Seven new deep-water Tetractinellida (Porifera: Demospongiae) from the Galápagos Islands – morphological descriptions and DNA barcodes

Seven new deep-water Tetractinellida (Porifera: Demospongiae) from the Galápagos Islands –... Abstract The Galápagos Islands, positioned in the confluence of warm and coldwater currents in the Eastern Pacific, is well known for the high degree of endemism of its marine invertebrate fauna. This fauna has been studied extensively in recent years: the echinoderms, corals and other benthic cnidarians, but little is known about the deep- and shallow-water sponge faunas. To date, only 70 sponge species have been described from the Galápagos Islands, 37 of which are endemic. Of these 70 species, only one shallow-water species of desma-bearing Tetractinellida (Demospongiae), Corallistes isabela, has been reported. In 1995, Harbor Branch Oceanographic Institution, Florida, led an expedition around the Galápagos archipelago, focussed on the collection of deep-water Porifera. Here, we describe seven new species and provide DNA barcodes for the tetractinellids from these collections. Phylogenetic relationships of these new species are discussed and compared with other material from the Caribbean, the Central and West Pacific Oceans. The new species represent five genera (Craniella, and desma-bearing Tetractinellida Neophrissospongia, Corallistes, Racodiscula and Scleritoderma). Phylogenetic reconstructions combining independent markers (mtDNA and rDNA) support the generic affiliation of these new species and confirm the separation of Eastern Pacific species from Caribbean and Central to West Pacific species. Astrophorina, COI, Corallistidae, DNA barcode, endemism, Galápagos Islands, lithistids, Scleritodermidae, Spirophorina, Theonellidae INTRODUCTION The Galápagos Islands are located on the Nazca Plate, in the Eastern equatorial Pacific Ocean, about 926 km west of Ecuador (Fig. 1). Plate tectonic analyses demonstrate proximity to the so-called ‘Galápagos Triple Junction’, where the Pacific, Cocos and Nazca plates concur (Holden & Dietz, 1972). The position of Galápagos archipelago at the confluence of three major cold oceanic currents (Peru Coastal, Peru Oceanic and Cromwell) and one warm (Panamá) oceanic current (Chavez & Brusca, 1991) is further influenced by anomalous events such as the El Niño and La Niña, which seasonally change the ocean surface temperatures and are known to impact shallow coral reef communities (Feingold, 2011). Due to its extremely isolated geographic position, diverse underwater geomorphological settings as a result of ongoing tectonic and volcanic activity (Geist et al., 2008), in combination with complex mixing of water masses (Chavez & Brusca, 1991), the Galápagos archipelago constitutes a unique marine laboratory to investigate the ecology, evolution and biogeography of marine animals in an isolated region of the Eastern Pacific. In 1978, the Galápagos Islands were protected by UNESCO and described as a ‘unique living museum and showcase of evolution’. In 2001, the status of World Heritage Site was granted to the Galápagos Marine Reserve (133 000 km2), which comprises the second largest Marine Reserve in the world (UNESCO, 1978). Figure 1. View largeDownload slide Map of the Galápagos archipelago with sampling locations and species collected. Colour code denotes species and records (stars = holotype location; circles = paratypes). Map made with GeoMapApp, http://www.geomapapp.org, using the implemented Global Multi-Resolution Topography (GMRT Grid Version 3.2) (Ryan et al., 2009). Figure 1. View largeDownload slide Map of the Galápagos archipelago with sampling locations and species collected. Colour code denotes species and records (stars = holotype location; circles = paratypes). Map made with GeoMapApp, http://www.geomapapp.org, using the implemented Global Multi-Resolution Topography (GMRT Grid Version 3.2) (Ryan et al., 2009). Despite the young age of the Galápagos platform (10–15 Myr) and its biogeographic isolation (e.g. Chavez & Brusca, 1991), the marine invertebrate fauna is relatively diverse and reveals large numbers of described endemic species, including echinoderms, molluscs, corals and other benthic cnidarians (James, 1991). However, little is known about the deep- and shallow-water sponge fauna; to date, only 70 demosponge species have been described from the Galápagos Islands, of which 37 (~50%) are endemic. Only seven deep-water species (>100 m) have been described (Wilson, 1904; de Laubenfels, 1939; Desqueyroux-Faúndez & Van Soest, 1997). Among these 70 species, 11 were reported as belonging to the order Tetractinellida Marshall, 1876 (Desqueyroux-Faúndez & Van Soest, 1997), of which ten belong to Astrophorina Sollas, 1887 (families Ancorinidae Schmidt, 1870; Pachastrellidae Carter, 1875; Geodiidae Gray, 1867; Corallistidae Sollas, 1888) and one (Cinachyrella globulosa Desqueyroux-Faúndez & Van Soest, 1997) to the Spirophorina Bergquist & Hogg, 1969 (Tetillidae Sollas, 1886). The Galápagos archipelago constitutes one of the largest island groups in the Pacific, but has the lowest demosponge diversity compared to the Central Indo-Pacific and Tropical Eastern Pacific (Van Soest et al., 2012). Besides the discovery of one shallow-water species of the family Corallistidae (Corallistes isabela Desqueyroux-Faúndez & Van Soest, 1997), no further desma-bearing tetractinellids have been described from this area. This study is focused primarily on the morphological description of seven new deep-water tetractinellids from the Galápagos archipelago, the majority of which are desma-bearing, and provides additional supportive molecular phylogenies for the generic affiliation of these new species using three markers (mtDNA COI and 28S, 18S rDNA). MATERIAL AND METHODS Sampling Sponges were collected using the Johnson-Sea-Link I research submersible, operated by Harbor Branch Oceanographic Institution (HBOI), during an expedition to the Galápagos Islands, conducted 14–31 October 1995. The objective of the expedition was to conduct a biodiversity inventory and carry out biodiscovery research on sponges, octocorals and algae. Sponges analyzed in the present study were collected from ten stations around the Galápagos archipelago between 21 and 396 m depth (Fig. 1). A detailed list of these specimens, with their corresponding vouchers, location, latitude, longitude and National Center for Biotechnology Information (NCBI) accession number is given in Supporting Information, Table S1. Frozen (−80 °C) as well as formalin and 70% ethanol-preserved specimens, stubs, spicule slides and skeletal sections of the type materials are archived at the Harbor Branch Oceanographic Museum (HBOM), Fort Pierce, Florida, USA. Molecular investigations Two independent molecular markers were used to amplify 18 samples from the Galápagos Islands and 16 comparative samples, following the protocol described in Schuster et al. (2015): the ‘Folmer’ fragment of mitochondrial COI (c. 650 bp) and the C1-D2 region of the nuclear 28S rDNA (c. 800 bp), using, respectively, the primers dgLCO1490 and dgHCO2198 (Meyer, Geller & Paulay, 2005) and C1’ASTR (Cárdenas et al., 2009) and D2 (Lê, Lecointre & Perasso, 1993). Additionally, we amplified and sequenced near-complete 18S rDNA sequences (~1800 bp) for all Galápagos Islands samples. Amplification of ~1800 bp of 18S was performed using primers SP18aF and SP18gR (Lavrov, Wang & Kelly, 2008) with PCR settings of 95 °C, 3 min (95 °C, 30 s; 50 °C, 40 s; 72 °C, 2.5 min) × 35 cycles; 72 °C, 7 min. Primers SP18aF, SP18gR (Lavrov et al., 2008), and 400F18S, 1200F18S, 600R18S and 1350R18S (Lavrov et al., 2008) were used to sequence ~1800 bp of overlapping fragments. In order to confirm the sponge origin of newly generated sequences, we assessed their homology by BLAST searches against NCBI GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were assembled into contigs using Geneious v.8.1.8 (http://www.geneious.com, Kearse et al., 2012). 28S (C1-D2) and COI were independently aligned against alignments from Schuster et al. (2015). In addition to these sequences, we included Stupenda singularisKelly & Cárdenas, 2016, in the alignment, taxonomic updates on the Antarctic Tetillidae made by Carella et al. (2016), Theonella species from Hall, Ekins & Hooper (2014), and corrected Isabella tanoa Ekins, Erpenbeck, Wörheide & Hooper, 2016, which was re-identified by Ekins et al. (2016) and previously named as Isabella mirabilis (Schuster et al., 2015). Isabella harborbranchi Carvalho, Pomponi & Xavier, 2015, was added from Carvalho et al. (2015) (see also Supporting Information, Table S1). A representative concatenated matrix was compiled from the independent COI and 28S alignments in Geneious, assuring that for most taxa, sequences of both markers were present. The concatenated alignment (28S rDNA and COI mtDNA) was 1440 bp long; 658 bp were constant, 105 bp parsimony uninformative and 677 bp parsimony informative. Sequences of 18S rDNA were only obtained from the Galápagos Islands material, thus we did not incorporate these sequences into the final concatenated alignment. Bayesian inference (BI) and maximum likelihood (ML) analyses were conducted as described in Schuster et al. (2015). Partitionfinder v.1.1.1 (Lanfear et al., 2012) was used to select the best-fit partitioning scheme and model, which was the generalizing GTR+G+I model for both markers. New sequences from this study are stored at GenBank (NCBI). GenBank accession numbers are: COI KY652805–KY652825; 28S C1-D2 KY652771–KY652804; 18S KY652826–KY652840. Additionally, COI sequences of all type material are deposited at the Sponge Barcoding Project (SBP) (http://www.spongebarcoding.org) (record numbers 1702–1733). The concatenated alignment from this study is freely available at OpenDataLMU (https://doi.org/10.5282/ubm/data.113). Morphological investigations In order to carry out scanning electron microscopy (SEM) of the sponge skeleton and spicules, a section of formalin-preserved sponge tissue, perpendicular to the sponge surface, was cut under a binocular microscope, dried and digested in 66% nitric acid (HNO3) for 15–20 min. The digested product was boiled until all organic material was dissolved. The remaining skeletons and spicules were rinsed at least five times with ultrapure water and twice with 99% ethanol, as described in Pisera & Pomponi (2015). The zygosed skeletons were mounted on SEM stubs using Carbon-Leit-C (PLANO, Germany) with the ectosomal surface uppermost. Loose spicules were prepared on glass coverslips attached to SEM stubs. All stubs were sputter-coated with gold and examined with a Leo 1430VP SEM at 5–25 kV at the Zoological State Collection (Munich). For thick histological sections, the same tissue orientation as described above was used. Tissues were resuspended overnight in 70% ethanol, dehydrated in graded ethanol series (1 h 90%, 12 h 99%) at room temperature and embedded into L.R. White (PLANO) after two to three changes. For polymerization, the blocks were placed at 46 °C for 1 h and 60 °C for 12 h. Thick sections (50–200 µm) were cut using a Leica SP1600 saw microtome (Germany) and examined under a Leica M165FC microscope. Measurements of spicules are given as minimum–mean–maximum length × minimum–mean–maximum width (µm) with total number of measured spicules (N) in parentheses. Measurements of the triaene cladome refer to the total diameter of the cladome, across the endpoints of the clades; measurements of the triaene rhabdome refer to the distance from just below the cladome to the tip. Microscleres in the desma-bearing Tetractinellida are predominantly streptasters, a general term for microscleres with rays that emanate from an elongate centrum (amphiasters, metasters, spirasters). Metasters have a few quite long rays emanating from a short, elongate centrum; spirasters have sparse or numerous rays emanating from a single bent or multiple spiraled elongate centrum; amphiasters have numerous rays emanating from either end of a straight shaft. Strict distinction between these microscleres is often not clear-cut, so we chose to cite combinations of forms used in the present study, for example, streptaster/spiraster or streptaster/amphiaster when distinction was difficult. Measurements of microscleres are given as the complete length of the microsclere and the maximum width of the actin. RESULTS Systematics The classification used here follows Morrow & Cárdenas (2015). The taxonomic authority for all new species described is restricted to the first author. Class Demospongiae Sollas, 1885, Subclass Heteroscleromorpha Cárdenas, Pérez & Boury-Esnault, 2012, Order Tetractinellida Marshall, 1876, Suborder Astrophorina Sollas, 1887, Family Corallistidae Sollas, 1888 Genus Neophrissospongia Pisera & Lévi, 2002 N eophrissospongia galapagoensis sp. nov. (Figs 2–4; Table 1) Diagnosis: Massive ear-shaped, flabellate, Neophrissospongia with rhabdome of dichotriaenes showing a large variation from 172 to 617 µm length with spined cladome and blunt to acute tips. Type material: Holotype: HBOM 003:02010, Coll. Johnson Sea-Link I (JSL-I) dive 3923 [27 October 1995, Galápagos, 4 nautical miles (NM) SE of Tortuga Island, 01°04′59″S, 90°51′56″W, 241 m]. Paratypes: HBOM 003:01097 (227 m), HBOM 003:01098 (189 m), Galápagos, NE of Rocas Gordin, Santa Cruz Island, 0°32′53″S, 90°7′47″W, Coll. JSL-I dive 3900. HBOM 003:02002 (204 m), HBOM 003:02003 (120 m), Galápagos, 5 NM NE of Kicker Rock at San Cristobal Island, 01°43′8″S, 89° 27′14″W, Coll. JSL-I dive 3905. HBOM 003:01099 (177 m), Galápagos, N-Side 1.25 NM NW of Cormorant PT at the Santa Maria Island (Floreana), 01°12′6″S, 90°26′10″W, Coll. JSL-I dive 3901. HBOM 003:02008 (235 m), Galápagos, West Coast 2 NM NW of PTA Tortuga at Isabela Island, 0°14′31″S, 91°24′56″W, Coll. JSL-I dive 3915. HBOM 003:02005 (328 m), Galápagos, NW Coast, 66 NM offshore, Isabela Island, 0°8′35″S, 91°23′42″W, Coll. JSL-I dive 3912. Comparative material: Neophrissospongia nolitangere Schmidt, 1870, HBOI 2-VI-91-1-005 (358 m), Portugal, Madeira, Savage Islands, Selvagem Grande, South Coast, 30°07′21″N, 15°52′7.8″W, Coll. JSL-I dive 3006, June 1991, identified by Michelle Kelly. Neophrissospongia microstylifera Lévi & Lévi, 1983 0CDN7083-J (111 m), Palau, Koror Reef, W-Side of Uchelbeluu Reef, wall, rock, 7°16′25″N, 134°31′25″E, 2001, identified by Michelle Kelly; 0CDN5043-Q (109 m), Palau, Koror Reef, W-Side of Uchelbeluu Reef, overhang, rock, 7°16′25″N, 134°31′26″E, 1997, identified by Michelle Kelly. Type locality: Tortuga Island, Galápagos Islands (241 m) (Fig. 1). Distribution: Tortuga Island, Isabela Island, Fernandina, Santa Cruz Island, San Cristobal, Floreana Island (Fig. 1). Habitat: Attached to hard substratum, depth range 120–328 m. Description: Morphology, in the shape of an ear, flared cup or vase, or foliose with folded margins when mature; walls 1–1.5 cm thick, margins flattened with rounded edges (Fig. 2); dimension of holotype, 12 cm diameter, 16 cm high (Fig. 2A); paratypes range 20–30 cm diameter, 30–45 cm high or larger (Fig. 2C, D). Surface, with horizontal growth lines; concave inhalant surface with numerous ostia, 25–35 µm diameter; convex (exhalent surface) has numerous, barely visible, oscules. Texture, stony, especially towards the base of the sponge. Colour, beige in life, brown in ethanol preservative. Ectosomal skeleton, 1.2–1.5 mm thick, outermost surface a densely packed, 100-µm-thick layer of microscleres, within which lies the cladomes of the dichotriaenes, the rhabdomes of which penetrate deeply into the choanosome (Fig. 4A, B). Choanosomal skeleton composed of zygosed dicranoclone desmas and acanthose microtylostyles, often located around the pore openings. Megascleres, desmas and triaenes; dicranoclone desmas, 280–450 µm (N15) maximum diameter, with a massive, arched, central core, from which four to six arms splay away from the underside of the arch, both the central core and the arms are covered irregularly with tubercles (Fig. 3B, C, F), the tubercules on the desma core are larger, fungiform and multituberculate; dichotriaenes, rhabdome 172–422–617 µm (N30) long, cladome, 175–227–372 µm (N30) wide, upper surface of clads irregularly spined (Fig. 4G, H). Microscleres, irregular amphiasters with short, thick rays, 15.2–11.2–18 µm long × 4.9–7.2–10.7 µm (N25) wide; acanthose microtylostyles, 44–91–133 µm (N25) long. Figure 2. View largeDownload slide In situ and deck pictures of Neophrissospongia galapagoensis sp. nov. A, B, cup-shaped holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. C, D, massive flabellate paratype HBOM 003:01099. Scale bar is 1 cm. Figure 2. View largeDownload slide In situ and deck pictures of Neophrissospongia galapagoensis sp. nov. A, B, cup-shaped holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. C, D, massive flabellate paratype HBOM 003:01099. Scale bar is 1 cm. Figure 3. View largeDownload slide Various views of the choanosome skeleton of Neophrissospongia galapagoensis sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, overview of dicranoclone desmas. B, dicranoclone desmas with a spinose dichotriaene in situ. C, details of the dicranoclones. D, in situ picture of a bundle of spinose dichotriaenes between the ectosome and choanosome. E, choanosome cut with disordered partly broken dicranoclones, arrows pointing to numerous microtylostyles. F, detailed view of dicranoclone articulation (zygome). Figure 3. View largeDownload slide Various views of the choanosome skeleton of Neophrissospongia galapagoensis sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, overview of dicranoclone desmas. B, dicranoclone desmas with a spinose dichotriaene in situ. C, details of the dicranoclones. D, in situ picture of a bundle of spinose dichotriaenes between the ectosome and choanosome. E, choanosome cut with disordered partly broken dicranoclones, arrows pointing to numerous microtylostyles. F, detailed view of dicranoclone articulation (zygome). Figure 4. View largeDownload slide Section and mega- and microscleres of Neophrissospongia galapagoensis sp. nov., holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, thin section of the ectosome and choanosome showing channel (ch) and spinose dichotriaenes (di) penetrating deeply into the choanosome. B, detailed thin section illustrating a thick layer of spiraster/streptaster (sp/st) on top of the ectosomal region. C–F, thick streptasters from the ectosomal regions. G, various ectosomal dichotriaenes. H, top view of tubercled cladomes of dichotriaenes. I, J, microspinose choanosomal microtylostyles, general view (I), detailed view (J). Figure 4. View largeDownload slide Section and mega- and microscleres of Neophrissospongia galapagoensis sp. nov., holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, thin section of the ectosome and choanosome showing channel (ch) and spinose dichotriaenes (di) penetrating deeply into the choanosome. B, detailed thin section illustrating a thick layer of spiraster/streptaster (sp/st) on top of the ectosomal region. C–F, thick streptasters from the ectosomal regions. G, various ectosomal dichotriaenes. H, top view of tubercled cladomes of dichotriaenes. I, J, microspinose choanosomal microtylostyles, general view (I), detailed view (J). Etymology: Named after the locality and distribution of the species, the Galápagos Islands. DNA barcodes: We sequenced partial COI, 28S (C1-D2) and complete 18S of the holotype and the paratypes from different localities of the Galápagos Islands (Fig. 1). COI sequences of all type material sequenced were identical with the exception of HBOM 003:02005 (no COI sequenced). 28S and 18S sequences of all type material were identical with the exception of HBOM 003:02005 (no 18S sequenced). GenBank accession numbers: COI KY652805–KY652811; 28S KY652771–KY652778; 18S KY652826–KY652832; SBP No. 1702–1709. Remarks: According to the World Porifera Database (WPD) (Van Soest et al., 2017, http://www.marinespecies.org/porifera/), only six different species of Neophrissospongia have been described: N. nolitangere (Table 1; Pisera & Vacelet, 2011) and N. tubulata Van Soest & Stentoft, 1988 from the tropical Western Atlantic; N. endoumensis Pisera & Vacelet, 2011, N. nana Manconi & Serusi, 2008 and N. radjae Pisera & Vacelet, 2011, from the Mediterranean Sea, and N. microstylifera Lévi & Lévi, 1983 from New Caledonia in the Pacific Ocean. Table 1. Outer morphology and spicule measurements of the known seven Neophrissospongia species including the new species N. galapagoensis sp. nov. Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) If not otherwise stated measurements are given in micrometres. Measurements are given for the holotype (in bold = new species) and indicated for N. nolitangere with ‘*’. Measurements and outer morphology are taken from Van Soest & Stentoft (1988), Pisera & Lévi (2002a), Manconi & Serusi (2008), Pisera & Vacelet (2011) and Carvalho et al. (2015). clad = cladome in diameter; deut = deuteroclade; prot = protoclade; rhabd = rhabdome. View Large Table 1. Outer morphology and spicule measurements of the known seven Neophrissospongia species including the new species N. galapagoensis sp. nov. Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) If not otherwise stated measurements are given in micrometres. Measurements are given for the holotype (in bold = new species) and indicated for N. nolitangere with ‘*’. Measurements and outer morphology are taken from Van Soest & Stentoft (1988), Pisera & Lévi (2002a), Manconi & Serusi (2008), Pisera & Vacelet (2011) and Carvalho et al. (2015). clad = cladome in diameter; deut = deuteroclade; prot = protoclade; rhabd = rhabdome. View Large Neophrissospongia galapagoensis sp. nov., which is cup-shaped to foliose, clearly differs from the encrusting N. nana, discovered in a Mediterranean marine cave. The club-shaped N. radjae (Table 1; Pisera & Vacelet, 2011) from a cave in the Mediterranean with an apical oscule is also distinguished from our species by gross morphology. Minor differences between the dichotriaenes of N. galapagoensis sp. nov. and N. radjae (Table 1) also distinguish both species: N. radjae has a more distinctly tuberculated cladome than in our new species. Neophrissospongia nolitangere, from the Azores, Cape Verde Islands, off Portugal (e.g. Pisera & Lévi, 2002a), Madeira (Carvalho et al., 2015), submarine caves in the Mediterranean (Table 1; Pisera & Vacelet, 2011) and the Bahamas (Pomponi et al., 2001), is distinguished from N. galapagoensis sp. nov. in the possession of smaller streptasters with longer rays; 28S sequences of N. nolitangere by Chombard, Boury-Esnault & Tillier (1998) and here (HBOI 2-VI-91-1-005) are both 100% identical and differ by 1% from our new species. Molecular analyses (Fig. 5) further separate N. galapagoensis sp. nov. from N. nolitangere. Sequence comparison with material from the West Pacific (WAM Z35946, 36053; probably both new species, A. Pisera, personal observation) disclose a 1.6% difference. Figure 5. View largeDownload slide Bayesian inference (BI) reconstruction of the concatenated dataset (COI and 28S) showing the relationship of the seven new species from the Galápagos to other tetractinellids from the Caribbean, Pacific and Atlantic. Maximum likelihood (ML) tree is mostly congruent, otherwise no support values are given. Newly generated sequences are in bold (red: new species, black: comparative samples). Bayesian posterior probabilities and ML bootstrap support values are indicated for clades >0.75/60, otherwise marked with a dash. Numbers following the taxon names are collection numbers or NCBI Genbank accession numbers. Markers sequenced are indicated in square brackets. Three letter code in dark blue indicates the biogeographical location. Figure 5. View largeDownload slide Bayesian inference (BI) reconstruction of the concatenated dataset (COI and 28S) showing the relationship of the seven new species from the Galápagos to other tetractinellids from the Caribbean, Pacific and Atlantic. Maximum likelihood (ML) tree is mostly congruent, otherwise no support values are given. Newly generated sequences are in bold (red: new species, black: comparative samples). Bayesian posterior probabilities and ML bootstrap support values are indicated for clades >0.75/60, otherwise marked with a dash. Numbers following the taxon names are collection numbers or NCBI Genbank accession numbers. Markers sequenced are indicated in square brackets. Three letter code in dark blue indicates the biogeographical location. Neophrissospongia tubulata from Barbados is characterized by small, single or clustered tubes, in which the dichotriaenes have short rhabds (30–50 µm) and a non-tuberculate cladome. Rather, the clads have dentate margins, clearly distinguishing them from the dichotriaenes of N. galapagoensis sp. nov. (Table 1) and all other members of the genus. This lateral dentation of the dichotriaene clads of N. tubulata points to the need for a revision of this species. The dichotrianes of N. endoumensis, from the Mediterranean (cladome width: 248–283 µm; rhabdome length: 531–615 µm) are larger and thicker and are less variable in size (Table 1; Pisera & Vacelet, 2011) compared to those of N. galapagoensis sp. nov. In the original description of tubular N. microstylifera from New Caledonia, Lévi & Lévi (1983) documented microstyles of 110–170 µm length, which are absent in our new species, and the dichotriaenes are more robust than those of N. galapagoensis sp. nov. Key to species of Neophrissospongia (after Manconi & Serusi, 2008 and the present study) (1) Growth form ear- to cup-shaped or large flabellate massive foliose with small inhalant and large exhalant apertures on opposite sides of the lamella, ectosomal skeleton of tuberculated dichotriaenes, styles/subtylostyles, smaller streptaster microscleres with long arms ……………………………..............................................................................................................N. nolitangere – Spinose microtylostyles and streptaster with massive arms as microscleres………… N. galapagoensis sp. nov. – Cup-shaped growth with spinose and massive cladome of dichotrianes and regular massive streptaster …………….……………………………………………………….....…………………………………. N. endoumensis – Growth form clavate to plate-like with inhalant and exhalant areas in different regions of the same sponge surface.................................………………………………………………………………………………………...... 2 (2) Growth form clavate, ectosomal skeleton of dichotriaenes and strongyle/ subtylostyles ...................................………………………………………………………………………............ N. microstylifera – Spined dichotriaenes and spinose microtylostyles………................................………………......…....N. radjae – Plate-like grow form, ectosomal skeleton of dichotriaenes, styles/subtylostyles and aster-like microscleres..................................………………………………………………………………................... N. nana The key to species of Neophrissospongia by Manconi & Serusi (2008) is updated from the work of Pisera & Vacelet (2011) and the present study. Genus C orallistes Schmidt, 1870 C orallistes floreana sp. nov. (Figs 6, 7; Table 2) Diagnosis: Corallistes with smooth dichotriaenes in a broad range of sizes (93–558 µm rhabd; 72–272 µm cladome) and amphioxeas of 327–887 µm length; choanosomal megascleres are tuberculate dicranoclone desmas, about 211–345 µm diameter. Type material: Holotype: HBOM 003:02000, Coll. JSL-I dive 3901 [16 October 1995, Galápagos, N-Side 1.25 NM NW of Cormorant Point, Floreana Island, Santa Maria, 01°12′5.88″S, 90°26′9.96″W, 121 m]. Comparative material: Corallistes isabela HBOM 003:02013 (203 m), Galápagos, Santiago Island, 0°11′29″S, 90°34′58″W, Coll. JSL-I dive 3927, identified by A. Schuster. Type locality: Floreana Island, Galápagos Islands (121 m) (Fig. 1). Distribution: Only known from type locality. Habitat: Attached to hard substratum (121 m). Description: Morphology, a shallow bowl with a slightly folded or indented margin, or ear-shaped, walls about 5–6 mm thick with rounded margins (Fig. 6A, B), holotype, about 8 cm diameter, about 6 cm high (Fig. 6A, B). Surface, uneven, partly covered with algae, coral and shell fragments, bundles of projecting spicules trap sediment on the upper surface, otherwise the surface is smooth, oscules are not visible (Fig. 6B). Texture, stony. Colour, beige in life (Fig. 6A), light brown to cream in ethanol preservative (Fig. 6B). Ectosomal skeleton, irregular, composed of dichotriaene cladomes, aligned tangentially to surface with rhabdome pointing in towards choanosome (Figs 6C, D, 7J, K), with scattered microscleres (Fig. 7B, E). Choanosomal skeleton composed of zygosed dicranoclone desmas (Fig. 6E, F) between which are large subdermal spaces and scattered microscleres (Fig. 7C–G). Megascleres, desmas, triaenes, microxeas; dicranoclone desmas, 211–354 × 176–261 µm (N15). Dichotriaenes in a large size range with long and short rhabdomes: smallest dichotriaenes, cladome, 72–82–106 µm, rhabdome 93–110–130 µm (N25), largest dichotriaenes, cladome, 130–186–278 µm, rhabdome, 208–314–558 µm (N25) (Figs 6D, 7J, K); microxeas, 327–630–887 × 4.8–6.6–10.8 µm (N6) (Fig. 7H, I). Microscleres, metasters with long, pointed, acanthose rays, 16.8–29.9–42 × 14.3–23.6–30.7 µm (N21), amphiasters and spirasters with short, pointed, microspined rays, 8.2–11.9–15.1 × 6.4–10.1–13.5 µm (N21). Figure 6. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, in situ picture (121 m). B, deck picture with inner surface: left part and outer surface: right part. C, D, thick sections of the ectosomal part showing diverse dichotriaenes penetrating to the choanosome. E, F, scanning electron microscopy pictures of cross-section showing articulated dicranoclone desmas. Figure 6. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, in situ picture (121 m). B, deck picture with inner surface: left part and outer surface: right part. C, D, thick sections of the ectosomal part showing diverse dichotriaenes penetrating to the choanosome. E, F, scanning electron microscopy pictures of cross-section showing articulated dicranoclone desmas. Figure 7. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, B, thick sections showing the accumulation of streptaster at the subectosomal region (A) and around channel openings (B). C, bundle of metasters/amphiasters. D–G, diverse streptaster with long pointed rays. H, I, amphioxea. J, K, ectosomal dichotriaene. Figure 7. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, B, thick sections showing the accumulation of streptaster at the subectosomal region (A) and around channel openings (B). C, bundle of metasters/amphiasters. D–G, diverse streptaster with long pointed rays. H, I, amphioxea. J, K, ectosomal dichotriaene. Etymology: Named after the type locality, Floreana Island. DNA barcodes: We sequenced COI, 28S (C1-D2) and 18S (1303 bp) from the holotype; GenBank accession numbers: COI KY652816, 28S KY652784, 18S KY652833. SBD record no. 1715. Remarks: Corallistes floreana sp. nov. resembles C. isabela, previously described from the Galápagos Islands, but there are notable differences in the size and ornamentation of the megascleres. The dicranoclone desmas in C. floreana sp. nov. are smaller and tuberculate rather than knobbed as in C. isabela (Table 2). Furthermore, the alternating sizes of the C. floreana sp. nov. cladomes differ by their ratio from C. isabela (Table 2). Amphiasters / spirasters are smaller in size as well (Table 2). Pairwise sequence differences of 1.2% (28S) and 0.8% (COI) between C. floreana sp. nov. and C. isabela (HBOM 003:02013) further confirm integrity of these two species. Table 2. Spicule sizes in micrometres of Corallistes floreana sp. nov. compared to different Corallistes typus material and Corallistes isabela Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Information for Corallistes isabela taken from table 9 of Desqueyroux-Faúndez & Van Soest (1997). clad = cladome; rhabd = rhabdome. View Large Table 2. Spicule sizes in micrometres of Corallistes floreana sp. nov. compared to different Corallistes typus material and Corallistes isabela Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Information for Corallistes isabela taken from table 9 of Desqueyroux-Faúndez & Van Soest (1997). clad = cladome; rhabd = rhabdome. View Large Family Theonellidae von Lendenfeld, 1903 Genus R acodiscula Zittel, 1878 R acodiscula radixa sp. nov. (Figs 8, 9; Table 3) Diagnosis: Racodiscula with a deep, apical, cavity/osculum, into which subdermal canals converge from the margins; desmas are irregular, loosely zygosed tetraclone desmas with root-like protrusions. Type material: Holotype: HBOM 003:02006, Coll. JSL-I dive 3913. [22 October 1995, Galápagos, N-Side 1.25 NM W of point Vincente Roca NW Coast, Isabela Island, 0°02′55″S, 91°35′16″W, 378 m]. Comparative material: Racodiscula seymoura sp. nov. HBOM 003:02014 (396 m) (see below), Galápagos, Seymour Island, 0°22′51″S, 90°16′06″W, Coll. JSL-I dive 3529; HBOM 003:02007 (319 m), Galápagos, Isabela Island, 0°14′31″S, 91°24′56″W, Coll. JSL-I dive 3015. Racodiscula borisi sp. nov. HBOM 003:02011 (242 m) (see below), Galápagos Island, Tortuga Island, 1°4′59″S, 90° 51′56″W, Coll. JSL-I dive 3923; HBOM 003:02001 (215 m), Galápagos Island, San Cristobal Island, 0°43′08″S, 89°27′14″W, Coll. JSL-I dive 3905. Type locality: Isabela Island, Galápagos Islands (378 m) (Fig. 1). Distribution: Only known from type locality. Habitat: Attached to hard substratum at a depth of 378 m. Description: Morphology, massive subspherical folded with a deep, apical, cavity/osculum, about 1 cm longest diameter, situated on the elevated apex (Fig. 8A, B); dimension of the holotype, about 7 cm diameter, about 5 cm high (Fig. 8A, B). Texture, medium hard. Surface, smooth, with branched, meandering, subdermal canals, converging from the margins (Fig. 8B) towards the apical cavity; lateral inhalant surface with pores, 450–500 µm diameter. Colour, white in cream (Fig. 8A), dark cream in ethanol preservative (Fig. 8B). Ectosomal skeleton, detachable in places due to large subdermal canals, composed of phyllotriaene cladomes, stacked and overlapping, (Figs 8C–F, 9G), interspersed with microrhabds and other microscleres (Fig. 9B, E) forming a thick outer layer; tylostyles are located at the subectosomal region (Fig. 8D–F). Choanosomal skeleton contains loosely zygosed tetraclone desmas (Fig. 8D), loose bundles of oxeas and scattered microscleres (Fig. 9C, D). Megascleres, tetraclone desmas, phyllotriaenes and oxeas: tetraclone desmas, relately smooth, from which emerge thin, root-like branches, 405–532 × 254–276 µm (N5) (Fig. 8C–F); phyllotriaenes with a leafy cladome, 305–389–494 µm (N20), short, irregular rhabdome, 93–116–160 × 32–42–55 µm (N20) (Fig. 9A, B, G); tylostyles, 450–315–605 µm (N15) (Fig. 9A). Microscleres, acanthose microrhabds with irregularly distributed spined tubercles, 21–25–30 × 4 µm (N30); metasters with long rays, 15.6–18.5–20.2 × 11.3–14.7–19.2 µm (N20). Figure 8. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, in situ picture (378 m). B, deck picture. C, D, thick section of the ecto- and choanosome, representing disorganized tetraclone, phyllotriaenes with rhabdomes protruding perpendicular from the surface and boundes of tylostyles (tylo) and oxeas (oxe). Gap between ecto- and choanosome (C) is an artefact from sectioning. E, F, thick section showing the poor articulation of tetraclone desmas and diverse streptasters. Figure 8. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, in situ picture (378 m). B, deck picture. C, D, thick section of the ecto- and choanosome, representing disorganized tetraclone, phyllotriaenes with rhabdomes protruding perpendicular from the surface and boundes of tylostyles (tylo) and oxeas (oxe). Gap between ecto- and choanosome (C) is an artefact from sectioning. E, F, thick section showing the poor articulation of tetraclone desmas and diverse streptasters. Figure 9. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in between (B), forming a thick crust on the surface. C, metaster. D, spiraster. E, acanthorhabds. F, tylostyles. G, ectosomal phyllotriaene. Figure 9. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in between (B), forming a thick crust on the surface. C, metaster. D, spiraster. E, acanthorhabds. F, tylostyles. G, ectosomal phyllotriaene. Etymology: Named for the root-like ornamentation of the tetraclone desmas in this species (radix, root; Latin). DNA barcodes: we sequenced COI, 28S (C1-D2) and 18S (1,623 bp) of the holotype. GenBank accession number: COI KY652819, 28S KY652788, 18 KY652836. SBD record no. 1720. Remarks: Racodiscula radixa sp. nov. is differentiated from the two other new Racodiscula species from the Galápagos Islands, R. seymoura sp. nov. and R. borisi sp. nov. (see species description below), in having the cavity/oscula at an elevated centre with vein-like furrows to the margins, while R. seymoura sp. nov. have the oscula in a centred depression and R. borisi sp. nov. has a perfectly circular and centred oscula on the surface without an elevated/compressed centre. The oscula of all three new Racodiscula species described are different to Racodiscula asteroidesZittel, 1878 in having only one opening, while R. asteroides has multiple oscules in a shallow depression (Pisera & Lévi, 2002b). However, intraspecific variations of this character cannot be excluded. Thick strongyles as found in R. seymoura sp. nov. and R. borisi sp. nov. (see below) could not be detected in any of the spicule preparations for R. radixa sp. nov., which may indicate that this second type of microsclere was secondarily lost. (Table 3). The cladome of phyllo- to discotriaenes is smaller than in R. seymoura sp. nov. and R. borisi sp. nov. The rhabdome is also slightly shorter than in R. seymoura sp. nov. but larger than in R. borisi sp. nov. (Table 3). R. radixa sp. nov. has irregularly rather loose root-like desmas with shorter branches than R. seymoura sp. nov. In contrast, R. borisi sp. nov. shows strong tuberculated tetraclone desmas similar to R. asteroides. Differences were also found in our molecular analyses (Fig. 5). R. radixa sp. nov. differ to R. seymoura sp. nov. with 3.3% (COI), 0.3% (28S) and 1.8% (18S) and to R. borisi sp. nov. with 4.8% (COI), 0.5% (28S), 2.3% (18S). The sister-group relationship of R. radixa sp. nov. and R. seymoura sp. nov. is supported [posterior probability (PP) = 0.97 and bootstrap support (BS) = 80]. The clade with the three new Galápagos Racodiscula species is well supported by PP = 0.90, but not by BS. The sister-group relationship of this clade with R. asteroides and Racodiscula sp. from the Caribbean is well supported (Fig. 5). Table 3. Morphology and spicule sizes of the three new Racodiscula species from the Galápagos Islands compared to Racodicula asteroides Zittel, 1878 (neotype) Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Measurements are given in micrometres, if not otherwise stated. Measurements for new species are given for the holotype (bold) and for R. asteroides (neotype) taken from Pisera & Lévi (2002b). View Large Table 3. Morphology and spicule sizes of the three new Racodiscula species from the Galápagos Islands compared to Racodicula asteroides Zittel, 1878 (neotype) Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Measurements are given in micrometres, if not otherwise stated. Measurements for new species are given for the holotype (bold) and for R. asteroides (neotype) taken from Pisera & Lévi (2002b). View Large Racodiscula seymoura sp. nov. (Figs 10, 11; Table 3) Diagnosis: Racodiscula with one oval to circular cavity/osculum (0.7 cm) opening in a shallow centred depression; choanosomal spicules are zygosed root-like tetraclone desmas with long and thin branches; choanosomal spicules are thick strongyles. Type material: Holotype: HBOM 003:02014, Coll. JSL-I dive 3529, [30 October 1995, Galápagos, 66 NM NE of Seymour Island, 0°22′51″S, 90°16′06″W, 396 m]. Paratype: HBOM 003:02007 (319 m), Galápagos, N-Side 1.25 NM W of PTA Vincente Roca NW Coast, Isabela Island, 0°14′31″S, 91°24′56″W, Coll. JSL-I dive 3915. Comparative material: Racodiscula radixa sp. nov. HBOM 003:02006. Racodiscula borisi sp. nov. HBOM 003:02011 (242 m), Galápagos Island, Tortuga Island, 1°4′59″S, 90°51′56″W, Coll. JSL-I dive 3923; HBOM 003:02001 (215 m), Galápagos Island, San Cristobal Island, 0°43′08″S, 89°27′14″W, Coll. JSL-I dive 3905. Type locality: Seymour Island, Galápagos Islands (396 m) (Fig. 1). Distribution: Known from type locality, Seymour Island, and Isabela Island, Galápagos Islands. Habitat: Attached to hard substratum, depth range, 319–396 m. Description: Morphology, massive subspherical with an apical cavity/osculum opening on top of the surface in a shallow centred depression (Fig. 10A, B). Dimension of the holotype is about 7 cm in diameter and about 5 cm in height (Fig. 10A, B). Texture, medium hard. Surface, smooth and uneven (Fig. 10B). Colour, white in vivo (Fig. 10A) and light brownish in ethanol (Fig. 10B). Ectosomal skeleton contains phyllo- to discotriaenes (Figs 10B, 11A, F) with acanthorhabds in between and on top of this layer (Fig. 11A, B, D). Tylostyles are located at the subectosomal region (Fig. 11A, B, I). Choanosomal skeleton contains articulated tetraclone desmas, which form a dense root-like skeleton (Fig. 10C–F). Spinose metasters (Fig. 11C) and amphiasters (Fig. 11E). Oxeas (Fig. 11A, G) with pointed tips and thick strongyles (Fig. 11H). Megascleres are tetraclone desmas with root-like structures, measuring 632–736 × 395–406 µm (N15) (Fig. 10C–F). Phyllo- to discotriaenes with cladomes 447–536–636 µm (N20) and rhabdomes of 121–150–185 × 22–37–40 µm (N20) (Figs 10D, 11F). Tylostyles are 434–317–719 µm long (N15) (Fig. 11I). Oxea with pointed tips 120–125–134 × 4–5 µm (N7). Thick strongyles with rounded ends 95–100–112 × 7–10 µm (N4); Microscleres are massive acanthorhabds with irregularly distributed spined tubercles 5–17–20 × 3–4 µm (N30). Spinose metaster/amphiasters with long rays 17.3–20.5–22.2 × 12.5–15.6–20.0 µm. Figure 10. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, in situ picture of the paratype (378 m). B, deck picture of the paratype. C, outer surface of the choanosomal skeleton from the holotype. D, tetraclone desmas with phyllotriaene in situ of the holotype. E, F, detailed view of the articulated tetraclone desmas from the holotype. Figure 10. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, in situ picture of the paratype (378 m). B, deck picture of the paratype. C, outer surface of the choanosomal skeleton from the holotype. D, tetraclone desmas with phyllotriaene in situ of the holotype. E, F, detailed view of the articulated tetraclone desmas from the holotype. Figure 11. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in subectosomal regions (B). C, metaster. E, amphiaster. D, acanthorhabds in various sizes. F, ectosomal phyllotriaene. G, ecto- to subectosomal oxea with pointed tips. H, thick strongyle. I, choanosomal style. Figure 11. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in subectosomal regions (B). C, metaster. E, amphiaster. D, acanthorhabds in various sizes. F, ectosomal phyllotriaene. G, ecto- to subectosomal oxea with pointed tips. H, thick strongyle. I, choanosomal style. Etymology: Named after the type locality: Seymour Island (Galápagos Islands). DNA barcodes: We sequenced all three markers of the holotype and 28S (C1-D2) and 18S of the paratype. 28S sequences of type material were identical as well as were the 18S. GenBank accession numbers: COI KY652820, 28S KY652789–KY652790, 18S KY652837–KY652838. SBD record no. 1721–1722. Remarks: Apart from the differences observed in the oscula opening (see remarks for R. radixa sp. nov. and Table 3) Racodiscula seymoura sp. nov. show clear spiculation differences compared to the two other Racodiscula species described in this study. The presence of oxeas and strongyles and the root-like tetraclone desmas with long thin branches (Fig. 10F) along with slightly differences observed in the sizes of phyllo- to discotrianes (Table 3) separates this species from R. radixa sp. nov. Tetraclone desmas, which are not tuberculated in R. seymoura sp. nov. but strongly tuberculated in R. borisi sp. nov., and the longer and thicker rhabdome size clearly separate this new species from R. borisi sp. nov. Molecular markers suggest that R. seymoura sp. nov. is more closely related to R. radixa sp. nov. than to R. borisi sp. nov. (Fig. 5), and positioned within the clade of Racodiscula from the Galápagos. This is in accordance with the observed morphological differences. R acodiscula borisi sp. nov. (Figs 12, 13; Table 3) Diagnosis: Racodiscula with one perfectly circular apical cavity/osculum (0.8 cm) located at the centre; choanosomal spicules are articulated tetraclone desmas with short thick and strongly tuberculated branches, choanosomal spicules are thick strongyles with rounded tips. Type material: Holotype: HBOM 003:02001, Coll. JSL-I dive 3905 [18 October 1995, Galápagos, 5NM NE of Kicker Rock, San Cristobal Island, 0°43′08″S, 89°27′14″W, 215 m]. Paratype: 003:02011 (242 m), Galápagos, 4 NM SE of Tortuga Island, 1°04′59″S, 90°51′56″W, Coll. JSL-I dive 3923. Comparative material: Racodiscula radixa sp. nov. HBOM 003:02006. Racodiscula seymoura sp. nov. HBOM 003:02014; HBOM 003:02007. Type locality: San Cristobal Island, Galápagos Islands (215 m) (Fig. 1). Distribution: Known from the type locality and Tortuga Island, Galápagos. Habitat: Attached to hard substratum, depth range 215–242 m. Description: Morphology, massive subspherical with a centralized circular apical cavity/osculum opening on top of the surface (Fig. 12A, B). Dimension of the holotype is 5.5 cm in diameter and about 4–5 cm high (Fig. 12A, B). Texture, medium hard. Surface, smooth (Fig. 12B). Colour, white in vivo (Fig. 12A) and beige in ethanol (Fig. 12B). Ectosomal skeleton contains phyllo- to discotriaenes (Fig. 13B, F) with acanthorhabds in between and on top of this layer (Fig. 13A, B, E). Tylostyles are located at the subectosomal region (Fig. 12A, B, I). Choanosomal skeleton contains articulated tetraclone desmas, which form a dense skeleton (Fig. 12C–F). Spinose metaster/amphiasters (Fig. 13C, D). Oxeas (Fig. 13G) with pointed tips and thick strongyles (Fig. 13H). Megascleres, tetraclone desmas with short branches measuring 540–650 × 385–400 µm (N15) (Fig. 12E). Phyllo- to discotriaenes with cladomes 395–504–605 µm (N25) and rhabdomes of 20–66–93 × 18–21–25 µm (N10) (Fig. 13B, F). Tylostyles are 440–410–904 µm (N15) (Fig. 13I). Oxeas with pointed tips 140–176–204 × 4–5 µm (N7). Thick strongyles with round ends 95–194–112 × 15–28 µm (N4); Microscleres are massive acanthorhabds with irregularly distributed spined tubercles 12–19–27 × 3–4 µm (N30). Spinose streptaster/amphiasters with long rays 12.0–14.5–17.2 × 11.5–15.4–20.0 µm. Figure 12. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, in situ picture of the holotype (215 m). B, deck picture of the holotype. C, outer surface of the choanosomal skeleton with pore openings. D, tetraclone desmas with tylostyles lying on top. E, F, detailed views of the articulated tetraclone desmas from the holotype. Figure 12. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, in situ picture of the holotype (215 m). B, deck picture of the holotype. C, outer surface of the choanosomal skeleton with pore openings. D, tetraclone desmas with tylostyles lying on top. E, F, detailed views of the articulated tetraclone desmas from the holotype. Figure 13. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, B, thick sections of the ectosomal to choanosomal region showing phyllotriaenes, with acanthorhabds and bundles of tylostyles. C, D, scanning electron microscopy (SEM) of diverse amphiaster/metaster. E, SEM of acanthorhabd. F, light microscopy picture of ectosomal phyllotriaene. G, light microscopy picture of ecto- to subectosomal oxea with pointed tips. H, thick strongyle with rounded ends. I, SEM of choanosomal style. Figure 13. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, B, thick sections of the ectosomal to choanosomal region showing phyllotriaenes, with acanthorhabds and bundles of tylostyles. C, D, scanning electron microscopy (SEM) of diverse amphiaster/metaster. E, SEM of acanthorhabd. F, light microscopy picture of ectosomal phyllotriaene. G, light microscopy picture of ecto- to subectosomal oxea with pointed tips. H, thick strongyle with rounded ends. I, SEM of choanosomal style. Etymology: Named in memory of the first author’s friend Dipl. Biol. Boris Knerr (†20 July 2012), for encouraging her scientific curiosity in sponge taxonomy. DNA barcodes: We sequenced COI, 28S (C1-D2) and 18S of the holotype and paratype. All three markers show identical sequences. GenBank accession numbers: COI KY652821–KY652822; 28S KY652791–KY652792; 18S KY652834–KY652835. SBD record no. 1723, 1724. Remarks: The most obvious key diagnostic character which differentiates this new species from the other two new Racodiscula species described here, is the presence of tetraclone desmas, which are strongly articulated with short root-like branches showing a strong tuberculation (Fig. 12C–F). This strong tuberculation of the tetraclone desmas is not present in the two other new species described. Racodiscula asteroides also shows sparsely tuberculated rays and strongly tuberculated tips of tetraclone desmas; however, it lacks oxeas and strongyles (Pisera & Lévi, 2002b). The rhabdome of phyllo- to discotrianes from R. borisi sp. nov. is much shorter and a bit thinner in size compared to the others newly described species (Table 3). Oxeas and strongyles appear larger in size compared to R. seymoura sp. nov. R. borisi sp. nov. groups within the Racodiscula clade from the Galápagos and forms a sister-group relationship to the other two species (Fig. 5). Suborder Spirophorina Bergquist & Hogg, 1969, Family Tetillidae Sollas, 1886 Genus C raniella Schmidt, 1870 C raniella wolfi sp. nov. (Fig. 14) Diagnosis: Craniella with worm-like long (62–91 µm) sigmaspires. Type material: Holotype: HBOM 003:02004, Coll. JSL-I dive 3909 [20 October 1995, Galápagos, S. Coast, approx. 5 NM NE of Wolf Island, 1°21′28″N, 91°48′41″W, 355 m]. Type locality: Wolf Island, Galápagos Islands (355 m) (Fig. 1). Distribution: Only known from type locality. Habitat: Attached to hard substratum, 355 m. Description: Morphology, globular small hispid sponge with small apical oscule and 1-mm-thick cortex (Fig. 14A, B). Dimension of the holotype is 3 cm in diameter and about 4–5 cm high (Fig. 14A, B). Texture, soft. Surface, conulose and hispid. Colour, white in vivo (Fig. 14A) and light brown in ethanol (Fig. 14B). Ectosomal skeleton contains bundles of oxeas with few protriaenes sticking out of these bundles. No anatriaenes detected. Choanosomal skeleton contains oxeas (Fig. 14C, D) and abandoned uncoiled sigmaspires. Megascleres are thick oxeas measuring 134–356–627 × 8–15–25 µm (N30) (Fig. 14C, D). Protriaenes, equal clads measuring 121–150–177 µm (N11) and rhabdome 3380–4864–5640 × 19–27–30 µm (N11). Anatriaenes are not present in our spicule preparations. Microscleres are thin and uncoiled long sigmaspires 62–79–91 × 1–3 µm (N30) (Fig. 14G). Figure 14. View largeDownload slide Craniella wolfi sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02004. A, in situ picture (355 m). B, deck picture. C, oxea. D, detailed view of oxea. E, protriaene with equal clads. F, details of protriaene. G, long sigmaspires. Figure 14. View largeDownload slide Craniella wolfi sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02004. A, in situ picture (355 m). B, deck picture. C, oxea. D, detailed view of oxea. E, protriaene with equal clads. F, details of protriaene. G, long sigmaspires. Etymology: Named after point of origin: Wolf Island (Latin, wolfi). DNA barcodes: We sequenced COI, 28S (C1-D2) and 18S (1,670 bp) of the holotype. GenBank accession number: COI KY652825, 28S KY652795, 18S KY652840. SBD record no. 1727. Remarks: The absence of porocalices and the presence of a conspicuous double-layered cortex clearly places this species into the genus Craniella. There are currently 34 valid Craniella species (P. Cárdenas, unpublished results), ten of which have lost their sigmaspires. The remaining 24 species have classical C- or S-shaped sigmaspires 8–30 µm long; none have these unusually worm-like long (62–91 µm) sigmaspires. Molecular investigations confirm that C. wolfi sp. nov. is closely related to Craniella aff. zetlandica (ZMBN 85239) from Western Norway (Korsfjord), a topology which is highly supported (PP = 1, BS = 100) (Fig. 5). Family Scleritodermidae Sollas, 1888 Genus S cleritoderma Sollas, 1888 Emended diagnosis: Scleritodermidae with ectosomal acanthostrongyles or acanthorhabds, occasionally with ectosomal tylostyles, and sigmaspires as microscleres (modified after Pisera & Lévi, 2002c). Remarks: Our new species has all the characters of typical Scleritoderma, for example, rhizoclone desmas, sigmaspire microscleres occurring in the choanosome; however, this is the first time tylostyles have been observed. Careful analysis demonstrated, that these are clearly not a contamination (see remarks below). The occurrence of tylostyles resulted in the emended diagnosis. S cleritoderma tortuga sp. nov. (Figs 15, 16) Diagnosis: Scleritoderma with tylostyles vertically protruding from the skeletons surface. Synonymy: Scleritodermida sp. 1 and Scleritodermida sp. 2 (Schuster et al., 2017, additional file 1, Fig. 4). Type material: Holotype: HBOM 003:02012, Coll. JSL-I dive 3923 [27 October 1995, Galápagos, SW Coast, Caleta Webb, 0.5 NM offshore of Tortuga Island, 1°04′59″S, 90°51′56″W, 242 m]. Paratype: HBOM 003:02009 (239 m), Galápagos, Isabela Island, 0°47′04″S, 91°26′32″W, Coll. JSL-I dive 3919. Comparative material: Scleritoderma camusi Lévi & Lévi, 1983, Holotype MNHN DCL 2787, paratypes: MNHN DCL 2788, 355–30 m, Norfolk Ridge. S. camusi (described in Schlacher-Hoenlinger, Pisera & Hooper, 2005), Norfolk Ridge 278–410 m, QMG318549, QMG318706. S. nodosum Thiele, 1900, MNH DCL 3233, 90–110 m, Philippines (described in Lévi & Lévi, 1989). S. flabelliforme Sollas, 1888 (described in Schlacher-Hoenlinger et al., 2005), Norfolk Ridge, 458–68 m, QMG318641, QMG318658, QMG318664. Type locality: Tortuga Island, Galápagos Islands (242 m) (Fig. 1). Distribution: Known from type locality and Isabela Island. Habitat: Attached to hard substratum, depth range 239–242 m. Description: Morphology, massive and flabelliform (Fig. 15A, B). Dimension of the holotype is 8 cm in diameter and about 3–5 cm in height (Fig. 15A, B). Texture, stony and hard. Surface, covered with small oscule openings 0.2–0.3 mm in diameter. Colour, beige in vivo (Fig. 15A) and light brown in ethanol (Fig. 15B). Ectosomal skeleton contains a thick layer of acanthorhabds (Fig. 15D) on the surface. The heads of tylostyles project beyond the surface layer (Fig. 15C, E). Very rare sigmaspires. Choanosomal skeleton composed of dense articulated slightly thorny rhizoclones (Fig. 16). Large oscula openings on the surface of the choanosomal skeleton 300–400 µm in diameter (Fig. 16A). Megascleres are rhizoclone desmas 225–350 µm in diameter and tylostyles measuring 153–256–316 µm (N15) (Fig. 15E, H). Microscleres are C-shaped thick spinose sigmaspires 9–13 × 1–2 µm (N3) (Fig. 15F) and slightly curved acanthostrongyles 61–84–101 × 5–6 µm (N30) (Fig. 15D, G). Figure 15. View largeDownload slide Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, in situ picture 242 m. B, deck picture. C, thick section of the ectosome and part of the choanosome. Arrows pointing to tylostyles (tylo). D, detailed view the ectosome with a layer of acanthorhabds (acant). E, ectosomal view with tylostyles (tylo). F, thick sigmaspire. G, spinose acanthorhabd. H, detailed views of tylostyles. Figure 15. View largeDownload slide Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, in situ picture 242 m. B, deck picture. C, thick section of the ectosome and part of the choanosome. Arrows pointing to tylostyles (tylo). D, detailed view the ectosome with a layer of acanthorhabds (acant). E, ectosomal view with tylostyles (tylo). F, thick sigmaspire. G, spinose acanthorhabd. H, detailed views of tylostyles. Figure 16. View largeDownload slide Diverse desmas of Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, overview of the choanosomal skeleton with several oscules. B, detailed view of one oscula opening. C, a loose rhizoclone desma. D, rhizoclone desmas with a tylostyle. E, F, detailed view of spines of rhizoclones with acanthorhabds. Figure 16. View largeDownload slide Diverse desmas of Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, overview of the choanosomal skeleton with several oscules. B, detailed view of one oscula opening. C, a loose rhizoclone desma. D, rhizoclone desmas with a tylostyle. E, F, detailed view of spines of rhizoclones with acanthorhabds. Etymology: Named after the type locality: Tortuga Island. DNA barcodes: COI sequences as in Schuster et al. (2017), 28S (C1-D2) of the holotype and paratype as well as 18S (1770 bp) of the paratype (GenBank accession numbers: 28S KY652801, KY652802; 18S KY652839, SBD record no. 1730, 1731). The 28S sequences were identical. The obtained COI sequence of HBOM 003:02012 was shorter (393 bp) compared to HBOM 003:02009 (630 bp). The 393 bp were identical (100%). Remarks: Our new species Scleritoderma tortuga sp. nov. differs from the four other Scleritoderma species, S. camusi Lévi & Lévi, 1983, S. cyaneum Van Soest & Stentoft, 1988, S. flabelliforme Sollas, 1888 and S. nodosum Thiele, 1900 (Van Soest et al., 2017), by the presence of tylostyles. Since tylostyles have never been observed in Scleritoderma species before, we first questioned if these could be due to a contamination from another sponge. However, we are convinced here that these are proper to the specimen, as we observed at least 15 tylostyles in both the holotype and paratype sections, all orientated in a vertical position protruding from the skeleton’s surface (Fig. 15C, E). Additionally, in all of our preparations, no other spicule types were found, which may have indicated a possible contamination from another sponge. Furthermore, our concatenated molecular analyses including sequences of all known Scleritoderma species (Fig. 5) indicate that Scleritoderma tortuga sp. nov. forms a sister-group relationship to S. nodosum from the Indo-Pacific (17–27 m). Additional supportive evidence of the new species is given by the presence of a 1109 bp long ‘intron 723’ located within the COI region (Schuster et al., 2017) in both specimens. Our concatenated phylogenetic analysis supports the monophyly of the genus Scleritoderma which was first reported by Schuster et al. (2017) on single mitochondrial data. Based on the presence of tylostyles in S. tortuga sp. nov. the definition of Scleritoderma was amended. Tylostyles are actually not uncommon within Scleritodermidae, for example, they are known to occur in Aciculites and may have been overlooked in other species. We therefore recommend that future revisions of this family reassess the presence of tylostyles within this group. DISCUSSION AND CONCLUSION Previous knowledge on tetractinellids from the Galápagos archipelago was scarce (about 11 species described) and mainly limited to shallow-water habitats (<100 m) (Wilson, 1904; de Laubenfels, 1939; Desqueyroux-Faúndez & Van Soest, 1997). In fact, only one single desma-bearing astrophorid species (Corallistes isabela) and one species from the suborder Spirophorina (Cinachyrella globulosa), both endemic to the Galápagos, were described by Desqueyroux-Faúndez & Van Soest (1997). Applying the morphological as well as the phylogenetic species concept we enlarge the current knowledge with the discovery of five new deep-water desma-bearing Astrophorina (Neophrissospongia galapagoensis sp. nov., Corallistes floreana sp. nov., Racodiscula radixa sp. nov., Racodiscula seymoura sp. nov. and Racodisucla borisi sp. nov.) and report for the second time the occurrence of Corallistes isabela. Additionally, Craniella wolfi sp. nov. of the family Tetillidae and Scleritoderma tortuga sp. nov. of the family Scleritodermidae (Spirophorina) are described. The definition of the genus Scleritoderma required subsequent amendment. All seven new species are most likely endemic to the Galápagos islands. Statistical methods such as Principle Component Analysis (PCA) or haplotype networks which may help discriminate the closely related species C. floreana np. nov. from C. isabela and R. radixa sp. nov. from R. seymoura sp. nov. were not feasible in this study, due to the low sample size (only one or two samples per species). A further statistical probabilistic based test is the coalescent theory which is based on the evolutionary history of alleles. The application of this test is widespread in integrative taxonomic studies to analyze species structures (Fujita et al., 2012). However, these methods recently have been shown to overestimate structure and consequently falsely suggest speciation events (Sukumaran & Knowles, 2017). This test again requires a higher sample size than given by our data. Therefore, we refrained from using multispecies coalescent analysis in this study as it may provide unambiguous results. Nevertheless, this study provides the first comprehensive dataset to investigate the deep-water ‘lithistid’ fauna of the Galápagos by providing descriptions of unique specimens collected in one of the most remote marine areas. Our phylogenetic analyses (Fig. 5) including other material from the Caribbean, Central and West Pacific provide strong evidence of biogeographical differences and support an apparent high level of demosponge endemism in this region (Desqueyroux-Faúndez & Van Soest, 1997), but should be corroborated by further sampling from other areas of the Eastern Pacific. The long paleontological history of most families like Corallistidae ~155 Myr and Theonellidae ~167 Myr, as well as rhizoclone desmas present since the Early Paleozoic (e.g. Pisera, 2002), may indicate that the current tetractinellid biodiversity in the Eastern Pacific is much higher than previously assumed. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Specimen list. [Version of Record, published online 23 March 2018; http://zoobank.org/urn:lsid:zoobank.org:pub:20FB9570-C49B-4B2A-ADFA-684F5495A0BF] ACKNOWLEDGEMENTS Financial support for this study was provided by the German Science Foundation (DFG ER 611/3-1, DFG Wo869/15-1). The LMUMentoring and the HELGE AX:Son JOHNSON STIFTELSE provided funding for AS to visit HBOI (Florida, USA) and NIWA (National Institute of Water and Atmospheric Research, Auckland and Wellington in New Zealand). We greatly thank the colleagues Amy Wright, John Reed and Megan Conkling from HBOI for sharing material, help in the collection, shipping from Florida and providing underwater and deck pictures for the new described species as well as the crew of the RV ‘Johnson Sea Link I’ submersible for collection of the samples. Specimens were supplied by the NIWA Invertebrate Collection (NIC); we are particularly grateful to Sadie Mills and Kareen Schnabel for their assistance in Wellington and diligent assistance with loans. Several specimens (NIWA 110983, 110214, 18823) were collected by the Coral Reef Research Foundation under contract to the US National Cancer Institute. This is a contribution of the Coral Reef Research Foundation, Palau. We thank Roland Melzer (ZSM, Zoologische Staatssammlung München) for the introduction to the SEM. We thank Gabrielle Büttner and Simone Schätzle (Department of Earth & Environmental Sciences, LMU Munich, Germany) for sequencing assistance. PC is supported by the European Union’s Horizon 2020 research and innovation program through the SponGES project (grant agreement no. 679849). MK’s participation was funded by NIWA under Coasts and Oceans Research Programme 2 Marine Biological Resources: Discovery and definition of the marine biota of New Zealand (2017/2018 SCI). REFERENCES Bergquist PR , Hogg JJ . 1969 . Free amino acid patterns in Demospongiae: a biochemical approach to sponge classification . Cahiers de Biologie Marine 10 : 205 – 220 . Cárdenas P , Pérez T , Boury-Esnault N . 2012 . Sponge systematics facing new challenges . Advances in Marine Biology 61 : 79 – 209 . Google Scholar CrossRef Search ADS PubMed Cárdenas P , Rapp HT , Schander C , Tendal O . 2009 . Molecular taxonomy and phylogeny of the Geodiidae (Porifera, Demospongiae, Astrophorida) – combining phylogenetic and Linnaean classification . Zoologica Scripta 39: 1 – 18 . Carella M , Agell G , Cárdenas P , Uriz MJ . 2016 . Phylogenetic reassessment of Antarctic Tetillidae (Demospongiae, Tetractinellida) reveals new genera and genetic similarity among morphologically distinct species . PloS ONE 11: e0160718 . Carvalho FC , Pomponi SA , Xavier JR . 2015 . Lithistid sponges of the upper bathyal of Madeira, Selvagens and Canary Islands, with description of a new species of Isabella . Journal of the Marine Biological Association of the United Kingdom 95 : 1287 – 1296 . Google Scholar CrossRef Search ADS Chavez FP , Brusca RC . 1991 . The Galápagos Islands and their relation to oceanographic processes in the tropical pacific . In: James M , ed. Galapagos marine invertebrates . New York : Plenum , 9 – 33 . Google Scholar CrossRef Search ADS Chombard C , Boury-Esnault N , Tillier S . 1998 . Reassessment of homology of morphological characters in tetractinellid sponges based on molecular data . Systematic Biology 47 : 351 – 366 . Google Scholar CrossRef Search ADS PubMed de Laubenfels MW . 1939 . Sponges collected on the presidential Cruise of 1938 . Smithsonian Miscellaneous Collections 98 : 1 – 7 . Google Scholar CrossRef Search ADS Desqueyroux-Faúndez R , Van Soest RWM . 1997 . Shallow waters demosponges of the Galápagos Islands . Revue Suisse de Zoologie 104 : 379 – 467 . Google Scholar CrossRef Search ADS Ekins M , Erpenbeck D , Wörheide G , Hooper JNA . 2016 . A new species of lithistid sponge hiding within the Isabella mirabilis species complex (Porifera: Demospongiae: Tetractinellida) from seamounts of the Norfolk Ridge . Zootaxa 4136 : 433 – 460 . Google Scholar CrossRef Search ADS PubMed Feingold JS . 2011 . El Niño, La Niña and ENSO . In: Hopley D , ed. Encyclopedia of modern coral reefs, part 5 . Dordrecht : Springer , 365 – 368 . Google Scholar CrossRef Search ADS Fujita MK , Leaché AD , Burbrink FT , McGuire JA , Moritz C . 2012 . Coalescent-based species delimitation in an integrative taxonomy . Trends in Ecology & Evolution 27 : 480 – 488 . Google Scholar CrossRef Search ADS PubMed Geist D , Diefenbach BA , Fornari DJ , Kurz MD , Harpp K , Blusztajn J . 2008 . Construction of the Galápagos platform by large submarine volcanic terraces . Geochemistry, Geophysics, Geosystems 9 : 1 – 27 . Google Scholar CrossRef Search ADS Hall KA , Ekins MG , Hooper JNA . 2014 . Two new desma-less species of Theonella Gray, 1868 (Demospongiae: Astrophorida: Theonellidae), from the Great Barrier Reef, Australia, and a re-evaluation of one species assigned previously to Dercitus Gray, 1867 . Zootaxa 3814 : 451 – 477 . Google Scholar CrossRef Search ADS Holden JC , Dietz RS . 1972 . Galápagos Gore, NazCoPac Triple Junction and Carnegie/Cocos Ridges . Nature 235 : 266 – 269 . Google Scholar CrossRef Search ADS James MJ (ed.). 1991 . Galápagos marine invertebrates. Taxonomy, biogeography, and evolution in Darwin’s Islands . New York : Springer Science+Business Media . Google Scholar CrossRef Search ADS Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , Sturrock S , Buxton S , Cooper A , Markowitz S , Duran C , Thierer T , Ashton B , Meintjes P , Drummond A . 2012 . Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics (Oxford, England) 28 : 1647 – 1649 . Google Scholar CrossRef Search ADS PubMed Kelly M , Cárdenas P . 2016 . An unprecedented new genus and family of Tetractinellida (Porifera, Demospongiae) from New Zealand’s Colville Ridge, with a new type of mitochondrial group I intron . Zoological Journal of the Linnean Society 177 : 335 – 352 . Google Scholar CrossRef Search ADS Lanfear R , Calcott B , Ho SY , Guindon S . 2012 . Partitionfinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses . Molecular Biology and Evolution 29 : 1695 – 1701 . Google Scholar CrossRef Search ADS PubMed Lavrov D , Wang X , Kelly M . 2008 . Reconstructing ordinal relationships in the Demospongiae using mitochondrial genomic data . Molecular Phylogenetics and Evolution 49 : 111 – 124 . Google Scholar CrossRef Search ADS PubMed Lê HLV , Lecointre G , Perasso R . 1993 . A 28S rRNA-based phylogeny of the gnathostomes: first steps in the analysis of conflict and congruence with morphologically based cladograms . Molecular Phylogenetics and Evolution 2 : 31 – 51 . Google Scholar CrossRef Search ADS PubMed Lévi C , Lévi P . 1983 . Eponges Tétractinellides et Lithistides bathyales de Nouvelle-Calédonie . Bulletin du Muséum national d’Histoire naturelle 5 : 101 – 168 . Lévi C , Lévi P . 1989 . Spongiaires (MUSORSTOM 1 and 2) . In: Forest J , ed. Résultats des Campagnes MUSORSTOM 4 Mémoires du Muséum National d’Histoire Naturelle. Zoologie 143 : 25 – 103 . Manconi R , Serusi A . 2008 . Rare sponges from marine caves: discovery of Neophrissospongia nana nov. sp. (Demospongiae, Corallistidae) from Sardinia with an annotated checklist of Mediterranean lithistids . ZooKeys 4 : 71 – 87 . Google Scholar CrossRef Search ADS Marshall W . 1876 . Ideen über die Verwandtschaftsverhältnisse der Hexactinelliden . Zeitschrift für wissenschaftliche Zoologie 21 : 113 – 136 . Meyer CP , Geller JB , Paulay G . 2005 . Fine scale endemism on coral reefs: Archipelagic differentiation in turbinid gastropods . Evolution 59 : 113 – 125 . Google Scholar CrossRef Search ADS PubMed Morrow C , Cárdenas P . 2015 . Proposal for a revised classification of the Demospongiae (Porifera) . Frontiers in Zoology 12 : 1 – 27 . Google Scholar CrossRef Search ADS PubMed Pisera A . 2002 . Fossil ‘Lithistids’: an overview . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 388 – 402 . Google Scholar CrossRef Search ADS Pisera A , Lévi C . 2002a . Family Corallistidae Sollas, 1888 . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 312 – 320 . Google Scholar CrossRef Search ADS Pisera A , Lévi C . 2002b . Family Theonellidae von Lendenfeld, 1903 . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 327 – 337 . Pisera A , Lévi C . 2002c . Family Scleritodermidae Sollas, 1888 . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 302 – 311 . Google Scholar CrossRef Search ADS Pisera A , Pomponi SA . 2015 . New data on lithistid sponges from the deep Florida shelf with description of a new species of Theonella . Journal of the Marine Biological Association of the United Kingdom 95 : 1297 – 1309 . Google Scholar CrossRef Search ADS Pisera A , Vacelet J . 2011 . Lithistid sponges from submarine caves in the Mediterranean: taxonomy and affinities . Scientia Marina 75 : 17 – 40 . Google Scholar CrossRef Search ADS Pomponi SA , Kelly M , Reed JK , Wright A . 2001 . Diversity and bathymetric distribution of lithistid sponges in the tropical western Atlantic region . Bulletin of the Biological Society of Washington 10 : 344 – 353 . Ryan WBF , Carbotte SM , Coplan JO , O’Hara S , Melkonian A , Arko R , Weissel RA , Ferrini V , Goodwillie A , Nitsche F , Bonczkowski J , Zemsky R . 2009 . Global multi-resolution topography synthesis . Geochemistry Geophysics Geosystem 10 : Q03014 . Google Scholar CrossRef Search ADS Schlacher-Hoenlinger MA , Pisera A , Hooper JNA . 2005 . Deep-sea ‘lithistid’ assemblages from the Norfolk Ridge (New Caledonia), with description of seven new species and a new genus (Porifera, Demospongiae) . Zoosystema 27 : 649 – 698 . Schmidt O . 1870 . Grundzüge einer Spongien-Fauna des atlantischen Gebietes . Leipzig : Verlag von Wilhelm Engelmann , 1 – 88 . Schuster A , Erpenbeck D , Pisera A , Hooper J , Bryce M , Fromont J , Wörheide G . 2015 . Deceptive Desmas: molecular phylogenetics suggests a new classification and uncovers convergent evolution of lithistid demosponges . PLoS ONE . 10 : e116038 . Google Scholar CrossRef Search ADS PubMed Schuster A , Lopez JV , Becking LE , Kelly M , Pomponi SA , Wörheide G , Erpenbeck D , Cárdenas P . 2017 . Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding . BMC Evolutionary Biology 17 : 1 – 21 . Google Scholar CrossRef Search ADS PubMed Sollas WJ . 1885 . A classification of the sponges . Journal of Natural History 16 : 395 – 395 . Google Scholar CrossRef Search ADS Sollas WJ . 1886 . Preliminary account of the Tetractinellid sponges Dredged by H.M.S. ‘Challenger’ 1872–76. Part I. The Choristida . The Scientific Proceedings of the Royal Dublin Society 5 : 177 – 199 . Sollas WJ . 1887 . Sponges . In: Black A & C , ed. Encyclopaedia Britannica, 9th edn . Edinburgh, 412 – 429 . Sollas WJ . 1888 . Report on the Tetractinellida collected by H.M.S. Challenger, during the years 1873–1876 . Zoology 25 : 1 – 458 . Sukumaran J , Knowles LL . 2017 . Multispecies coalescent delimits structure, not species . Proceedings of the National Academy of Sciences 114 : 1607 – 1612 . Google Scholar CrossRef Search ADS UNESCO . 1978 . Galápagos Islands . World Heritage Convention . Available at: http://whc.unesco.org/en/list/1 Van Soest RWM , Boury-Esnault N , Vacelet J , Dohrmann M , Erpenbeck D , De Voogd NJ , Santodomingo N , Vanhoorne B , Kelly M , Hooper JNA . 2012 . Global diversity of sponges (Porifera) . PloS ONE 7 : e35105 . Google Scholar CrossRef Search ADS PubMed Van Soest RWM , Boury-Esnault N , Hooper JNA , Rützler K , de Voogd NJ , Alvarez de Glasby B , Hajdu E , Pisera A , Manconi R , Schoenberg C , Klautau M , Picton B , Kelly M , Vacelet J , Dohrmann M , Díaz M-C , Cárdenas P , Carballo JL . 2017 . WorldPorifera database . Available at: http://www.marinespecies.org/Porifera on 2017-02-27 Van Soest RWM , Stentoft N . 1988 . Barbados deep-water sponges . Studies on the Fauna of Curaçao and other Caribbean Islands 70 : 1 – 175 . von Lendenfeld R . 1903 . Tetraxonia . In: Schulze FE , ed. Das Tierreich . Berlin : Friedländer , 1 – 168 . Wilson HV . 1904 . Reports on an exploration off the West Coasts of Mexico, Central and South America, and off the Galápagos Islands, in charge of Alexander Agassiz, by the U.S. Fish Commission Steamer ‘Albatross’ during 1891, Lieut. Commander Z.L. Tanner, U.S.S., commanding. XXX. The Sponges . Memoirs of the Museum of Comparative Zoology at Harvard Collegee 30 : 1 – 164 . Zittel KA . 1878 . Studien über fossile Spongien. II. Lithistidae . Abhandlungen der Mathematisch-Physikalischen Classe der Königlich-Bayerischen Akademie der Wissenschaften 13 : 65 – 154 . © 2018 The Linnean Society of London, Zoological Journal of the Linnean Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Zoological Journal of the Linnean Society Oxford University Press

Seven new deep-water Tetractinellida (Porifera: Demospongiae) from the Galápagos Islands – morphological descriptions and DNA barcodes

Loading next page...
 
/lp/ou_press/seven-new-deep-water-tetractinellida-porifera-demospongiae-from-the-I0eQKjKP09
Publisher
Oxford University Press
Copyright
© 2018 The Linnean Society of London, Zoological Journal of the Linnean Society
ISSN
0024-4082
eISSN
1096-3642
D.O.I.
10.1093/zoolinnean/zlx110
Publisher site
See Article on Publisher Site

Abstract

Abstract The Galápagos Islands, positioned in the confluence of warm and coldwater currents in the Eastern Pacific, is well known for the high degree of endemism of its marine invertebrate fauna. This fauna has been studied extensively in recent years: the echinoderms, corals and other benthic cnidarians, but little is known about the deep- and shallow-water sponge faunas. To date, only 70 sponge species have been described from the Galápagos Islands, 37 of which are endemic. Of these 70 species, only one shallow-water species of desma-bearing Tetractinellida (Demospongiae), Corallistes isabela, has been reported. In 1995, Harbor Branch Oceanographic Institution, Florida, led an expedition around the Galápagos archipelago, focussed on the collection of deep-water Porifera. Here, we describe seven new species and provide DNA barcodes for the tetractinellids from these collections. Phylogenetic relationships of these new species are discussed and compared with other material from the Caribbean, the Central and West Pacific Oceans. The new species represent five genera (Craniella, and desma-bearing Tetractinellida Neophrissospongia, Corallistes, Racodiscula and Scleritoderma). Phylogenetic reconstructions combining independent markers (mtDNA and rDNA) support the generic affiliation of these new species and confirm the separation of Eastern Pacific species from Caribbean and Central to West Pacific species. Astrophorina, COI, Corallistidae, DNA barcode, endemism, Galápagos Islands, lithistids, Scleritodermidae, Spirophorina, Theonellidae INTRODUCTION The Galápagos Islands are located on the Nazca Plate, in the Eastern equatorial Pacific Ocean, about 926 km west of Ecuador (Fig. 1). Plate tectonic analyses demonstrate proximity to the so-called ‘Galápagos Triple Junction’, where the Pacific, Cocos and Nazca plates concur (Holden & Dietz, 1972). The position of Galápagos archipelago at the confluence of three major cold oceanic currents (Peru Coastal, Peru Oceanic and Cromwell) and one warm (Panamá) oceanic current (Chavez & Brusca, 1991) is further influenced by anomalous events such as the El Niño and La Niña, which seasonally change the ocean surface temperatures and are known to impact shallow coral reef communities (Feingold, 2011). Due to its extremely isolated geographic position, diverse underwater geomorphological settings as a result of ongoing tectonic and volcanic activity (Geist et al., 2008), in combination with complex mixing of water masses (Chavez & Brusca, 1991), the Galápagos archipelago constitutes a unique marine laboratory to investigate the ecology, evolution and biogeography of marine animals in an isolated region of the Eastern Pacific. In 1978, the Galápagos Islands were protected by UNESCO and described as a ‘unique living museum and showcase of evolution’. In 2001, the status of World Heritage Site was granted to the Galápagos Marine Reserve (133 000 km2), which comprises the second largest Marine Reserve in the world (UNESCO, 1978). Figure 1. View largeDownload slide Map of the Galápagos archipelago with sampling locations and species collected. Colour code denotes species and records (stars = holotype location; circles = paratypes). Map made with GeoMapApp, http://www.geomapapp.org, using the implemented Global Multi-Resolution Topography (GMRT Grid Version 3.2) (Ryan et al., 2009). Figure 1. View largeDownload slide Map of the Galápagos archipelago with sampling locations and species collected. Colour code denotes species and records (stars = holotype location; circles = paratypes). Map made with GeoMapApp, http://www.geomapapp.org, using the implemented Global Multi-Resolution Topography (GMRT Grid Version 3.2) (Ryan et al., 2009). Despite the young age of the Galápagos platform (10–15 Myr) and its biogeographic isolation (e.g. Chavez & Brusca, 1991), the marine invertebrate fauna is relatively diverse and reveals large numbers of described endemic species, including echinoderms, molluscs, corals and other benthic cnidarians (James, 1991). However, little is known about the deep- and shallow-water sponge fauna; to date, only 70 demosponge species have been described from the Galápagos Islands, of which 37 (~50%) are endemic. Only seven deep-water species (>100 m) have been described (Wilson, 1904; de Laubenfels, 1939; Desqueyroux-Faúndez & Van Soest, 1997). Among these 70 species, 11 were reported as belonging to the order Tetractinellida Marshall, 1876 (Desqueyroux-Faúndez & Van Soest, 1997), of which ten belong to Astrophorina Sollas, 1887 (families Ancorinidae Schmidt, 1870; Pachastrellidae Carter, 1875; Geodiidae Gray, 1867; Corallistidae Sollas, 1888) and one (Cinachyrella globulosa Desqueyroux-Faúndez & Van Soest, 1997) to the Spirophorina Bergquist & Hogg, 1969 (Tetillidae Sollas, 1886). The Galápagos archipelago constitutes one of the largest island groups in the Pacific, but has the lowest demosponge diversity compared to the Central Indo-Pacific and Tropical Eastern Pacific (Van Soest et al., 2012). Besides the discovery of one shallow-water species of the family Corallistidae (Corallistes isabela Desqueyroux-Faúndez & Van Soest, 1997), no further desma-bearing tetractinellids have been described from this area. This study is focused primarily on the morphological description of seven new deep-water tetractinellids from the Galápagos archipelago, the majority of which are desma-bearing, and provides additional supportive molecular phylogenies for the generic affiliation of these new species using three markers (mtDNA COI and 28S, 18S rDNA). MATERIAL AND METHODS Sampling Sponges were collected using the Johnson-Sea-Link I research submersible, operated by Harbor Branch Oceanographic Institution (HBOI), during an expedition to the Galápagos Islands, conducted 14–31 October 1995. The objective of the expedition was to conduct a biodiversity inventory and carry out biodiscovery research on sponges, octocorals and algae. Sponges analyzed in the present study were collected from ten stations around the Galápagos archipelago between 21 and 396 m depth (Fig. 1). A detailed list of these specimens, with their corresponding vouchers, location, latitude, longitude and National Center for Biotechnology Information (NCBI) accession number is given in Supporting Information, Table S1. Frozen (−80 °C) as well as formalin and 70% ethanol-preserved specimens, stubs, spicule slides and skeletal sections of the type materials are archived at the Harbor Branch Oceanographic Museum (HBOM), Fort Pierce, Florida, USA. Molecular investigations Two independent molecular markers were used to amplify 18 samples from the Galápagos Islands and 16 comparative samples, following the protocol described in Schuster et al. (2015): the ‘Folmer’ fragment of mitochondrial COI (c. 650 bp) and the C1-D2 region of the nuclear 28S rDNA (c. 800 bp), using, respectively, the primers dgLCO1490 and dgHCO2198 (Meyer, Geller & Paulay, 2005) and C1’ASTR (Cárdenas et al., 2009) and D2 (Lê, Lecointre & Perasso, 1993). Additionally, we amplified and sequenced near-complete 18S rDNA sequences (~1800 bp) for all Galápagos Islands samples. Amplification of ~1800 bp of 18S was performed using primers SP18aF and SP18gR (Lavrov, Wang & Kelly, 2008) with PCR settings of 95 °C, 3 min (95 °C, 30 s; 50 °C, 40 s; 72 °C, 2.5 min) × 35 cycles; 72 °C, 7 min. Primers SP18aF, SP18gR (Lavrov et al., 2008), and 400F18S, 1200F18S, 600R18S and 1350R18S (Lavrov et al., 2008) were used to sequence ~1800 bp of overlapping fragments. In order to confirm the sponge origin of newly generated sequences, we assessed their homology by BLAST searches against NCBI GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were assembled into contigs using Geneious v.8.1.8 (http://www.geneious.com, Kearse et al., 2012). 28S (C1-D2) and COI were independently aligned against alignments from Schuster et al. (2015). In addition to these sequences, we included Stupenda singularisKelly & Cárdenas, 2016, in the alignment, taxonomic updates on the Antarctic Tetillidae made by Carella et al. (2016), Theonella species from Hall, Ekins & Hooper (2014), and corrected Isabella tanoa Ekins, Erpenbeck, Wörheide & Hooper, 2016, which was re-identified by Ekins et al. (2016) and previously named as Isabella mirabilis (Schuster et al., 2015). Isabella harborbranchi Carvalho, Pomponi & Xavier, 2015, was added from Carvalho et al. (2015) (see also Supporting Information, Table S1). A representative concatenated matrix was compiled from the independent COI and 28S alignments in Geneious, assuring that for most taxa, sequences of both markers were present. The concatenated alignment (28S rDNA and COI mtDNA) was 1440 bp long; 658 bp were constant, 105 bp parsimony uninformative and 677 bp parsimony informative. Sequences of 18S rDNA were only obtained from the Galápagos Islands material, thus we did not incorporate these sequences into the final concatenated alignment. Bayesian inference (BI) and maximum likelihood (ML) analyses were conducted as described in Schuster et al. (2015). Partitionfinder v.1.1.1 (Lanfear et al., 2012) was used to select the best-fit partitioning scheme and model, which was the generalizing GTR+G+I model for both markers. New sequences from this study are stored at GenBank (NCBI). GenBank accession numbers are: COI KY652805–KY652825; 28S C1-D2 KY652771–KY652804; 18S KY652826–KY652840. Additionally, COI sequences of all type material are deposited at the Sponge Barcoding Project (SBP) (http://www.spongebarcoding.org) (record numbers 1702–1733). The concatenated alignment from this study is freely available at OpenDataLMU (https://doi.org/10.5282/ubm/data.113). Morphological investigations In order to carry out scanning electron microscopy (SEM) of the sponge skeleton and spicules, a section of formalin-preserved sponge tissue, perpendicular to the sponge surface, was cut under a binocular microscope, dried and digested in 66% nitric acid (HNO3) for 15–20 min. The digested product was boiled until all organic material was dissolved. The remaining skeletons and spicules were rinsed at least five times with ultrapure water and twice with 99% ethanol, as described in Pisera & Pomponi (2015). The zygosed skeletons were mounted on SEM stubs using Carbon-Leit-C (PLANO, Germany) with the ectosomal surface uppermost. Loose spicules were prepared on glass coverslips attached to SEM stubs. All stubs were sputter-coated with gold and examined with a Leo 1430VP SEM at 5–25 kV at the Zoological State Collection (Munich). For thick histological sections, the same tissue orientation as described above was used. Tissues were resuspended overnight in 70% ethanol, dehydrated in graded ethanol series (1 h 90%, 12 h 99%) at room temperature and embedded into L.R. White (PLANO) after two to three changes. For polymerization, the blocks were placed at 46 °C for 1 h and 60 °C for 12 h. Thick sections (50–200 µm) were cut using a Leica SP1600 saw microtome (Germany) and examined under a Leica M165FC microscope. Measurements of spicules are given as minimum–mean–maximum length × minimum–mean–maximum width (µm) with total number of measured spicules (N) in parentheses. Measurements of the triaene cladome refer to the total diameter of the cladome, across the endpoints of the clades; measurements of the triaene rhabdome refer to the distance from just below the cladome to the tip. Microscleres in the desma-bearing Tetractinellida are predominantly streptasters, a general term for microscleres with rays that emanate from an elongate centrum (amphiasters, metasters, spirasters). Metasters have a few quite long rays emanating from a short, elongate centrum; spirasters have sparse or numerous rays emanating from a single bent or multiple spiraled elongate centrum; amphiasters have numerous rays emanating from either end of a straight shaft. Strict distinction between these microscleres is often not clear-cut, so we chose to cite combinations of forms used in the present study, for example, streptaster/spiraster or streptaster/amphiaster when distinction was difficult. Measurements of microscleres are given as the complete length of the microsclere and the maximum width of the actin. RESULTS Systematics The classification used here follows Morrow & Cárdenas (2015). The taxonomic authority for all new species described is restricted to the first author. Class Demospongiae Sollas, 1885, Subclass Heteroscleromorpha Cárdenas, Pérez & Boury-Esnault, 2012, Order Tetractinellida Marshall, 1876, Suborder Astrophorina Sollas, 1887, Family Corallistidae Sollas, 1888 Genus Neophrissospongia Pisera & Lévi, 2002 N eophrissospongia galapagoensis sp. nov. (Figs 2–4; Table 1) Diagnosis: Massive ear-shaped, flabellate, Neophrissospongia with rhabdome of dichotriaenes showing a large variation from 172 to 617 µm length with spined cladome and blunt to acute tips. Type material: Holotype: HBOM 003:02010, Coll. Johnson Sea-Link I (JSL-I) dive 3923 [27 October 1995, Galápagos, 4 nautical miles (NM) SE of Tortuga Island, 01°04′59″S, 90°51′56″W, 241 m]. Paratypes: HBOM 003:01097 (227 m), HBOM 003:01098 (189 m), Galápagos, NE of Rocas Gordin, Santa Cruz Island, 0°32′53″S, 90°7′47″W, Coll. JSL-I dive 3900. HBOM 003:02002 (204 m), HBOM 003:02003 (120 m), Galápagos, 5 NM NE of Kicker Rock at San Cristobal Island, 01°43′8″S, 89° 27′14″W, Coll. JSL-I dive 3905. HBOM 003:01099 (177 m), Galápagos, N-Side 1.25 NM NW of Cormorant PT at the Santa Maria Island (Floreana), 01°12′6″S, 90°26′10″W, Coll. JSL-I dive 3901. HBOM 003:02008 (235 m), Galápagos, West Coast 2 NM NW of PTA Tortuga at Isabela Island, 0°14′31″S, 91°24′56″W, Coll. JSL-I dive 3915. HBOM 003:02005 (328 m), Galápagos, NW Coast, 66 NM offshore, Isabela Island, 0°8′35″S, 91°23′42″W, Coll. JSL-I dive 3912. Comparative material: Neophrissospongia nolitangere Schmidt, 1870, HBOI 2-VI-91-1-005 (358 m), Portugal, Madeira, Savage Islands, Selvagem Grande, South Coast, 30°07′21″N, 15°52′7.8″W, Coll. JSL-I dive 3006, June 1991, identified by Michelle Kelly. Neophrissospongia microstylifera Lévi & Lévi, 1983 0CDN7083-J (111 m), Palau, Koror Reef, W-Side of Uchelbeluu Reef, wall, rock, 7°16′25″N, 134°31′25″E, 2001, identified by Michelle Kelly; 0CDN5043-Q (109 m), Palau, Koror Reef, W-Side of Uchelbeluu Reef, overhang, rock, 7°16′25″N, 134°31′26″E, 1997, identified by Michelle Kelly. Type locality: Tortuga Island, Galápagos Islands (241 m) (Fig. 1). Distribution: Tortuga Island, Isabela Island, Fernandina, Santa Cruz Island, San Cristobal, Floreana Island (Fig. 1). Habitat: Attached to hard substratum, depth range 120–328 m. Description: Morphology, in the shape of an ear, flared cup or vase, or foliose with folded margins when mature; walls 1–1.5 cm thick, margins flattened with rounded edges (Fig. 2); dimension of holotype, 12 cm diameter, 16 cm high (Fig. 2A); paratypes range 20–30 cm diameter, 30–45 cm high or larger (Fig. 2C, D). Surface, with horizontal growth lines; concave inhalant surface with numerous ostia, 25–35 µm diameter; convex (exhalent surface) has numerous, barely visible, oscules. Texture, stony, especially towards the base of the sponge. Colour, beige in life, brown in ethanol preservative. Ectosomal skeleton, 1.2–1.5 mm thick, outermost surface a densely packed, 100-µm-thick layer of microscleres, within which lies the cladomes of the dichotriaenes, the rhabdomes of which penetrate deeply into the choanosome (Fig. 4A, B). Choanosomal skeleton composed of zygosed dicranoclone desmas and acanthose microtylostyles, often located around the pore openings. Megascleres, desmas and triaenes; dicranoclone desmas, 280–450 µm (N15) maximum diameter, with a massive, arched, central core, from which four to six arms splay away from the underside of the arch, both the central core and the arms are covered irregularly with tubercles (Fig. 3B, C, F), the tubercules on the desma core are larger, fungiform and multituberculate; dichotriaenes, rhabdome 172–422–617 µm (N30) long, cladome, 175–227–372 µm (N30) wide, upper surface of clads irregularly spined (Fig. 4G, H). Microscleres, irregular amphiasters with short, thick rays, 15.2–11.2–18 µm long × 4.9–7.2–10.7 µm (N25) wide; acanthose microtylostyles, 44–91–133 µm (N25) long. Figure 2. View largeDownload slide In situ and deck pictures of Neophrissospongia galapagoensis sp. nov. A, B, cup-shaped holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. C, D, massive flabellate paratype HBOM 003:01099. Scale bar is 1 cm. Figure 2. View largeDownload slide In situ and deck pictures of Neophrissospongia galapagoensis sp. nov. A, B, cup-shaped holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. C, D, massive flabellate paratype HBOM 003:01099. Scale bar is 1 cm. Figure 3. View largeDownload slide Various views of the choanosome skeleton of Neophrissospongia galapagoensis sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, overview of dicranoclone desmas. B, dicranoclone desmas with a spinose dichotriaene in situ. C, details of the dicranoclones. D, in situ picture of a bundle of spinose dichotriaenes between the ectosome and choanosome. E, choanosome cut with disordered partly broken dicranoclones, arrows pointing to numerous microtylostyles. F, detailed view of dicranoclone articulation (zygome). Figure 3. View largeDownload slide Various views of the choanosome skeleton of Neophrissospongia galapagoensis sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, overview of dicranoclone desmas. B, dicranoclone desmas with a spinose dichotriaene in situ. C, details of the dicranoclones. D, in situ picture of a bundle of spinose dichotriaenes between the ectosome and choanosome. E, choanosome cut with disordered partly broken dicranoclones, arrows pointing to numerous microtylostyles. F, detailed view of dicranoclone articulation (zygome). Figure 4. View largeDownload slide Section and mega- and microscleres of Neophrissospongia galapagoensis sp. nov., holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, thin section of the ectosome and choanosome showing channel (ch) and spinose dichotriaenes (di) penetrating deeply into the choanosome. B, detailed thin section illustrating a thick layer of spiraster/streptaster (sp/st) on top of the ectosomal region. C–F, thick streptasters from the ectosomal regions. G, various ectosomal dichotriaenes. H, top view of tubercled cladomes of dichotriaenes. I, J, microspinose choanosomal microtylostyles, general view (I), detailed view (J). Figure 4. View largeDownload slide Section and mega- and microscleres of Neophrissospongia galapagoensis sp. nov., holotype Harbor Branch Oceanographic Museum (HBOM) 003:02010. A, thin section of the ectosome and choanosome showing channel (ch) and spinose dichotriaenes (di) penetrating deeply into the choanosome. B, detailed thin section illustrating a thick layer of spiraster/streptaster (sp/st) on top of the ectosomal region. C–F, thick streptasters from the ectosomal regions. G, various ectosomal dichotriaenes. H, top view of tubercled cladomes of dichotriaenes. I, J, microspinose choanosomal microtylostyles, general view (I), detailed view (J). Etymology: Named after the locality and distribution of the species, the Galápagos Islands. DNA barcodes: We sequenced partial COI, 28S (C1-D2) and complete 18S of the holotype and the paratypes from different localities of the Galápagos Islands (Fig. 1). COI sequences of all type material sequenced were identical with the exception of HBOM 003:02005 (no COI sequenced). 28S and 18S sequences of all type material were identical with the exception of HBOM 003:02005 (no 18S sequenced). GenBank accession numbers: COI KY652805–KY652811; 28S KY652771–KY652778; 18S KY652826–KY652832; SBP No. 1702–1709. Remarks: According to the World Porifera Database (WPD) (Van Soest et al., 2017, http://www.marinespecies.org/porifera/), only six different species of Neophrissospongia have been described: N. nolitangere (Table 1; Pisera & Vacelet, 2011) and N. tubulata Van Soest & Stentoft, 1988 from the tropical Western Atlantic; N. endoumensis Pisera & Vacelet, 2011, N. nana Manconi & Serusi, 2008 and N. radjae Pisera & Vacelet, 2011, from the Mediterranean Sea, and N. microstylifera Lévi & Lévi, 1983 from New Caledonia in the Pacific Ocean. Table 1. Outer morphology and spicule measurements of the known seven Neophrissospongia species including the new species N. galapagoensis sp. nov. Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) If not otherwise stated measurements are given in micrometres. Measurements are given for the holotype (in bold = new species) and indicated for N. nolitangere with ‘*’. Measurements and outer morphology are taken from Van Soest & Stentoft (1988), Pisera & Lévi (2002a), Manconi & Serusi (2008), Pisera & Vacelet (2011) and Carvalho et al. (2015). clad = cladome in diameter; deut = deuteroclade; prot = protoclade; rhabd = rhabdome. View Large Table 1. Outer morphology and spicule measurements of the known seven Neophrissospongia species including the new species N. galapagoensis sp. nov. Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) Species Distribution Depth Grow form/shape Dicranoclone desma Dichotriane Cladome tuberculated/spinose Dichotriaene smooth Monaxial spicule Streptaster/amphiaster N. nolitangere Schmidt, 1870* Portugal Depth unknown Flabellate massive 350–500 Tuberculate clad 230–310* clad 450–1200* rhabd Not recorded Oxea not measured, not recorded* 10–13 × 9–10 N. nolitangere Schmidt, 1870 Azores, Fayal 365 m Flabellate massive 35 thick Tuberculated clad 45–50 prot 90–100 deut 450–1200 rhabd Not recorded Oxea 70–100 10–13 N. nolitangere Schmidt, 1870 Azores, Fayal MNHN DT 781, re-examined in: Manconi & Serusi (2008) 440 m Flabellate massive Not measured Tuberculate clad 151–280 clad (N36) 35–50 × 18–20 prot 50–100 × 18–20 deut 366–1030 × 28 rhabd (N15) Not measured Style/subtylostyle, slightly bent/sinuous 51–87 × < 3 (N50) Not measured N. nolitangere Azores, AZR 71 Pulitzer-Finali collection re-examined in Manconi & Serusi (2008) Flabellate massive Not measured Tuberculated clad 47–58 prot (N20) 70–116 deut (N20) 478–1200 × 23–35 rhabd (N16) 47–120 prot 47–151 deut Style-like 52–96 × <3 (N30) 9–14 (N15) N. nolitangerevon Lendenfeld, 1903 Sao Tiago, Cape Verde Island Flabellate massive 35 thick Tuberculated clad 550 rhabd Not recorded Not measured Not recorded N. nolitangere (Carvalho et al., 2015) Selvagens (358 m) Madeira (317 m) Canaries (295–450 m) Flabellate massive Not recorded Not recorded Not recorded Not recorded Not recorded N. microstylifer Lévi & Lévi, 1983 MNHN-DCL 2773 New Caledonia 355–360 m Clavate 250–300 250–350 clad 30–40 prot 100 deut 350–600 × 30–45 rhabd Not recorded Strongyle 25–45 × 3 Subtylostyle 110–170 × 2 Not recorded N. nana Manconi & Serusi, 2008 MSNG 54599 W-Sardinia Terrazze Cave 6 m Encrusting plate-like 280 145–270 clad (N30) 20–50 × 10–20 prot 50–100 × 10–20 deut 290–540 rhabd (N30) 93–172 clad 59–86 × < 3 (N30) Subtylostyle 5–15 (N30) N. endoumensis Pisera & Vacelet, 2011 MNHN-DJV-120 Endoume Cave (France) Depth not reported Cup-shaped 318–490 248–283 massive and densely spined clad 320–615 × 32–45 rhabd Not recorded Spinose microtylostyle 68.8–123 × 1.6–1.7 12.7–20.1 × 11.4–16.6 N. radjae Pisera & Vacelet, 2011 MNHN-DJV-119 Dalmatian Cave (Croatia) Depth not reported Clavate 45 mm high and 30 mm in diameter 280–500 160–340 spined clad 220–639 × 20–30 rhabd Not recorded Spinose microtylostyles 80–140 × 0.8–2.1 9.55–15.5 × 8.35–12 N. galapagoensis sp. nov. HBOM 003:02010 Tortuga Island (Galápagos Islands) 241 m Cup- to ear-shaped, flabellate massive, 12 cm in diameter and 16 cm in height 280–450 (N15) 175–227–372 spined clad (N30) 172–422–617 rhabd (N30) Not recorded Spinose microtylostyles 44–91–133 (N25) 15.2–11.2–80 × 4.9–7.2–10.7 (N25) If not otherwise stated measurements are given in micrometres. Measurements are given for the holotype (in bold = new species) and indicated for N. nolitangere with ‘*’. Measurements and outer morphology are taken from Van Soest & Stentoft (1988), Pisera & Lévi (2002a), Manconi & Serusi (2008), Pisera & Vacelet (2011) and Carvalho et al. (2015). clad = cladome in diameter; deut = deuteroclade; prot = protoclade; rhabd = rhabdome. View Large Neophrissospongia galapagoensis sp. nov., which is cup-shaped to foliose, clearly differs from the encrusting N. nana, discovered in a Mediterranean marine cave. The club-shaped N. radjae (Table 1; Pisera & Vacelet, 2011) from a cave in the Mediterranean with an apical oscule is also distinguished from our species by gross morphology. Minor differences between the dichotriaenes of N. galapagoensis sp. nov. and N. radjae (Table 1) also distinguish both species: N. radjae has a more distinctly tuberculated cladome than in our new species. Neophrissospongia nolitangere, from the Azores, Cape Verde Islands, off Portugal (e.g. Pisera & Lévi, 2002a), Madeira (Carvalho et al., 2015), submarine caves in the Mediterranean (Table 1; Pisera & Vacelet, 2011) and the Bahamas (Pomponi et al., 2001), is distinguished from N. galapagoensis sp. nov. in the possession of smaller streptasters with longer rays; 28S sequences of N. nolitangere by Chombard, Boury-Esnault & Tillier (1998) and here (HBOI 2-VI-91-1-005) are both 100% identical and differ by 1% from our new species. Molecular analyses (Fig. 5) further separate N. galapagoensis sp. nov. from N. nolitangere. Sequence comparison with material from the West Pacific (WAM Z35946, 36053; probably both new species, A. Pisera, personal observation) disclose a 1.6% difference. Figure 5. View largeDownload slide Bayesian inference (BI) reconstruction of the concatenated dataset (COI and 28S) showing the relationship of the seven new species from the Galápagos to other tetractinellids from the Caribbean, Pacific and Atlantic. Maximum likelihood (ML) tree is mostly congruent, otherwise no support values are given. Newly generated sequences are in bold (red: new species, black: comparative samples). Bayesian posterior probabilities and ML bootstrap support values are indicated for clades >0.75/60, otherwise marked with a dash. Numbers following the taxon names are collection numbers or NCBI Genbank accession numbers. Markers sequenced are indicated in square brackets. Three letter code in dark blue indicates the biogeographical location. Figure 5. View largeDownload slide Bayesian inference (BI) reconstruction of the concatenated dataset (COI and 28S) showing the relationship of the seven new species from the Galápagos to other tetractinellids from the Caribbean, Pacific and Atlantic. Maximum likelihood (ML) tree is mostly congruent, otherwise no support values are given. Newly generated sequences are in bold (red: new species, black: comparative samples). Bayesian posterior probabilities and ML bootstrap support values are indicated for clades >0.75/60, otherwise marked with a dash. Numbers following the taxon names are collection numbers or NCBI Genbank accession numbers. Markers sequenced are indicated in square brackets. Three letter code in dark blue indicates the biogeographical location. Neophrissospongia tubulata from Barbados is characterized by small, single or clustered tubes, in which the dichotriaenes have short rhabds (30–50 µm) and a non-tuberculate cladome. Rather, the clads have dentate margins, clearly distinguishing them from the dichotriaenes of N. galapagoensis sp. nov. (Table 1) and all other members of the genus. This lateral dentation of the dichotriaene clads of N. tubulata points to the need for a revision of this species. The dichotrianes of N. endoumensis, from the Mediterranean (cladome width: 248–283 µm; rhabdome length: 531–615 µm) are larger and thicker and are less variable in size (Table 1; Pisera & Vacelet, 2011) compared to those of N. galapagoensis sp. nov. In the original description of tubular N. microstylifera from New Caledonia, Lévi & Lévi (1983) documented microstyles of 110–170 µm length, which are absent in our new species, and the dichotriaenes are more robust than those of N. galapagoensis sp. nov. Key to species of Neophrissospongia (after Manconi & Serusi, 2008 and the present study) (1) Growth form ear- to cup-shaped or large flabellate massive foliose with small inhalant and large exhalant apertures on opposite sides of the lamella, ectosomal skeleton of tuberculated dichotriaenes, styles/subtylostyles, smaller streptaster microscleres with long arms ……………………………..............................................................................................................N. nolitangere – Spinose microtylostyles and streptaster with massive arms as microscleres………… N. galapagoensis sp. nov. – Cup-shaped growth with spinose and massive cladome of dichotrianes and regular massive streptaster …………….……………………………………………………….....…………………………………. N. endoumensis – Growth form clavate to plate-like with inhalant and exhalant areas in different regions of the same sponge surface.................................………………………………………………………………………………………...... 2 (2) Growth form clavate, ectosomal skeleton of dichotriaenes and strongyle/ subtylostyles ...................................………………………………………………………………………............ N. microstylifera – Spined dichotriaenes and spinose microtylostyles………................................………………......…....N. radjae – Plate-like grow form, ectosomal skeleton of dichotriaenes, styles/subtylostyles and aster-like microscleres..................................………………………………………………………………................... N. nana The key to species of Neophrissospongia by Manconi & Serusi (2008) is updated from the work of Pisera & Vacelet (2011) and the present study. Genus C orallistes Schmidt, 1870 C orallistes floreana sp. nov. (Figs 6, 7; Table 2) Diagnosis: Corallistes with smooth dichotriaenes in a broad range of sizes (93–558 µm rhabd; 72–272 µm cladome) and amphioxeas of 327–887 µm length; choanosomal megascleres are tuberculate dicranoclone desmas, about 211–345 µm diameter. Type material: Holotype: HBOM 003:02000, Coll. JSL-I dive 3901 [16 October 1995, Galápagos, N-Side 1.25 NM NW of Cormorant Point, Floreana Island, Santa Maria, 01°12′5.88″S, 90°26′9.96″W, 121 m]. Comparative material: Corallistes isabela HBOM 003:02013 (203 m), Galápagos, Santiago Island, 0°11′29″S, 90°34′58″W, Coll. JSL-I dive 3927, identified by A. Schuster. Type locality: Floreana Island, Galápagos Islands (121 m) (Fig. 1). Distribution: Only known from type locality. Habitat: Attached to hard substratum (121 m). Description: Morphology, a shallow bowl with a slightly folded or indented margin, or ear-shaped, walls about 5–6 mm thick with rounded margins (Fig. 6A, B), holotype, about 8 cm diameter, about 6 cm high (Fig. 6A, B). Surface, uneven, partly covered with algae, coral and shell fragments, bundles of projecting spicules trap sediment on the upper surface, otherwise the surface is smooth, oscules are not visible (Fig. 6B). Texture, stony. Colour, beige in life (Fig. 6A), light brown to cream in ethanol preservative (Fig. 6B). Ectosomal skeleton, irregular, composed of dichotriaene cladomes, aligned tangentially to surface with rhabdome pointing in towards choanosome (Figs 6C, D, 7J, K), with scattered microscleres (Fig. 7B, E). Choanosomal skeleton composed of zygosed dicranoclone desmas (Fig. 6E, F) between which are large subdermal spaces and scattered microscleres (Fig. 7C–G). Megascleres, desmas, triaenes, microxeas; dicranoclone desmas, 211–354 × 176–261 µm (N15). Dichotriaenes in a large size range with long and short rhabdomes: smallest dichotriaenes, cladome, 72–82–106 µm, rhabdome 93–110–130 µm (N25), largest dichotriaenes, cladome, 130–186–278 µm, rhabdome, 208–314–558 µm (N25) (Figs 6D, 7J, K); microxeas, 327–630–887 × 4.8–6.6–10.8 µm (N6) (Fig. 7H, I). Microscleres, metasters with long, pointed, acanthose rays, 16.8–29.9–42 × 14.3–23.6–30.7 µm (N21), amphiasters and spirasters with short, pointed, microspined rays, 8.2–11.9–15.1 × 6.4–10.1–13.5 µm (N21). Figure 6. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, in situ picture (121 m). B, deck picture with inner surface: left part and outer surface: right part. C, D, thick sections of the ectosomal part showing diverse dichotriaenes penetrating to the choanosome. E, F, scanning electron microscopy pictures of cross-section showing articulated dicranoclone desmas. Figure 6. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, in situ picture (121 m). B, deck picture with inner surface: left part and outer surface: right part. C, D, thick sections of the ectosomal part showing diverse dichotriaenes penetrating to the choanosome. E, F, scanning electron microscopy pictures of cross-section showing articulated dicranoclone desmas. Figure 7. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, B, thick sections showing the accumulation of streptaster at the subectosomal region (A) and around channel openings (B). C, bundle of metasters/amphiasters. D–G, diverse streptaster with long pointed rays. H, I, amphioxea. J, K, ectosomal dichotriaene. Figure 7. View largeDownload slide Corallistes floreana sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02000. A, B, thick sections showing the accumulation of streptaster at the subectosomal region (A) and around channel openings (B). C, bundle of metasters/amphiasters. D–G, diverse streptaster with long pointed rays. H, I, amphioxea. J, K, ectosomal dichotriaene. Etymology: Named after the type locality, Floreana Island. DNA barcodes: We sequenced COI, 28S (C1-D2) and 18S (1303 bp) from the holotype; GenBank accession numbers: COI KY652816, 28S KY652784, 18S KY652833. SBD record no. 1715. Remarks: Corallistes floreana sp. nov. resembles C. isabela, previously described from the Galápagos Islands, but there are notable differences in the size and ornamentation of the megascleres. The dicranoclone desmas in C. floreana sp. nov. are smaller and tuberculate rather than knobbed as in C. isabela (Table 2). Furthermore, the alternating sizes of the C. floreana sp. nov. cladomes differ by their ratio from C. isabela (Table 2). Amphiasters / spirasters are smaller in size as well (Table 2). Pairwise sequence differences of 1.2% (28S) and 0.8% (COI) between C. floreana sp. nov. and C. isabela (HBOM 003:02013) further confirm integrity of these two species. Table 2. Spicule sizes in micrometres of Corallistes floreana sp. nov. compared to different Corallistes typus material and Corallistes isabela Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Information for Corallistes isabela taken from table 9 of Desqueyroux-Faúndez & Van Soest (1997). clad = cladome; rhabd = rhabdome. View Large Table 2. Spicule sizes in micrometres of Corallistes floreana sp. nov. compared to different Corallistes typus material and Corallistes isabela Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Species Locality Desmas Dichotriaenes, alternating in sizes of: I: large and II: small Oxeas Amphiasters/ spirasters/metaster (in diameter) Dicranoclone Corallistes typus (Schmidt, 1870) Florida Not recorded Not recorded Not recorded Not recorded Corallistes typus (Sollas, 1888) Pernambuco Tuberculated Rhabd: 238–320 × 32 701 × 4 Spirasters: 20–24 Corallistes typus (Van Soest & Stentoft, 1988) Barbados 300–360 × 15–24 Rhabd: 130–380 × 15–24 Clad: 90–300 700–1260 × 4–8 Spirasters: 14–26 Corallistes isabela (Desqueyroux-Faúndez & Van Soest, 1997) ZMA POR 11237 MHNG 20599 HBOM 003:02013 Galápagos (Isabela Island) Knobbe-like 526–959 × 129–327 I: Rhabd: 14–40 Clad: 329–540 II: Rhabd: 120 Clad: 229–269 × 23 >1000 × 1–5 Amphiasters: 42 Spirasters: 23 Corallistes floreana sp. nov. HBOM 003:02000 Galápagos (Floreana Island) Tuberculated 211–354 × 176–261 I: Rhabd: 208–558 Clad: 130–278 II: Rhabd: 93–130 Clad: 72–106 327–887 × 4–10 Amphiasters: 29 Metaster: 10 Information for Corallistes isabela taken from table 9 of Desqueyroux-Faúndez & Van Soest (1997). clad = cladome; rhabd = rhabdome. View Large Family Theonellidae von Lendenfeld, 1903 Genus R acodiscula Zittel, 1878 R acodiscula radixa sp. nov. (Figs 8, 9; Table 3) Diagnosis: Racodiscula with a deep, apical, cavity/osculum, into which subdermal canals converge from the margins; desmas are irregular, loosely zygosed tetraclone desmas with root-like protrusions. Type material: Holotype: HBOM 003:02006, Coll. JSL-I dive 3913. [22 October 1995, Galápagos, N-Side 1.25 NM W of point Vincente Roca NW Coast, Isabela Island, 0°02′55″S, 91°35′16″W, 378 m]. Comparative material: Racodiscula seymoura sp. nov. HBOM 003:02014 (396 m) (see below), Galápagos, Seymour Island, 0°22′51″S, 90°16′06″W, Coll. JSL-I dive 3529; HBOM 003:02007 (319 m), Galápagos, Isabela Island, 0°14′31″S, 91°24′56″W, Coll. JSL-I dive 3015. Racodiscula borisi sp. nov. HBOM 003:02011 (242 m) (see below), Galápagos Island, Tortuga Island, 1°4′59″S, 90° 51′56″W, Coll. JSL-I dive 3923; HBOM 003:02001 (215 m), Galápagos Island, San Cristobal Island, 0°43′08″S, 89°27′14″W, Coll. JSL-I dive 3905. Type locality: Isabela Island, Galápagos Islands (378 m) (Fig. 1). Distribution: Only known from type locality. Habitat: Attached to hard substratum at a depth of 378 m. Description: Morphology, massive subspherical folded with a deep, apical, cavity/osculum, about 1 cm longest diameter, situated on the elevated apex (Fig. 8A, B); dimension of the holotype, about 7 cm diameter, about 5 cm high (Fig. 8A, B). Texture, medium hard. Surface, smooth, with branched, meandering, subdermal canals, converging from the margins (Fig. 8B) towards the apical cavity; lateral inhalant surface with pores, 450–500 µm diameter. Colour, white in cream (Fig. 8A), dark cream in ethanol preservative (Fig. 8B). Ectosomal skeleton, detachable in places due to large subdermal canals, composed of phyllotriaene cladomes, stacked and overlapping, (Figs 8C–F, 9G), interspersed with microrhabds and other microscleres (Fig. 9B, E) forming a thick outer layer; tylostyles are located at the subectosomal region (Fig. 8D–F). Choanosomal skeleton contains loosely zygosed tetraclone desmas (Fig. 8D), loose bundles of oxeas and scattered microscleres (Fig. 9C, D). Megascleres, tetraclone desmas, phyllotriaenes and oxeas: tetraclone desmas, relately smooth, from which emerge thin, root-like branches, 405–532 × 254–276 µm (N5) (Fig. 8C–F); phyllotriaenes with a leafy cladome, 305–389–494 µm (N20), short, irregular rhabdome, 93–116–160 × 32–42–55 µm (N20) (Fig. 9A, B, G); tylostyles, 450–315–605 µm (N15) (Fig. 9A). Microscleres, acanthose microrhabds with irregularly distributed spined tubercles, 21–25–30 × 4 µm (N30); metasters with long rays, 15.6–18.5–20.2 × 11.3–14.7–19.2 µm (N20). Figure 8. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, in situ picture (378 m). B, deck picture. C, D, thick section of the ecto- and choanosome, representing disorganized tetraclone, phyllotriaenes with rhabdomes protruding perpendicular from the surface and boundes of tylostyles (tylo) and oxeas (oxe). Gap between ecto- and choanosome (C) is an artefact from sectioning. E, F, thick section showing the poor articulation of tetraclone desmas and diverse streptasters. Figure 8. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, in situ picture (378 m). B, deck picture. C, D, thick section of the ecto- and choanosome, representing disorganized tetraclone, phyllotriaenes with rhabdomes protruding perpendicular from the surface and boundes of tylostyles (tylo) and oxeas (oxe). Gap between ecto- and choanosome (C) is an artefact from sectioning. E, F, thick section showing the poor articulation of tetraclone desmas and diverse streptasters. Figure 9. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in between (B), forming a thick crust on the surface. C, metaster. D, spiraster. E, acanthorhabds. F, tylostyles. G, ectosomal phyllotriaene. Figure 9. View largeDownload slide Racodiscula radixa sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02006. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in between (B), forming a thick crust on the surface. C, metaster. D, spiraster. E, acanthorhabds. F, tylostyles. G, ectosomal phyllotriaene. Etymology: Named for the root-like ornamentation of the tetraclone desmas in this species (radix, root; Latin). DNA barcodes: we sequenced COI, 28S (C1-D2) and 18S (1,623 bp) of the holotype. GenBank accession number: COI KY652819, 28S KY652788, 18 KY652836. SBD record no. 1720. Remarks: Racodiscula radixa sp. nov. is differentiated from the two other new Racodiscula species from the Galápagos Islands, R. seymoura sp. nov. and R. borisi sp. nov. (see species description below), in having the cavity/oscula at an elevated centre with vein-like furrows to the margins, while R. seymoura sp. nov. have the oscula in a centred depression and R. borisi sp. nov. has a perfectly circular and centred oscula on the surface without an elevated/compressed centre. The oscula of all three new Racodiscula species described are different to Racodiscula asteroidesZittel, 1878 in having only one opening, while R. asteroides has multiple oscules in a shallow depression (Pisera & Lévi, 2002b). However, intraspecific variations of this character cannot be excluded. Thick strongyles as found in R. seymoura sp. nov. and R. borisi sp. nov. (see below) could not be detected in any of the spicule preparations for R. radixa sp. nov., which may indicate that this second type of microsclere was secondarily lost. (Table 3). The cladome of phyllo- to discotriaenes is smaller than in R. seymoura sp. nov. and R. borisi sp. nov. The rhabdome is also slightly shorter than in R. seymoura sp. nov. but larger than in R. borisi sp. nov. (Table 3). R. radixa sp. nov. has irregularly rather loose root-like desmas with shorter branches than R. seymoura sp. nov. In contrast, R. borisi sp. nov. shows strong tuberculated tetraclone desmas similar to R. asteroides. Differences were also found in our molecular analyses (Fig. 5). R. radixa sp. nov. differ to R. seymoura sp. nov. with 3.3% (COI), 0.3% (28S) and 1.8% (18S) and to R. borisi sp. nov. with 4.8% (COI), 0.5% (28S), 2.3% (18S). The sister-group relationship of R. radixa sp. nov. and R. seymoura sp. nov. is supported [posterior probability (PP) = 0.97 and bootstrap support (BS) = 80]. The clade with the three new Galápagos Racodiscula species is well supported by PP = 0.90, but not by BS. The sister-group relationship of this clade with R. asteroides and Racodiscula sp. from the Caribbean is well supported (Fig. 5). Table 3. Morphology and spicule sizes of the three new Racodiscula species from the Galápagos Islands compared to Racodicula asteroides Zittel, 1878 (neotype) Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Measurements are given in micrometres, if not otherwise stated. Measurements for new species are given for the holotype (bold) and for R. asteroides (neotype) taken from Pisera & Lévi (2002b). View Large Table 3. Morphology and spicule sizes of the three new Racodiscula species from the Galápagos Islands compared to Racodicula asteroides Zittel, 1878 (neotype) Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Species Locality Cavity/oscula Tetraclone desma Phyllo- to discotriaenes Tylostyles I: oxeas Microscleres I: cladome II: strongyles I: streptaster II: rhabdome II: acanthorhabds Racodiscula radixa sp. nov. Isabela Island One oval oscula at elevated centre, vein-like furrows from the oscula to the margins; diameter: 1.0 cm Irregular, loosely zygosed tetraclone desmas with root-like protrusions, not tuberculated, 405–532 by 254–276 I: 305–389–494 450–315–605 I: present, but difficult to measure as only seen in the sections (Fig. 9C–F) between desmas; II: not present I: 15.6–18.5–20.2 × 11.3–14.7–19.2 HBOM 003:02006 II: 93–116–160 × 32–42–55 II: 21–25–30 × 4 Racodiscula seymoura sp. nov. Seymour Island, Isabela Island One oval to circular oscula in a shallow centred depression; diameter: 0.7 cm Zygosed root-like with long thin branches, not tuberculated, 632–736 by 395–406 I: 447–536–636 434–317–719 I: 120–125–134 × 4–5 I: 17.3–20.5–22.2 × 12.5–15.6–20.0 HBOM 003:02014, HBOM 003:02007 II: 121–150–185 × 22–37–40 II: 95–100–112 × 7–10 II: 5–17–20 × 3–4 Racodiscula borisi sp. nov. San Cristobal Island, Tortuga Island One perfectly circular oscula located at the centre; diameter: 0.8 cm Zygosed with short thick root-like branches, strongly tuberculated, 540–650 by 385–400 I: 395–504–605 440–410–904 I: 140–176–204 × 4–5 I: 12.0–14.5–17.2 × 11.5–15.4–20.0 HBOM 003:02001, HBOM 003:02011 II: 20–66–93 × 18–21–25 II: 95–194–112 × 15–28 II: 12–19–27 × 3–4 Racodiscula asteroides Zittel, 1878 Barbados Multiple oscules located at shallow depression, iris-like diaphragm Sparsely tuberculated rays and strongly tuberculated tips I: 250–420 Present, but no measurement given Not present I: 14.7–20.7 × 11.2–19.4 II: 6.7–10.6 × 4.8–5.3 Neotype ZMA POR5264 (=ZPAL Pf.15/1) Pseudospherasters: 6.53–7.41 Measurements are given in micrometres, if not otherwise stated. Measurements for new species are given for the holotype (bold) and for R. asteroides (neotype) taken from Pisera & Lévi (2002b). View Large Racodiscula seymoura sp. nov. (Figs 10, 11; Table 3) Diagnosis: Racodiscula with one oval to circular cavity/osculum (0.7 cm) opening in a shallow centred depression; choanosomal spicules are zygosed root-like tetraclone desmas with long and thin branches; choanosomal spicules are thick strongyles. Type material: Holotype: HBOM 003:02014, Coll. JSL-I dive 3529, [30 October 1995, Galápagos, 66 NM NE of Seymour Island, 0°22′51″S, 90°16′06″W, 396 m]. Paratype: HBOM 003:02007 (319 m), Galápagos, N-Side 1.25 NM W of PTA Vincente Roca NW Coast, Isabela Island, 0°14′31″S, 91°24′56″W, Coll. JSL-I dive 3915. Comparative material: Racodiscula radixa sp. nov. HBOM 003:02006. Racodiscula borisi sp. nov. HBOM 003:02011 (242 m), Galápagos Island, Tortuga Island, 1°4′59″S, 90°51′56″W, Coll. JSL-I dive 3923; HBOM 003:02001 (215 m), Galápagos Island, San Cristobal Island, 0°43′08″S, 89°27′14″W, Coll. JSL-I dive 3905. Type locality: Seymour Island, Galápagos Islands (396 m) (Fig. 1). Distribution: Known from type locality, Seymour Island, and Isabela Island, Galápagos Islands. Habitat: Attached to hard substratum, depth range, 319–396 m. Description: Morphology, massive subspherical with an apical cavity/osculum opening on top of the surface in a shallow centred depression (Fig. 10A, B). Dimension of the holotype is about 7 cm in diameter and about 5 cm in height (Fig. 10A, B). Texture, medium hard. Surface, smooth and uneven (Fig. 10B). Colour, white in vivo (Fig. 10A) and light brownish in ethanol (Fig. 10B). Ectosomal skeleton contains phyllo- to discotriaenes (Figs 10B, 11A, F) with acanthorhabds in between and on top of this layer (Fig. 11A, B, D). Tylostyles are located at the subectosomal region (Fig. 11A, B, I). Choanosomal skeleton contains articulated tetraclone desmas, which form a dense root-like skeleton (Fig. 10C–F). Spinose metasters (Fig. 11C) and amphiasters (Fig. 11E). Oxeas (Fig. 11A, G) with pointed tips and thick strongyles (Fig. 11H). Megascleres are tetraclone desmas with root-like structures, measuring 632–736 × 395–406 µm (N15) (Fig. 10C–F). Phyllo- to discotriaenes with cladomes 447–536–636 µm (N20) and rhabdomes of 121–150–185 × 22–37–40 µm (N20) (Figs 10D, 11F). Tylostyles are 434–317–719 µm long (N15) (Fig. 11I). Oxea with pointed tips 120–125–134 × 4–5 µm (N7). Thick strongyles with rounded ends 95–100–112 × 7–10 µm (N4); Microscleres are massive acanthorhabds with irregularly distributed spined tubercles 5–17–20 × 3–4 µm (N30). Spinose metaster/amphiasters with long rays 17.3–20.5–22.2 × 12.5–15.6–20.0 µm. Figure 10. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, in situ picture of the paratype (378 m). B, deck picture of the paratype. C, outer surface of the choanosomal skeleton from the holotype. D, tetraclone desmas with phyllotriaene in situ of the holotype. E, F, detailed view of the articulated tetraclone desmas from the holotype. Figure 10. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, in situ picture of the paratype (378 m). B, deck picture of the paratype. C, outer surface of the choanosomal skeleton from the holotype. D, tetraclone desmas with phyllotriaene in situ of the holotype. E, F, detailed view of the articulated tetraclone desmas from the holotype. Figure 11. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in subectosomal regions (B). C, metaster. E, amphiaster. D, acanthorhabds in various sizes. F, ectosomal phyllotriaene. G, ecto- to subectosomal oxea with pointed tips. H, thick strongyle. I, choanosomal style. Figure 11. View largeDownload slide Racodiscula seymoura sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02014. A, B, thick sections showing phyllotriaenes with acanthorhabds on top (A) and in subectosomal regions (B). C, metaster. E, amphiaster. D, acanthorhabds in various sizes. F, ectosomal phyllotriaene. G, ecto- to subectosomal oxea with pointed tips. H, thick strongyle. I, choanosomal style. Etymology: Named after the type locality: Seymour Island (Galápagos Islands). DNA barcodes: We sequenced all three markers of the holotype and 28S (C1-D2) and 18S of the paratype. 28S sequences of type material were identical as well as were the 18S. GenBank accession numbers: COI KY652820, 28S KY652789–KY652790, 18S KY652837–KY652838. SBD record no. 1721–1722. Remarks: Apart from the differences observed in the oscula opening (see remarks for R. radixa sp. nov. and Table 3) Racodiscula seymoura sp. nov. show clear spiculation differences compared to the two other Racodiscula species described in this study. The presence of oxeas and strongyles and the root-like tetraclone desmas with long thin branches (Fig. 10F) along with slightly differences observed in the sizes of phyllo- to discotrianes (Table 3) separates this species from R. radixa sp. nov. Tetraclone desmas, which are not tuberculated in R. seymoura sp. nov. but strongly tuberculated in R. borisi sp. nov., and the longer and thicker rhabdome size clearly separate this new species from R. borisi sp. nov. Molecular markers suggest that R. seymoura sp. nov. is more closely related to R. radixa sp. nov. than to R. borisi sp. nov. (Fig. 5), and positioned within the clade of Racodiscula from the Galápagos. This is in accordance with the observed morphological differences. R acodiscula borisi sp. nov. (Figs 12, 13; Table 3) Diagnosis: Racodiscula with one perfectly circular apical cavity/osculum (0.8 cm) located at the centre; choanosomal spicules are articulated tetraclone desmas with short thick and strongly tuberculated branches, choanosomal spicules are thick strongyles with rounded tips. Type material: Holotype: HBOM 003:02001, Coll. JSL-I dive 3905 [18 October 1995, Galápagos, 5NM NE of Kicker Rock, San Cristobal Island, 0°43′08″S, 89°27′14″W, 215 m]. Paratype: 003:02011 (242 m), Galápagos, 4 NM SE of Tortuga Island, 1°04′59″S, 90°51′56″W, Coll. JSL-I dive 3923. Comparative material: Racodiscula radixa sp. nov. HBOM 003:02006. Racodiscula seymoura sp. nov. HBOM 003:02014; HBOM 003:02007. Type locality: San Cristobal Island, Galápagos Islands (215 m) (Fig. 1). Distribution: Known from the type locality and Tortuga Island, Galápagos. Habitat: Attached to hard substratum, depth range 215–242 m. Description: Morphology, massive subspherical with a centralized circular apical cavity/osculum opening on top of the surface (Fig. 12A, B). Dimension of the holotype is 5.5 cm in diameter and about 4–5 cm high (Fig. 12A, B). Texture, medium hard. Surface, smooth (Fig. 12B). Colour, white in vivo (Fig. 12A) and beige in ethanol (Fig. 12B). Ectosomal skeleton contains phyllo- to discotriaenes (Fig. 13B, F) with acanthorhabds in between and on top of this layer (Fig. 13A, B, E). Tylostyles are located at the subectosomal region (Fig. 12A, B, I). Choanosomal skeleton contains articulated tetraclone desmas, which form a dense skeleton (Fig. 12C–F). Spinose metaster/amphiasters (Fig. 13C, D). Oxeas (Fig. 13G) with pointed tips and thick strongyles (Fig. 13H). Megascleres, tetraclone desmas with short branches measuring 540–650 × 385–400 µm (N15) (Fig. 12E). Phyllo- to discotriaenes with cladomes 395–504–605 µm (N25) and rhabdomes of 20–66–93 × 18–21–25 µm (N10) (Fig. 13B, F). Tylostyles are 440–410–904 µm (N15) (Fig. 13I). Oxeas with pointed tips 140–176–204 × 4–5 µm (N7). Thick strongyles with round ends 95–194–112 × 15–28 µm (N4); Microscleres are massive acanthorhabds with irregularly distributed spined tubercles 12–19–27 × 3–4 µm (N30). Spinose streptaster/amphiasters with long rays 12.0–14.5–17.2 × 11.5–15.4–20.0 µm. Figure 12. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, in situ picture of the holotype (215 m). B, deck picture of the holotype. C, outer surface of the choanosomal skeleton with pore openings. D, tetraclone desmas with tylostyles lying on top. E, F, detailed views of the articulated tetraclone desmas from the holotype. Figure 12. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, in situ picture of the holotype (215 m). B, deck picture of the holotype. C, outer surface of the choanosomal skeleton with pore openings. D, tetraclone desmas with tylostyles lying on top. E, F, detailed views of the articulated tetraclone desmas from the holotype. Figure 13. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, B, thick sections of the ectosomal to choanosomal region showing phyllotriaenes, with acanthorhabds and bundles of tylostyles. C, D, scanning electron microscopy (SEM) of diverse amphiaster/metaster. E, SEM of acanthorhabd. F, light microscopy picture of ectosomal phyllotriaene. G, light microscopy picture of ecto- to subectosomal oxea with pointed tips. H, thick strongyle with rounded ends. I, SEM of choanosomal style. Figure 13. View largeDownload slide Racodiscula borisi sp. nov. Harbor Branch Oceanographic Museum (HBOM) 003:02001. A, B, thick sections of the ectosomal to choanosomal region showing phyllotriaenes, with acanthorhabds and bundles of tylostyles. C, D, scanning electron microscopy (SEM) of diverse amphiaster/metaster. E, SEM of acanthorhabd. F, light microscopy picture of ectosomal phyllotriaene. G, light microscopy picture of ecto- to subectosomal oxea with pointed tips. H, thick strongyle with rounded ends. I, SEM of choanosomal style. Etymology: Named in memory of the first author’s friend Dipl. Biol. Boris Knerr (†20 July 2012), for encouraging her scientific curiosity in sponge taxonomy. DNA barcodes: We sequenced COI, 28S (C1-D2) and 18S of the holotype and paratype. All three markers show identical sequences. GenBank accession numbers: COI KY652821–KY652822; 28S KY652791–KY652792; 18S KY652834–KY652835. SBD record no. 1723, 1724. Remarks: The most obvious key diagnostic character which differentiates this new species from the other two new Racodiscula species described here, is the presence of tetraclone desmas, which are strongly articulated with short root-like branches showing a strong tuberculation (Fig. 12C–F). This strong tuberculation of the tetraclone desmas is not present in the two other new species described. Racodiscula asteroides also shows sparsely tuberculated rays and strongly tuberculated tips of tetraclone desmas; however, it lacks oxeas and strongyles (Pisera & Lévi, 2002b). The rhabdome of phyllo- to discotrianes from R. borisi sp. nov. is much shorter and a bit thinner in size compared to the others newly described species (Table 3). Oxeas and strongyles appear larger in size compared to R. seymoura sp. nov. R. borisi sp. nov. groups within the Racodiscula clade from the Galápagos and forms a sister-group relationship to the other two species (Fig. 5). Suborder Spirophorina Bergquist & Hogg, 1969, Family Tetillidae Sollas, 1886 Genus C raniella Schmidt, 1870 C raniella wolfi sp. nov. (Fig. 14) Diagnosis: Craniella with worm-like long (62–91 µm) sigmaspires. Type material: Holotype: HBOM 003:02004, Coll. JSL-I dive 3909 [20 October 1995, Galápagos, S. Coast, approx. 5 NM NE of Wolf Island, 1°21′28″N, 91°48′41″W, 355 m]. Type locality: Wolf Island, Galápagos Islands (355 m) (Fig. 1). Distribution: Only known from type locality. Habitat: Attached to hard substratum, 355 m. Description: Morphology, globular small hispid sponge with small apical oscule and 1-mm-thick cortex (Fig. 14A, B). Dimension of the holotype is 3 cm in diameter and about 4–5 cm high (Fig. 14A, B). Texture, soft. Surface, conulose and hispid. Colour, white in vivo (Fig. 14A) and light brown in ethanol (Fig. 14B). Ectosomal skeleton contains bundles of oxeas with few protriaenes sticking out of these bundles. No anatriaenes detected. Choanosomal skeleton contains oxeas (Fig. 14C, D) and abandoned uncoiled sigmaspires. Megascleres are thick oxeas measuring 134–356–627 × 8–15–25 µm (N30) (Fig. 14C, D). Protriaenes, equal clads measuring 121–150–177 µm (N11) and rhabdome 3380–4864–5640 × 19–27–30 µm (N11). Anatriaenes are not present in our spicule preparations. Microscleres are thin and uncoiled long sigmaspires 62–79–91 × 1–3 µm (N30) (Fig. 14G). Figure 14. View largeDownload slide Craniella wolfi sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02004. A, in situ picture (355 m). B, deck picture. C, oxea. D, detailed view of oxea. E, protriaene with equal clads. F, details of protriaene. G, long sigmaspires. Figure 14. View largeDownload slide Craniella wolfi sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02004. A, in situ picture (355 m). B, deck picture. C, oxea. D, detailed view of oxea. E, protriaene with equal clads. F, details of protriaene. G, long sigmaspires. Etymology: Named after point of origin: Wolf Island (Latin, wolfi). DNA barcodes: We sequenced COI, 28S (C1-D2) and 18S (1,670 bp) of the holotype. GenBank accession number: COI KY652825, 28S KY652795, 18S KY652840. SBD record no. 1727. Remarks: The absence of porocalices and the presence of a conspicuous double-layered cortex clearly places this species into the genus Craniella. There are currently 34 valid Craniella species (P. Cárdenas, unpublished results), ten of which have lost their sigmaspires. The remaining 24 species have classical C- or S-shaped sigmaspires 8–30 µm long; none have these unusually worm-like long (62–91 µm) sigmaspires. Molecular investigations confirm that C. wolfi sp. nov. is closely related to Craniella aff. zetlandica (ZMBN 85239) from Western Norway (Korsfjord), a topology which is highly supported (PP = 1, BS = 100) (Fig. 5). Family Scleritodermidae Sollas, 1888 Genus S cleritoderma Sollas, 1888 Emended diagnosis: Scleritodermidae with ectosomal acanthostrongyles or acanthorhabds, occasionally with ectosomal tylostyles, and sigmaspires as microscleres (modified after Pisera & Lévi, 2002c). Remarks: Our new species has all the characters of typical Scleritoderma, for example, rhizoclone desmas, sigmaspire microscleres occurring in the choanosome; however, this is the first time tylostyles have been observed. Careful analysis demonstrated, that these are clearly not a contamination (see remarks below). The occurrence of tylostyles resulted in the emended diagnosis. S cleritoderma tortuga sp. nov. (Figs 15, 16) Diagnosis: Scleritoderma with tylostyles vertically protruding from the skeletons surface. Synonymy: Scleritodermida sp. 1 and Scleritodermida sp. 2 (Schuster et al., 2017, additional file 1, Fig. 4). Type material: Holotype: HBOM 003:02012, Coll. JSL-I dive 3923 [27 October 1995, Galápagos, SW Coast, Caleta Webb, 0.5 NM offshore of Tortuga Island, 1°04′59″S, 90°51′56″W, 242 m]. Paratype: HBOM 003:02009 (239 m), Galápagos, Isabela Island, 0°47′04″S, 91°26′32″W, Coll. JSL-I dive 3919. Comparative material: Scleritoderma camusi Lévi & Lévi, 1983, Holotype MNHN DCL 2787, paratypes: MNHN DCL 2788, 355–30 m, Norfolk Ridge. S. camusi (described in Schlacher-Hoenlinger, Pisera & Hooper, 2005), Norfolk Ridge 278–410 m, QMG318549, QMG318706. S. nodosum Thiele, 1900, MNH DCL 3233, 90–110 m, Philippines (described in Lévi & Lévi, 1989). S. flabelliforme Sollas, 1888 (described in Schlacher-Hoenlinger et al., 2005), Norfolk Ridge, 458–68 m, QMG318641, QMG318658, QMG318664. Type locality: Tortuga Island, Galápagos Islands (242 m) (Fig. 1). Distribution: Known from type locality and Isabela Island. Habitat: Attached to hard substratum, depth range 239–242 m. Description: Morphology, massive and flabelliform (Fig. 15A, B). Dimension of the holotype is 8 cm in diameter and about 3–5 cm in height (Fig. 15A, B). Texture, stony and hard. Surface, covered with small oscule openings 0.2–0.3 mm in diameter. Colour, beige in vivo (Fig. 15A) and light brown in ethanol (Fig. 15B). Ectosomal skeleton contains a thick layer of acanthorhabds (Fig. 15D) on the surface. The heads of tylostyles project beyond the surface layer (Fig. 15C, E). Very rare sigmaspires. Choanosomal skeleton composed of dense articulated slightly thorny rhizoclones (Fig. 16). Large oscula openings on the surface of the choanosomal skeleton 300–400 µm in diameter (Fig. 16A). Megascleres are rhizoclone desmas 225–350 µm in diameter and tylostyles measuring 153–256–316 µm (N15) (Fig. 15E, H). Microscleres are C-shaped thick spinose sigmaspires 9–13 × 1–2 µm (N3) (Fig. 15F) and slightly curved acanthostrongyles 61–84–101 × 5–6 µm (N30) (Fig. 15D, G). Figure 15. View largeDownload slide Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, in situ picture 242 m. B, deck picture. C, thick section of the ectosome and part of the choanosome. Arrows pointing to tylostyles (tylo). D, detailed view the ectosome with a layer of acanthorhabds (acant). E, ectosomal view with tylostyles (tylo). F, thick sigmaspire. G, spinose acanthorhabd. H, detailed views of tylostyles. Figure 15. View largeDownload slide Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, in situ picture 242 m. B, deck picture. C, thick section of the ectosome and part of the choanosome. Arrows pointing to tylostyles (tylo). D, detailed view the ectosome with a layer of acanthorhabds (acant). E, ectosomal view with tylostyles (tylo). F, thick sigmaspire. G, spinose acanthorhabd. H, detailed views of tylostyles. Figure 16. View largeDownload slide Diverse desmas of Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, overview of the choanosomal skeleton with several oscules. B, detailed view of one oscula opening. C, a loose rhizoclone desma. D, rhizoclone desmas with a tylostyle. E, F, detailed view of spines of rhizoclones with acanthorhabds. Figure 16. View largeDownload slide Diverse desmas of Scleritoderma tortuga sp. nov. holotype Harbor Branch Oceanographic Museum (HBOM) 003:02012. A, overview of the choanosomal skeleton with several oscules. B, detailed view of one oscula opening. C, a loose rhizoclone desma. D, rhizoclone desmas with a tylostyle. E, F, detailed view of spines of rhizoclones with acanthorhabds. Etymology: Named after the type locality: Tortuga Island. DNA barcodes: COI sequences as in Schuster et al. (2017), 28S (C1-D2) of the holotype and paratype as well as 18S (1770 bp) of the paratype (GenBank accession numbers: 28S KY652801, KY652802; 18S KY652839, SBD record no. 1730, 1731). The 28S sequences were identical. The obtained COI sequence of HBOM 003:02012 was shorter (393 bp) compared to HBOM 003:02009 (630 bp). The 393 bp were identical (100%). Remarks: Our new species Scleritoderma tortuga sp. nov. differs from the four other Scleritoderma species, S. camusi Lévi & Lévi, 1983, S. cyaneum Van Soest & Stentoft, 1988, S. flabelliforme Sollas, 1888 and S. nodosum Thiele, 1900 (Van Soest et al., 2017), by the presence of tylostyles. Since tylostyles have never been observed in Scleritoderma species before, we first questioned if these could be due to a contamination from another sponge. However, we are convinced here that these are proper to the specimen, as we observed at least 15 tylostyles in both the holotype and paratype sections, all orientated in a vertical position protruding from the skeleton’s surface (Fig. 15C, E). Additionally, in all of our preparations, no other spicule types were found, which may have indicated a possible contamination from another sponge. Furthermore, our concatenated molecular analyses including sequences of all known Scleritoderma species (Fig. 5) indicate that Scleritoderma tortuga sp. nov. forms a sister-group relationship to S. nodosum from the Indo-Pacific (17–27 m). Additional supportive evidence of the new species is given by the presence of a 1109 bp long ‘intron 723’ located within the COI region (Schuster et al., 2017) in both specimens. Our concatenated phylogenetic analysis supports the monophyly of the genus Scleritoderma which was first reported by Schuster et al. (2017) on single mitochondrial data. Based on the presence of tylostyles in S. tortuga sp. nov. the definition of Scleritoderma was amended. Tylostyles are actually not uncommon within Scleritodermidae, for example, they are known to occur in Aciculites and may have been overlooked in other species. We therefore recommend that future revisions of this family reassess the presence of tylostyles within this group. DISCUSSION AND CONCLUSION Previous knowledge on tetractinellids from the Galápagos archipelago was scarce (about 11 species described) and mainly limited to shallow-water habitats (<100 m) (Wilson, 1904; de Laubenfels, 1939; Desqueyroux-Faúndez & Van Soest, 1997). In fact, only one single desma-bearing astrophorid species (Corallistes isabela) and one species from the suborder Spirophorina (Cinachyrella globulosa), both endemic to the Galápagos, were described by Desqueyroux-Faúndez & Van Soest (1997). Applying the morphological as well as the phylogenetic species concept we enlarge the current knowledge with the discovery of five new deep-water desma-bearing Astrophorina (Neophrissospongia galapagoensis sp. nov., Corallistes floreana sp. nov., Racodiscula radixa sp. nov., Racodiscula seymoura sp. nov. and Racodisucla borisi sp. nov.) and report for the second time the occurrence of Corallistes isabela. Additionally, Craniella wolfi sp. nov. of the family Tetillidae and Scleritoderma tortuga sp. nov. of the family Scleritodermidae (Spirophorina) are described. The definition of the genus Scleritoderma required subsequent amendment. All seven new species are most likely endemic to the Galápagos islands. Statistical methods such as Principle Component Analysis (PCA) or haplotype networks which may help discriminate the closely related species C. floreana np. nov. from C. isabela and R. radixa sp. nov. from R. seymoura sp. nov. were not feasible in this study, due to the low sample size (only one or two samples per species). A further statistical probabilistic based test is the coalescent theory which is based on the evolutionary history of alleles. The application of this test is widespread in integrative taxonomic studies to analyze species structures (Fujita et al., 2012). However, these methods recently have been shown to overestimate structure and consequently falsely suggest speciation events (Sukumaran & Knowles, 2017). This test again requires a higher sample size than given by our data. Therefore, we refrained from using multispecies coalescent analysis in this study as it may provide unambiguous results. Nevertheless, this study provides the first comprehensive dataset to investigate the deep-water ‘lithistid’ fauna of the Galápagos by providing descriptions of unique specimens collected in one of the most remote marine areas. Our phylogenetic analyses (Fig. 5) including other material from the Caribbean, Central and West Pacific provide strong evidence of biogeographical differences and support an apparent high level of demosponge endemism in this region (Desqueyroux-Faúndez & Van Soest, 1997), but should be corroborated by further sampling from other areas of the Eastern Pacific. The long paleontological history of most families like Corallistidae ~155 Myr and Theonellidae ~167 Myr, as well as rhizoclone desmas present since the Early Paleozoic (e.g. Pisera, 2002), may indicate that the current tetractinellid biodiversity in the Eastern Pacific is much higher than previously assumed. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. Specimen list. [Version of Record, published online 23 March 2018; http://zoobank.org/urn:lsid:zoobank.org:pub:20FB9570-C49B-4B2A-ADFA-684F5495A0BF] ACKNOWLEDGEMENTS Financial support for this study was provided by the German Science Foundation (DFG ER 611/3-1, DFG Wo869/15-1). The LMUMentoring and the HELGE AX:Son JOHNSON STIFTELSE provided funding for AS to visit HBOI (Florida, USA) and NIWA (National Institute of Water and Atmospheric Research, Auckland and Wellington in New Zealand). We greatly thank the colleagues Amy Wright, John Reed and Megan Conkling from HBOI for sharing material, help in the collection, shipping from Florida and providing underwater and deck pictures for the new described species as well as the crew of the RV ‘Johnson Sea Link I’ submersible for collection of the samples. Specimens were supplied by the NIWA Invertebrate Collection (NIC); we are particularly grateful to Sadie Mills and Kareen Schnabel for their assistance in Wellington and diligent assistance with loans. Several specimens (NIWA 110983, 110214, 18823) were collected by the Coral Reef Research Foundation under contract to the US National Cancer Institute. This is a contribution of the Coral Reef Research Foundation, Palau. We thank Roland Melzer (ZSM, Zoologische Staatssammlung München) for the introduction to the SEM. We thank Gabrielle Büttner and Simone Schätzle (Department of Earth & Environmental Sciences, LMU Munich, Germany) for sequencing assistance. PC is supported by the European Union’s Horizon 2020 research and innovation program through the SponGES project (grant agreement no. 679849). MK’s participation was funded by NIWA under Coasts and Oceans Research Programme 2 Marine Biological Resources: Discovery and definition of the marine biota of New Zealand (2017/2018 SCI). REFERENCES Bergquist PR , Hogg JJ . 1969 . Free amino acid patterns in Demospongiae: a biochemical approach to sponge classification . Cahiers de Biologie Marine 10 : 205 – 220 . Cárdenas P , Pérez T , Boury-Esnault N . 2012 . Sponge systematics facing new challenges . Advances in Marine Biology 61 : 79 – 209 . Google Scholar CrossRef Search ADS PubMed Cárdenas P , Rapp HT , Schander C , Tendal O . 2009 . Molecular taxonomy and phylogeny of the Geodiidae (Porifera, Demospongiae, Astrophorida) – combining phylogenetic and Linnaean classification . Zoologica Scripta 39: 1 – 18 . Carella M , Agell G , Cárdenas P , Uriz MJ . 2016 . Phylogenetic reassessment of Antarctic Tetillidae (Demospongiae, Tetractinellida) reveals new genera and genetic similarity among morphologically distinct species . PloS ONE 11: e0160718 . Carvalho FC , Pomponi SA , Xavier JR . 2015 . Lithistid sponges of the upper bathyal of Madeira, Selvagens and Canary Islands, with description of a new species of Isabella . Journal of the Marine Biological Association of the United Kingdom 95 : 1287 – 1296 . Google Scholar CrossRef Search ADS Chavez FP , Brusca RC . 1991 . The Galápagos Islands and their relation to oceanographic processes in the tropical pacific . In: James M , ed. Galapagos marine invertebrates . New York : Plenum , 9 – 33 . Google Scholar CrossRef Search ADS Chombard C , Boury-Esnault N , Tillier S . 1998 . Reassessment of homology of morphological characters in tetractinellid sponges based on molecular data . Systematic Biology 47 : 351 – 366 . Google Scholar CrossRef Search ADS PubMed de Laubenfels MW . 1939 . Sponges collected on the presidential Cruise of 1938 . Smithsonian Miscellaneous Collections 98 : 1 – 7 . Google Scholar CrossRef Search ADS Desqueyroux-Faúndez R , Van Soest RWM . 1997 . Shallow waters demosponges of the Galápagos Islands . Revue Suisse de Zoologie 104 : 379 – 467 . Google Scholar CrossRef Search ADS Ekins M , Erpenbeck D , Wörheide G , Hooper JNA . 2016 . A new species of lithistid sponge hiding within the Isabella mirabilis species complex (Porifera: Demospongiae: Tetractinellida) from seamounts of the Norfolk Ridge . Zootaxa 4136 : 433 – 460 . Google Scholar CrossRef Search ADS PubMed Feingold JS . 2011 . El Niño, La Niña and ENSO . In: Hopley D , ed. Encyclopedia of modern coral reefs, part 5 . Dordrecht : Springer , 365 – 368 . Google Scholar CrossRef Search ADS Fujita MK , Leaché AD , Burbrink FT , McGuire JA , Moritz C . 2012 . Coalescent-based species delimitation in an integrative taxonomy . Trends in Ecology & Evolution 27 : 480 – 488 . Google Scholar CrossRef Search ADS PubMed Geist D , Diefenbach BA , Fornari DJ , Kurz MD , Harpp K , Blusztajn J . 2008 . Construction of the Galápagos platform by large submarine volcanic terraces . Geochemistry, Geophysics, Geosystems 9 : 1 – 27 . Google Scholar CrossRef Search ADS Hall KA , Ekins MG , Hooper JNA . 2014 . Two new desma-less species of Theonella Gray, 1868 (Demospongiae: Astrophorida: Theonellidae), from the Great Barrier Reef, Australia, and a re-evaluation of one species assigned previously to Dercitus Gray, 1867 . Zootaxa 3814 : 451 – 477 . Google Scholar CrossRef Search ADS Holden JC , Dietz RS . 1972 . Galápagos Gore, NazCoPac Triple Junction and Carnegie/Cocos Ridges . Nature 235 : 266 – 269 . Google Scholar CrossRef Search ADS James MJ (ed.). 1991 . Galápagos marine invertebrates. Taxonomy, biogeography, and evolution in Darwin’s Islands . New York : Springer Science+Business Media . Google Scholar CrossRef Search ADS Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , Sturrock S , Buxton S , Cooper A , Markowitz S , Duran C , Thierer T , Ashton B , Meintjes P , Drummond A . 2012 . Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics (Oxford, England) 28 : 1647 – 1649 . Google Scholar CrossRef Search ADS PubMed Kelly M , Cárdenas P . 2016 . An unprecedented new genus and family of Tetractinellida (Porifera, Demospongiae) from New Zealand’s Colville Ridge, with a new type of mitochondrial group I intron . Zoological Journal of the Linnean Society 177 : 335 – 352 . Google Scholar CrossRef Search ADS Lanfear R , Calcott B , Ho SY , Guindon S . 2012 . Partitionfinder: combined selection of partitioning schemes and substitution models for phylogenetic analyses . Molecular Biology and Evolution 29 : 1695 – 1701 . Google Scholar CrossRef Search ADS PubMed Lavrov D , Wang X , Kelly M . 2008 . Reconstructing ordinal relationships in the Demospongiae using mitochondrial genomic data . Molecular Phylogenetics and Evolution 49 : 111 – 124 . Google Scholar CrossRef Search ADS PubMed Lê HLV , Lecointre G , Perasso R . 1993 . A 28S rRNA-based phylogeny of the gnathostomes: first steps in the analysis of conflict and congruence with morphologically based cladograms . Molecular Phylogenetics and Evolution 2 : 31 – 51 . Google Scholar CrossRef Search ADS PubMed Lévi C , Lévi P . 1983 . Eponges Tétractinellides et Lithistides bathyales de Nouvelle-Calédonie . Bulletin du Muséum national d’Histoire naturelle 5 : 101 – 168 . Lévi C , Lévi P . 1989 . Spongiaires (MUSORSTOM 1 and 2) . In: Forest J , ed. Résultats des Campagnes MUSORSTOM 4 Mémoires du Muséum National d’Histoire Naturelle. Zoologie 143 : 25 – 103 . Manconi R , Serusi A . 2008 . Rare sponges from marine caves: discovery of Neophrissospongia nana nov. sp. (Demospongiae, Corallistidae) from Sardinia with an annotated checklist of Mediterranean lithistids . ZooKeys 4 : 71 – 87 . Google Scholar CrossRef Search ADS Marshall W . 1876 . Ideen über die Verwandtschaftsverhältnisse der Hexactinelliden . Zeitschrift für wissenschaftliche Zoologie 21 : 113 – 136 . Meyer CP , Geller JB , Paulay G . 2005 . Fine scale endemism on coral reefs: Archipelagic differentiation in turbinid gastropods . Evolution 59 : 113 – 125 . Google Scholar CrossRef Search ADS PubMed Morrow C , Cárdenas P . 2015 . Proposal for a revised classification of the Demospongiae (Porifera) . Frontiers in Zoology 12 : 1 – 27 . Google Scholar CrossRef Search ADS PubMed Pisera A . 2002 . Fossil ‘Lithistids’: an overview . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 388 – 402 . Google Scholar CrossRef Search ADS Pisera A , Lévi C . 2002a . Family Corallistidae Sollas, 1888 . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 312 – 320 . Google Scholar CrossRef Search ADS Pisera A , Lévi C . 2002b . Family Theonellidae von Lendenfeld, 1903 . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 327 – 337 . Pisera A , Lévi C . 2002c . Family Scleritodermidae Sollas, 1888 . In: Hooper JNA , Van Soest RWM , eds. Systema Porifera. A guide to the classification of sponges . New York, Boston, Dordrecht, London, Moscow : Kluwer Academic/ Plenum Publishers , 302 – 311 . Google Scholar CrossRef Search ADS Pisera A , Pomponi SA . 2015 . New data on lithistid sponges from the deep Florida shelf with description of a new species of Theonella . Journal of the Marine Biological Association of the United Kingdom 95 : 1297 – 1309 . Google Scholar CrossRef Search ADS Pisera A , Vacelet J . 2011 . Lithistid sponges from submarine caves in the Mediterranean: taxonomy and affinities . Scientia Marina 75 : 17 – 40 . Google Scholar CrossRef Search ADS Pomponi SA , Kelly M , Reed JK , Wright A . 2001 . Diversity and bathymetric distribution of lithistid sponges in the tropical western Atlantic region . Bulletin of the Biological Society of Washington 10 : 344 – 353 . Ryan WBF , Carbotte SM , Coplan JO , O’Hara S , Melkonian A , Arko R , Weissel RA , Ferrini V , Goodwillie A , Nitsche F , Bonczkowski J , Zemsky R . 2009 . Global multi-resolution topography synthesis . Geochemistry Geophysics Geosystem 10 : Q03014 . Google Scholar CrossRef Search ADS Schlacher-Hoenlinger MA , Pisera A , Hooper JNA . 2005 . Deep-sea ‘lithistid’ assemblages from the Norfolk Ridge (New Caledonia), with description of seven new species and a new genus (Porifera, Demospongiae) . Zoosystema 27 : 649 – 698 . Schmidt O . 1870 . Grundzüge einer Spongien-Fauna des atlantischen Gebietes . Leipzig : Verlag von Wilhelm Engelmann , 1 – 88 . Schuster A , Erpenbeck D , Pisera A , Hooper J , Bryce M , Fromont J , Wörheide G . 2015 . Deceptive Desmas: molecular phylogenetics suggests a new classification and uncovers convergent evolution of lithistid demosponges . PLoS ONE . 10 : e116038 . Google Scholar CrossRef Search ADS PubMed Schuster A , Lopez JV , Becking LE , Kelly M , Pomponi SA , Wörheide G , Erpenbeck D , Cárdenas P . 2017 . Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding . BMC Evolutionary Biology 17 : 1 – 21 . Google Scholar CrossRef Search ADS PubMed Sollas WJ . 1885 . A classification of the sponges . Journal of Natural History 16 : 395 – 395 . Google Scholar CrossRef Search ADS Sollas WJ . 1886 . Preliminary account of the Tetractinellid sponges Dredged by H.M.S. ‘Challenger’ 1872–76. Part I. The Choristida . The Scientific Proceedings of the Royal Dublin Society 5 : 177 – 199 . Sollas WJ . 1887 . Sponges . In: Black A & C , ed. Encyclopaedia Britannica, 9th edn . Edinburgh, 412 – 429 . Sollas WJ . 1888 . Report on the Tetractinellida collected by H.M.S. Challenger, during the years 1873–1876 . Zoology 25 : 1 – 458 . Sukumaran J , Knowles LL . 2017 . Multispecies coalescent delimits structure, not species . Proceedings of the National Academy of Sciences 114 : 1607 – 1612 . Google Scholar CrossRef Search ADS UNESCO . 1978 . Galápagos Islands . World Heritage Convention . Available at: http://whc.unesco.org/en/list/1 Van Soest RWM , Boury-Esnault N , Vacelet J , Dohrmann M , Erpenbeck D , De Voogd NJ , Santodomingo N , Vanhoorne B , Kelly M , Hooper JNA . 2012 . Global diversity of sponges (Porifera) . PloS ONE 7 : e35105 . Google Scholar CrossRef Search ADS PubMed Van Soest RWM , Boury-Esnault N , Hooper JNA , Rützler K , de Voogd NJ , Alvarez de Glasby B , Hajdu E , Pisera A , Manconi R , Schoenberg C , Klautau M , Picton B , Kelly M , Vacelet J , Dohrmann M , Díaz M-C , Cárdenas P , Carballo JL . 2017 . WorldPorifera database . Available at: http://www.marinespecies.org/Porifera on 2017-02-27 Van Soest RWM , Stentoft N . 1988 . Barbados deep-water sponges . Studies on the Fauna of Curaçao and other Caribbean Islands 70 : 1 – 175 . von Lendenfeld R . 1903 . Tetraxonia . In: Schulze FE , ed. Das Tierreich . Berlin : Friedländer , 1 – 168 . Wilson HV . 1904 . Reports on an exploration off the West Coasts of Mexico, Central and South America, and off the Galápagos Islands, in charge of Alexander Agassiz, by the U.S. Fish Commission Steamer ‘Albatross’ during 1891, Lieut. Commander Z.L. Tanner, U.S.S., commanding. XXX. The Sponges . Memoirs of the Museum of Comparative Zoology at Harvard Collegee 30 : 1 – 164 . Zittel KA . 1878 . Studien über fossile Spongien. II. Lithistidae . Abhandlungen der Mathematisch-Physikalischen Classe der Königlich-Bayerischen Akademie der Wissenschaften 13 : 65 – 154 . © 2018 The Linnean Society of London, Zoological Journal of the Linnean Society

Journal

Zoological Journal of the Linnean SocietyOxford University Press

Published: Mar 23, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off