P203T (=DSM 19153T=NCIMB 14288T)] and Bacillus murimartini [type strain LMG 21005T (=NCIMB 14102T)].

A close examination of Mehta and Kundu's synthesis of an analogue of spiculoic acid A revealed discrepancies between their proposed structure and corresponding data. A revised structure is proposed for Mehta and Kundu's analogue after a careful analysis of their key synthetic transformations and published data. [reaction: see text].

A collection of an undescribed marine sponge of the genus Plakortis yielded four new "polyketide-derived" metabolites, lehualides A-D (1-4). The structures of compounds 1-4 were elucidated by interpretation of spectral data. Compound 2 demonstrated cytotoxicity against an ovarian cancer cell line, while compound 4 was active against both ovarian cancer and leukemia cell lines.

Simplextone E (1), a new metabolite of polyketide origin, was isolated with eight known analogues (2–9) from the South China Sea sponge Plakortis sp. The relative configuration of the new compound was elucidated by a detailed analysis of the spectroscopic data and quantum mechanical calculation of NMR chemical shifts, aided by the newly reported DP4+ approach. Its absolute configuration was determined by the TDDFT/ECD calculation. Simplextone E (1) is proven to be one of the isomers of simplextone D. The absolute configuration at C-8 in alkyl chain of plakortone Q (2) was also assigned based on the NMR calculation. In the preliminary in vitro bioassay, compounds 6 and 7 showed a selective growth inhibitory activity against HCT-116 human colon cancer cells with IC50 values of 8.3 ± 2.4 and 8.4 ± 2.3 μM, corresponding to that of the positive control, adriamycin (IC50 4.1 μM). The two compounds also showed selective activities towards MCF-7 human breast cancer and K562 human erythroleukemia cells while compound 3 only displayed weak activity against K562 cells.

Plakortis zyggompha (de Laubenfels, 1934) Synonymy and references. Plakortis zyggompha (de Laubenfels, 1934): Diaz & van Soest (1994): 104. Material. USNM 1228902, 1228903, 1228904, Carrie Bow Cay, back reef near reef crest, underside of Acropora palmata coral rubble, 0.3 m; K. Ruetzler col. 23 Apr 1974. USNM 1228905, Carrie Bow Cay south back reef, lower surface of coral rock, 1m; K. Ruetzler col. 13 Mar 0 9. External morphology. Small crusts or cushions (to 5 mm thick), covering up to 5 cm 2. Small (1 mm) oscula, located on the thicker lobes. Smooth surface, firm-elastic consistency. Color cream to olive, brown, and blackish brown. Skeleton structure. Spicules densely packed throughout. Spicules. Diods, small but of considerable size range: 18–138 x 3–8 (85 x 5) Μm; triods, common: 38–110 (55) Μm. Ecology. Under platy coral rubble in the back reef, 0.3–1 m, reported from below 100 m. Distribution. Gulf of Mexico and Caribbean., Published as part of Rützler, Klaus, Piantoni, Carla, Van, Rob W. M. & Díaz, Cristina, 2014, Diversity of sponges (Porifera) from cryptic habitats on the Belize barrier reef near Carrie Bow Cay, pp. 1-129 in Zootaxa 3805 (1) on page 16, DOI: 10.11646/zootaxa.3805.1.1, http://zenodo.org/record/249983, {"references":["de Laubenfels, M. W. (1934) New sponges from the Puerto Rican Deep. Smithsonian Miscellaneous Collections, 91, 1 - 28."]}

Plakortis angulospiculatus (Carter, 1882) Synonymy and references. Plakortis angulospiculatus (Carter, 1879): Zea (1987): 222, fig. 81, pl. 13: 6, 7. Material. USNM 1228898, Carrie Bow Cay, back reef near reef crest, underside of Acropora palmata coral rubble, 0.3 m; K. Ruetzler col. 23 Apr 1974. USNM 1228899, 1228900 Curlew Bank, forereef cave, 20 m; C. Piantoni col. 28 Jun 2007. USNM 1228901, Carrie Bow Cay forereef cave, 26 m; M. C. Diaz col. 8 Sep 2009. External morphology. Crustose to cushion-shaped (1–12 mm thick), covering up to 120 cm 2 substratum; with a few small (2 mm), elevated oscula. Surface rough, may be completely covered by another sponge; consistency firm but brittle. Color pale orange to grayish brown and medium-dark brown. Skeleton structure. Reticulate spicule tracts in the ectosome, densely packed spicules without orientation in the choanosome. Spicules. Straight, bent, and flexuous diods: 38–300 x 1–10 (165 x 6) Μm; triods generally common, many deformed, with one ray smaller than the others or in odd angles (such as Y-shaped): 53–130 (84) Μm. Ecology. Under coral plates in the back reef and inside forereef caves, 0.3– 26 m. Tends to be overgrown by other sponges, for instance, Xestospongia deweerdtae (see the description there). Distribution. Gulf of Mexico and throughout the Caribbean. Comments. The spicule size range in our specimens is larger than that reported by Zea (1987), close to that given by this author for Plakortis halichondroides (Wilson), but the latter species is black in color and without triactines. However, recent molecular analyses (cox 1, 100 % similarity) suggest that these two morphotypes of Plakortis are conspecific (Ereskovsky et al., 2013). P. zygompha (described below) can be distinguished in the field by greenish-brown color and it has considerably smaller spicules. Three new species of Plakortis recently described from cave habitats in Jamaica (Ereskovsky et al., 2013; Table 2) differ clearly in color and spicule characteristics and dimensions; none of them occurred in our samples., Published as part of Rützler, Klaus, Piantoni, Carla, Van, Rob W. M. & Díaz, Cristina, 2014, Diversity of sponges (Porifera) from cryptic habitats on the Belize barrier reef near Carrie Bow Cay, pp. 1-129 in Zootaxa 3805 (1) on page 16, DOI: 10.11646/zootaxa.3805.1.1, http://zenodo.org/record/249983, {"references":["Carter, H. J. (1882) Some sponges from the West Indies and Acapulco in the Liverpool Free Museum described, with general and classificatory remarks. Annals and Magazine of Natural History, 5 (9), 266 - 309, 346 - 368, pls. XI - XII.","Carter, H. J. (1879) Contributions to our knowledge of the Spongida. Annals and Magazine of Natural History, Series 5, 3, 284 - 304, 343 - 360, pls. XXV - XXVII.","Zea, S. (1987) Esponjas del Caribe Colombiano: Dictyoceratida, Dendroceratida, Verongida, Haplosclerida, Poecilosclerida, Halichondrida, Axinellida, Desmophorida y Homosclerophorida. Editorial Catalogo Cientifico, Colombia, pp. 1 - 286.","Ereskovsky, A. V., Lavrov, D. & Willenz, P. (2013) Five New Species of Homoscleromorpha (Porifera) from the Caribbean Sea and re-description of Plakina jamaicensis. Journal of the Marine Biological Association of United Kingdom, 94 (2), pp. 285 - 307."]}

Chemical investigation of the organic extract obtained from the sponge Plakortis simplex collected in the South China Sea afforded five new polyketide endoperoxides ( 2 and 4 – 7 ), along with two known analogues ( 1 and 3 ). The stereostructures of these metabolites have been deduced on the basis of spectroscopic analysis and chemical conversion. The isolated endoperoxide derivatives have been tested for their in vitro antimalarial activity against Plasmodium falciparum strains, showing IC 50 values in the low micromolar range. The structure–activity relationships were analyzed by means of a detailed computational investigation and rationalized in the light of the mechanism of action proposed for this class of simple antimalarials. The relative orientation of the atoms involved in the putative radical generation and transfer reaction was demonstrated to have a great impact on the antimalarial activity. The resulting 3D pharmacophoric model can be a useful guide to design simple and effective antimalarial lead compounds belonging to the class of 1,2-dioxanes.

Sponge-associated microorganisms are able to assemble the complex machinery for the production of secondary metabolites such as polyketides, the most important class of marine natural products from a drug discovery perspective. A comprehensive overview of polyketide biosynthetic genes of the sponge Plakortis halichondrioides and its symbionts was obtained in the present study by massively parallel 454 pyrosequencing of complex and heterogeneous PCR (Polymerase Chain Reaction) products amplified from the metagenomic DNA of a specimen of P. halichondrioides collected in the Caribbean Sea. This was accompanied by a survey of the bacterial diversity within the sponge. In line with previous studies, sequences belonging to supA and swfA, two widespread sponge-specific groups of polyketide synthase (PKS) genes were dominant. While they have been previously reported as belonging to Poribacteria (a novel bacterial phylum found exclusively in sponges), re-examination of current genomic sequencing data showed supA and swfA not to be present in the poribacterial genome. Several non-supA, non-swfA type-I PKS fragments were also identified. A significant portion of these fragments resembled type-I PKSs from protists, suggesting that bacteria may not be the only source of polyketides from P. halichondrioides, and that protistan PKSs should receive further investigation as a source of novel polyketides.