New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae: Sphaerocladina) reveal an ancient relict fauna with Tethys origin

New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae:... Abstract Vetulina Schmidt, 1879 (Demospongiae, Sphaerocladina, Vetulinidae) currently constitutes the only living representative of a once diverse Mesozoic group. Molecular data place Vetulina as a sister taxon to freshwater sponges (Spongillida) despite different skeletal composition. To date, only three extant species of this desma-bearing ‘rock sponge’ have been described from the Caribbean and Indian Ocean, all with similar growth forms and spiculation, but different desma and surface details. Comparison of these genetically very similar species was not possible until the present study. The distribution of Vetulina is taken to be a consequence of the closure of the Tethyan Seaway in the Early Miocene, suggesting a more widely distributed population with its origin in the Tethys Sea. To support this hypothesis in a molecular palaeobiological framework, we first increased the taxon sampling by describing and sequencing two new species of Vetulina from the Bahamas and Philippines and report Vetulina stalactites from nine additional locations in the Tropical Western Atlantic. A robust, dated phylogeny was calculated from the combined dataset and amended by five representative fossils. Our results point to an Eocene origin for Vetulina, even before the closure of the Tethyan Seaway in the Miocene, supporting the hypothesis that Vetulina presents a relict fauna with its origin in the Tethys Sea. cox1, DNA barcode, freshwater sponges, molecular clocks, Porifera, relict fauna, Sphaerocladina, Tethys Sea, Vetulina INTRODUCTION Demosponges forming a rock-like skeleton of articulated megascleres (desmas) have historically been called ‘lithistid’ sponges. Morphological and molecular studies assigned 11 out of 13 ‘lithistid’ families to the order Tetractinellida Marshall, 1876 (e.g. Pisera & Lévi, 2002a; Cárdenas et al., 2011; Morrow & Cárdenas, 2015; Schuster et al., 2015). Desmanthidae Topsent, 1893, Vetulinidae von Lendenfeld, 1903 and Crambeidae Lévi, 1963 represent the only desma-bearing group of families outside the Tetractinellida, mostly comprising only one desma-bearing genus (e.g. Schuster et al., 2015): Desmanthidae is allocated to the order Bubarida Morrow and Cárdenas, 2015 (Morrow & Cárdenas, 2015) and Crambeidae to Poecilosclerida Topsent, 1928 (e.g. Van Soest, 2002), whereas Vetulinidae constitutes its own order, Sphaerocladina Schrammen, 1924, which already exists in the fossil classification (Morrow & Cárdenas, 2015; Schuster et al., 2015). Among all extant valid marine demosponges, Sphaerocladina has been recovered as sister taxon to the freshwater sponges (Spongillida; Manconi & Pronzato, 2002) by independent ribosomal (28S and 18S) and mitochondrial (cox1 and complete mitogenomes) markers (Kelly-Borges & Pomponi, 1994; McInerney, Adams, & Kelly, 1999; Redmond et al., 2013; Schuster et al., 2015). However, these two orders clearly differ in their skeleton and spicule composition. Spongillida is mainly characterized by smooth or spined monaxonic megascleres and may possess distinct gemmule microscleres (Manconi & Pronzato, 2002). In contrast, the skeleton of Sphaerocladina is built up by astro- to sphaeroclonar desmas (Pisera & Lévi, 2002b) and various microstyles (Schmidt, 1879; Pisera et al., 2017). Thus, their molecularly close but morphologically and ecologically disparate and enigmatic sister relationship to freshwater sponges remains a matter of further investigation. Within the monogeneric family Vetulinidae, the genus Vetulina Schmidt, 1879 (Pisera & Lévi, 2002b) is described as a flabellate or vase-shaped demosponge, with acrepid polyaxial astro- to sphaeroclonar megascleres (Pisera & Lévi, 2002b) and microscleric styles (Schmidt, 1879; Pisera et al., 2017). Before the recently discovered two new species from the Indian Ocean (Vetulina rugosa Pisera, Łukowiak, Fromont & Schuster, 2017 and Vetulina indica Pisera, Łukowiak, Fromont & Schuster, 2017; Pisera et al., 2017), this genus was monospecific, with Vetulina stalactites Schmidt, 1879 from Barbados (Schmidt, 1879; Van Soest & Stentoft, 1988). All extant Vetulina species were collected from similar rocky substrates and/or sandy slopes at depths between 95 and 220 m (Schmidt, 1879; Pomponi et al., 2001; Pisera et al., 2017). Nevertheless, the extant diversity of Sphaerocladina, with three valid species and one genus, appears considerably low compared with their fossil record, with ten genera known, e.g. from the Jurassic, Cretaceous and Eocene of Europe (Reid, 2004; Frisone, Pisera & Preto, 2016). The known Mesozoic Sphaerocladina fauna and their Recent disjunct occurrences in the Caribbean and Southwest Pacific led Pisera et al. (2017) to conclude that Vetulina represents an example of a relict fauna with a possible origin in the Tethys Sea, which was a large ocean during the Mesozoic Era that separated the supercontinent of Laurasia in the north from Gondwana in the south until its closure in the Miocene (Serravallian; 13–11 Mya; Rögl, 1998). The closure of the Tethyan Seaway in the Miocene is considered to be a key factor for the Recent rare occurrences and disjunct distribution of extant Vetulina species (Pisera et al., 2017). A correlation between the biogeographical distribution of modern and Mesozoic ‘lithistid’ demosponges to the Tethys Sea was already noticed and discussed by Reid (1967). He recognized that the ‘lithistid’ sponge fauna from the Upper Cretaceous of Northern Europe, which, at that time, represented a subtropical/warm-temperate region, included several representatives from the western end of the Tethys Sea, which was by that time a tropical/subtropical region still connected with the Atlantic Ocean. Thus, the Tethys Sea was climatically very similar to the present known regions of the Tropical Western Atlantic and the Pacific Ocean. Both of these regions are known for their rich extant ‘lithistid’ fauna (see e.g. Pomponi et al., 2001; Kelly, 2007). Further Recent ‘lithistid’ species, similar to Vetulina, also appear to have a disjunct distribution. The genus Isabella Schlacher-Hoenlinger, Pisera and Hooper, 2005 (Family Corallistidae Sollas, 1888) was first described in the southwest Pacific and recently discovered in the north-east Atlantic (Carvalho, Pomponi & Xavier, 2015). Corallistidae has a diverse (11 genera) and well-known Mesozoic fossil record, and was recently discovered from the Eocene of Europe (e.g. Pisera, 2002; Świerczewska-Gładysz, 2017). Likewise, Boury-Esnault, Pansini & Uriz (1992) noticed the similarity between the Recent Mediterranean Discorhabdella hindei Boury-Esnault, Pansini & Uriz, 1992 species and the fossil pre-Messinian Indo-Pacific species, but clearly disclosed any affinity to the Atlantic Discorhabdella species. Further support for a Tethyan origin is provided by the study of Maldonado et al. (2001), who reported the first occurrences of Discorhabdella Dendy, 1924 and Crambe Vosmaer, 1880 (desma bearing) from the eastern Pacific, which until then were known only from the Mediterranean Sea and the Atlantic. Further examples of non-‘lithistid’ demosponges and their hypothesized Tethyan origin were discussed and proposed in the studies of Łukowiak, Pisera & Schlögl (2014) and Łukowiak (2016), who compared the late Early Miocene fossil sponges from the central Paratethys in Slovakia as well as the Eocene fossil and modern sponge fauna of southern Australia. Although several examples of relict faunas with a possible origin in the Tethys Sea are predicted, not only among sponges (see e.g. Bitner & Motchurova-Dekova, 2016 for brachiopods; and Obura, 2016 for scleractinian corals), no attempt has yet been made to test this hypothesis for any of these groups in a molecular palaeobiological approach using fossil data. Its application requires not only a well-studied fossil record, but also a comprehensive molecular dataset. The availability of suitable Sphaerocladina sequences in GenBank and other sources is currently poor. At present, no common gene regions of the Caribbean and the Indian Ocean Vetulina species have been sequenced. Only one 28S (D3–D5) and three 18S sequences of V. stalactites from the Caribbean and one cox1 sequence of V. rugosa and V. indica, both from the Indian Ocean (Redmond et al., 2013; Schuster et al., 2015; Pisera et al., 2017; McInerney et al., 1999; Kelly-Borges & Pomponi, 1994) are available, hampering an integrative molecular palaeobiological approach. As a result, all hypotheses of a Tethys origin for Vetulina and other sponges (desma and non-desma bearing), are currently based only on empirical data from distribution patterns and fossils (see examples above), but in-depth molecular dating analyses are needed to corroborate current hypotheses about these processes. The aims of the present study, therefore, were as follows: (1) to provide a morphological description and illustrate V. stalactites from the Tropical Western Atlantic and to describe two new species, Vetulina incrustans sp. nov. from the Philippines and Vetulina tholiformis sp. nov. from the Bahamas; (2) to discriminate all valid Vetulina species with genetic markers and to calculate a robust phylogenetic tree that can be used as a basis for dating analyses; and (3) to combine the phylogenies of two independent markers (28S and cox1) with selected representative fossils in a relaxed molecular clock analysis, with the objective of testing the hypothesis of whether Vetulina constitutes another example of a relict fauna with its origin in the Tethys Sea. MATERIAL AND METHODS Specimen collection and morphological investigations Vetulina stalactites (13 specimens) and V. tholiformis sp. nov. (four specimens) were collected by the Harbor Branch Oceanographic Institution (HBOI) during expeditions to the Bahamas, Curaçao, Turks and Caicos, Jamaica, Bonaire and St Vincent Island from 1989 to 2003. Samples were collected at 12 stations from water depths ranging from 115 to 569 m (Fig. 1A, Table 1). Vetulina incrustans sp. nov. was collected from the Philippines during a Coral Reef Research Foundation (CRRF; Republic of Palau) expedition in 1996, by scuba diving, from 7 m (Fig. 1B). Upon collection, specimens were preserved in 70–80% ethanol. All samples collected from the Tropical Western Atlantic regions are accessioned within the Harbor Branch Oceanographic Museum (HBOM), Fort Pierce, FL, USA, although microscope slides and scanning electron microscopy (SEM) stubs of the skeleton and a small piece of the holotype and paratypes of V. tholiformis sp. nov. are stored at the Bavarian State Collection for Palaeontology and Geology (BSPG) in Munich, Germany. The holotype of V. incrustans sp. nov. is accessioned within the collections of the National Institute of Water & Atmospheric Research (NIWA) Invertebrate collection (NIC), and a small piece of the holotype is archived at the BSPG with stubs and slides. A small piece of the holotype of V. incrustans is also accessioned into the collections of the National Museum of Natural History (USNM) Smithsonian Institute, Washington, DC, USA. A detailed list of all Vetulina specimens from this study with their corresponding museum numbers, locations and accession numbers, is given in Table 1. Table 1. Species list, accession numbers and Sponge Barcoding Project (SBP) numbers of Vetulina specimens used in this study Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  View Large Table 1. Species list, accession numbers and Sponge Barcoding Project (SBP) numbers of Vetulina specimens used in this study Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  View Large Figure 1. View largeDownload slide Maps illustrating the disjunct occurrences of currently valid Vetulina species. A, distribution of Vetulina stalactites (red) and Vetulina tholiformis sp. nov. (blue) in the Tropical Western Atlantic. B, distribution of Vetulina indica (pink) and Vetulina rugosa (brown) from the Indian Ocean, and Vetulina incrustans sp. nov. (yellow) from the Philippine Sea. Holotype locations are indicated as stars and paratypes as circles. The map was made with GeoMapApp 3.6.3 http://www.geomapapp.org (Ryan et al. 2009). Figure 1. View largeDownload slide Maps illustrating the disjunct occurrences of currently valid Vetulina species. A, distribution of Vetulina stalactites (red) and Vetulina tholiformis sp. nov. (blue) in the Tropical Western Atlantic. B, distribution of Vetulina indica (pink) and Vetulina rugosa (brown) from the Indian Ocean, and Vetulina incrustans sp. nov. (yellow) from the Philippine Sea. Holotype locations are indicated as stars and paratypes as circles. The map was made with GeoMapApp 3.6.3 http://www.geomapapp.org (Ryan et al. 2009). A vertically oriented piece of sponge tissue from the surface to the choanosome was cut, dried and prepared for SEM as outlined by Pisera & Pomponi (2015). Additionally, for V. stalactites, a small piece of the surface was cut and mounted on the stub without chemical (70% nitric acid) treatment to check for rare microscleres (styles), which are supposed to occur around pores (Pisera et al., 2017). All stubs were sputter-coated with gold or platinum and examined with a Hitachi SU 5000 SEM at the Department for Earth and Environmental Sciences (Munich, Germany) and on a Philips L-20 SEM at the Institute of Paleobiology (Warsaw, Poland). Taxonomic authority is limited to Schuster, Pisera and Kelly. Molecular systematics A small piece of sponge tissue from the choanosome of each specimen was cut, and genomic DNA was isolated using the NucleoSpin (Machery-Nagel) or the DNeasy (Quiagen) Blood and Tissue Kit according to the manufacturer’s protocol. An additional centrifugation step, immediately before transferring the lysate to the spin column, was inserted to avoid clogging of the membrane by sponge spicules. The isolated genomic DNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Amplifications of independent mitochondrial (cox1) and nuclear markers [28S, 18S and internal transcribed spacer (ITS)] were obtained by using different primers and PCR conditions. The mitochondrial cox1 gene (‘Folmer’ fragment, 659 bp) was amplified using the primers dgLCO1490 and dgHCO2198 (Meyer, Geller & Paulay, 2005). The ribosomal 28S gene (C1–D2 fragment, 802 bp) was amplified using the primers C1’ASTR (Cárdenas et al., 2010) and D2 (Le, Lecointre & Perasso, 1993). The PCR reagents and settings for cox1 and 28S were the same as those described by Schuster et al. (2015). Primers SP18aF and SP18gR (Lavrov, Wang & Kelly, 2008) were used to amplify a nearly complete fragment of 18S (~1800 bp) using the following PCR settings: 95 °C for 3 min; (95 °C for 30 s; 50°C for 40 s; 72 °C for 2 min 50 s) × 35–40 cycles; and 72 °C for 10 min. Additionally, full-length ITS 1 and 2, 5.8S and partial 28S rDNA were amplified with primers ITS-RA2-fwd and ITS2.2-rvse (Wörheide, 1998) and the following PCR settings: 95 °C for 3 min; (95 °C for 30 s; 50 °C for 30 s; 72 °C for 20 s) × 35 cycles; and 72 °C for 3 min. The success of the PCR was checked visually on a 1% agarose gel. A PCR dilution of 1:10 together with BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA) chemicals and the same PCR primers was mixed for cycle sequencing, except for 18S, for which multiple primers (400F18S, 1200F18S, 600R18S and 1350R18S; Lavrov et al., 2008) were needed. Sequencing was performed on an ABI 3730 Genetic Analyzer at the Sequencing Service of the Department of Biology (LMU München, Germany). Sponge origin of newly generated Vetulina spp. sequences were assessed by BLAST searches against NCBI GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Raw trace files were base-called using CodonCode Aligner v.3.7.1.1 (CodonCode Corporation). The assembly of forward and reverse reads was performed using Geneious v.8.1.8 (http://www.geneious.com;Kearse et al., 2012). Owing to heterogeneous taxa sets and sequences available in GenBank for cox1, 28S, 18S and ITS, in particular for freshwater sponges and outgroup taxa, single-gene alignments were generated and analysed separately. Sequences of the 28S fragment C1–D2 for Spongilla lacustris Linnaeus, 1759 and Ephydatia muelleri Lieberkühn, 1856 were obtained from transcriptomic data by BLASTN search [S. lacustris (Riesgo et al., 2014; Ludeman et al., 2014); E. muelleri: Compagen http://www.compagen.org/datasets.html]. For Xestospongia testudinaria Lamarck, 1815, raw reads were downloaded from the Sequence Read Archive (SRA) of NCBI and assembled using the software Trinity v2.0.6 before BLAST search was undertaken (Eitel M., personal communication). Further sequences of freshwater sponges and Vetulina specimens (not sequenced in the present study), as well as all outgroup taxa were downloaded from GenBank and aligned to the new sequences from this study using the implemented Muscle v.3.6 (Edgar, 2004) aligner in AliView v1.18 (Larsson, 2014). A statistical summary of all four alignments, comprising the total number of sequences, new sequences, alignment length, constant and parsimony uninformative/informative characters, is given in Supporting Information, Table 1. The alignments from the present study are freely available at OpenDataLMU (https://doi.org/10.5282/ubm/data.111). Bayesian inference (BI) of phylogenies was executed separately for each dataset on a parallel version of MrBayes v.3.2.4 (Ronquist et al., 2012) on a Linux cluster. The generalised time-reversible + Gamma + Invariant sites (GTR + G + I) evolutionary model was chosen as calculated from jModelTest v.2.1.7 (Darriba et al., 2012). Analyses were run in two concurrent runs of four Metropolis-coupled Markov chains (MCMC) for 100000000 generations and stopped when the average standard deviation of split frequencies reached 0.01. For further analysis, 25% (burn-in) of the sampled trees were removed. Maximum likelihood (ML) and bootstrap analyses (1000 replicates) were performed under the gernalised time-reversible + Gamma (GTR + G) model as results from jModelTest v.2.1.7 (Darriba et al., 2012) using RAxML v8.0.26 (Stamatakis, 2014) on a Linux cluster. Tree topologies resulting from BI and ML analyses were compared and visualized using Figtree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). Molecular clock approach In this study, we considered Mesozoic fossils only within Sphaerocladina and freshwater sponges (see Table 2). Palaeozoic sponges with sphaeroclonar desma skeletons are known from the Ordovician to Permian (Astylospongiidae Zittel, 1877), but their relationship to younger Mesozoic and Recent taxa is debated, owing to their more massively formed skeleton and the large (~200 Myr) stratigraphic gap (Mostler & Balogh, 1994; Pisera, 2002). Owing to these rather doubtful fossils, we considered Jumarella astrorhiza Mehl & Fürsich, 1997 (Bathonian) as the oldest reliable fossil representing Recent Sphearocladina. The currently oldest freshwater sponge spicules were described from the Permo-Carboniferous by Schindler & Wuttke (2008); however, this record is doubted by Schultze (2009), who interprets the stratigraphic level including these sponge spicules as marine or marine influenced. Therefore, in the present study, Eospongilla morrisonensis Dunagan, 1999 from the Upper Jurassic is considered as the oldest and most reliable fossil representing Recent freshwater sponges (see Table 2). Table 2. Fossil species used to calibrate divergence times under the fossilized birth–death (FBD) model in BEAST v2.4.3 Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  View Large Table 2. Fossil species used to calibrate divergence times under the fossilized birth–death (FBD) model in BEAST v2.4.3 Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  View Large In order to use the advantage of having several representative fossil taxa available, hence not only the oldest fossils, we applied a Bayesian relaxed molecular clock analysis under the fossilized birth–death (FBD) model (Heath, Huelsenbeck & Stadler, 2014) using BEAST v2.4.3 (Bouckaert et al., 2014). The FBD model allows the assignment of multiple fossils of different ages to a clade without requiring morphological information in the analysis (Heath et al., 2014). For the performance of this method, we took two datasets comprising the unlinked mitochondrial cox1 and ribosomal 28S (C1–D2) genes. Owing to heterogeneous datasets available, chimeric sequences were built for E. muelleri, S. lacustris (freshwater sponges) and X. testudinaria (outgroup taxa). The final dataset was complemented by fossil taxa (Table 2) and their ages. As the FBD model requires the specification of point fossil ages (Heath et al., 2014), the youngest stratigraphic age for each of the five fossils was taken (see Table 2). The fossils used were either considered ancestor or extinct sister taxa and associated with their appropriate subclades, based on results from BI and ML trees (see Results Figs 8, 9). The software BEAUti v.2.4.3 (integrated in the BEAST 2 package) was used to configure the FBD model parameters and set up the XML file for the analysis. We used an uncorrelated relaxed clock model. An earlier study has shown that partitioning on data matrices had no influence on the divergence time estimates; therefore, no partitioning of our data matrix was applied (Ma & Yang, 2016). For the molecular sequence data, the GTR substitution model was specified with the ‘shape’ and ‘substitution rate’ parameters set to ‘estimate’ for both genes. A gamma prior for rates (rateAC.s; rateAG.s; rateAT.s; rateCG.s; rateCT.s; rateGT.s) on both genes (cox1 and 28S) was parameterized by setting α = 2 and β = 0.25. The root of the tree was set to 1000 Myr, because previous molecular clock analyses (Sperling et al., 2010; Gold et al., 2016; Schuster et al. 2017) suggested a deep origin of demosponges. A lognormal prior was induced on this root (mean = 241 Myr, SD minimum = 168 Myr, maximum = 796 Myr) with two hyperparameters, ucldMean.c (set to exponential < 1) and ucldStdev.c (set to exponential 0.333). A ‘uniform’ (0,1) distribution was chosen for the sampling proportion and turnover of the FBD. Sampling proportion was set to a β distribution with Alpha 2.0. Two independent Markov chain analyses were run for 400 million generations, sampling every 5000th generation. Runs were evaluated using Tracer v.1.6 (Rambaut et al., 2014) to assure stationarity of each Markov chain, an effective sample size (EES) for all parameters > 200, and convergence of the independent runs. The first 25% of the sampled tree topologies from both analyses were discarded as burn-in, and the remaining trees were combined in LogCombiner and summarized in TreeAnnotator (both programs were implemented in the BEAST 2 package) with mean divergence times and 95% highest posterior density (HPD). Before this, all fossils were removed from the tree using the FullToExtantTreeConverter tool (a tool implemented in BEAUti). Possible prior influences on the posterior distribution estimates were checked by specifying the sampling from the prior only, followed by re-running of the analysis. RESULTS Species descriptions Class Demospongiae Sollas, 1885 Subclass Heteroscleromorpha Cárdenas, Pérez & Boury-Esnault, 2012 Order Sphaerocladina Schrammen, 1924 Family Vetulinidae Lendenfeld, 1903 Genus Vetulina Schmidt, 1879 Vetulina Schmidt, 1879: 19. Vetulina—Sollas, 1885: 486; Sollas, 1888: 354; de Laubenfels, 1955: E63; Van Soest & Stentoft, 1988 : 71; Gruber, 1993: 49, pl. 14: 4–8. Type species: Vetulina stalactites Schmidt, 1879 (by monotypy). Emended diagnosis: Polymorphic lithistid Demospongiae: encrusting to hemispherical, lamellate or vase shaped; acrepid polyaxial desmas (sphaeroclone to astroclone) as megascleres; microscleres if present are styles, (sub)tylostyles and strongyles (modified from Pisera et al., 2017). Remarks: Only lamellate or vase-shaped growth forms are published in the diagnosis of the genus (Pisera & Lévi, 2002b; Pisera et al., 2017). However, in the present study we describe two new species, the first with a thick encrusting, spreading morphology, and the second forming a thick hemispherical encrustation with a spherical outline, requiring amendment to the diagnosis of the genus. In addition, (sub)tylostyles and strongyles of various sizes (75–125 µm) are discovered and considered here as microstyles, despite their size, following the same argument as Pisera et al. (2017), who considered the location/function of these styles in the skeleton as being microscleres. Vetulina stalactites Schmidt, 1879 (Figs 2A–F, 3A–F) Vetulina stalactites Schmidt, 1879: 19, pl.1: 1, pl. 2: 9. Vetulina stalactites—Sollas, 1885: 486: pls 3, 4; Sollas, 1888: 454. von Lendenfeld, 1903: 150; de Laubenfels, 1955: E63, fig. 46: 4; Van Soest & Stentoft, 1988: 71, text-fig. P.43; Gruber, 1993: 49, pl. 14: 4–8; McInerney et al., 1999: p. 346. Vetulina sp.,— Redmond et al., 2013: p. 400. Material examined: HBOM 003:01011 (HBOI 21-V-00-1-004): Johnson Sea-Link II (JSL-II) dive 3226, South Central Coast, off Piscadera Bay, Curaçao, 12°6′38.77″N, 68°58′43.25″W, 212 m, May 2000. HBOI 11-V-00-3-006: JSL-II dive 3210, South Central Coast, Seamount off Porto Mari Bay, Curaçao, 12°12′51.19″N, 69°5′50.21″W, 180 m, May 2000. HBOI 11-V-00-3-004: JSL-II dive 3210, South Central Coast, Seamount off Porto Mari Bay, Curaçao, 12°12′51.19″N, 69°5′50.21″W, 219 m, May 2000. HBOI 31-VIII-93-4-004: JSL-I dive 3601, North Coast, 4 Nautical miles (NM) SE of Galina Port, Jamaica, 18.355°N, 76.833°W, 350 m, August 1993. HBOI 14-V-00-1-007: JSL-II dive 3214, West Coast, Wecua Port, Bonaire, 12°13′20.35″N, 68°25′2.93″W, 256 m, May 2000. HBOI 13-XI-98-3-003: JSL-I dive 4098, Andros Island, East of Stafford Creek, Bahamas, 24°54′49.79″N, 77°52′12.61″W, 498 m, June 1998. Type locality: Eastern Caribbean, Barbados (13°10′0″N, 59°40′0″W), 183–329 m, RV Hassler Expedition (Fig. 1A). Distribution: Barbados (13°10′0″N, 59°40′0″W), 135–601 m (Schmidt, 1879); Curaçao, 12°6′–12°12′N, 68°58′–69°05′W, 137–219 m (present study); Jamaica, 18°21′18.07″N, 76°49′59.41″W, 350 m (present study); Turks and Caicos, 21°31′– 21°52′N, 71°8′–72°20′W, 520–550 m (present study); Bahamas, 23°38′–24°54′N, 74°56′–77°52′W, 498–569 m (present study); St Vincent Island, 13°9′38.99″N, 61°16′31.19″W, 247 m (present study); Bonaire, 12°13′20.35″N, 68°25′2.93″W, 256 m (present study); Martinique 14°30′18″N, 61°6′12″W, 220 m (Pomponi et al., 2001). Description: The gross morphology is irregularly lamellate to undulating and foliose, occasionally with tubular outgrowth (Fig. 2A–E) or cup to vase shaped (Fig. 2F); the base is attached to hard substratum. Dimensions of the cups are ~5–20 cm in diameter and ~7–26 cm high, with walls of 0.6–1 cm thickness and round, flattened margins. Outer surfaces contain well-developed, regularly concentric growth lines (Fig. 2A–F) and irregularly distributed pores of ~0.1 mm in diameter (Fig. 3A, B), while the inner surface is rather smooth and finely porous (Fig. 2E). Texture stony. Colour in life is pale yellow, yellowish brown to tan in ethanol. Figure 2. View largeDownload slide Deck photographs of Vetulina stalactites (A–F) and Vetulina tholiformis sp. nov. (G–J). Abbreviations behind the (HBOI) voucher number: BAH, Bahamas; bas, base; BON, Bonaire; CUR, Curaçao; dep, depression; JAM, Jamaica; osc, osculum. Figure 2. View largeDownload slide Deck photographs of Vetulina stalactites (A–F) and Vetulina tholiformis sp. nov. (G–J). Abbreviations behind the (HBOI) voucher number: BAH, Bahamas; bas, base; BON, Bonaire; CUR, Curaçao; dep, depression; JAM, Jamaica; osc, osculum. The skeleton is composed of a dense and regular network of sphaeroclone desmas (Fig. 3D–F) stacked upon one another (Fig. 3D). Figure 3. View largeDownload slide Vetulina stalactites (HBOI 21-V-00-1-004). A–C, view of outer surface before removing the organic material by acid treatment. A, several small and large possible oscula openings. B, detail of the ectosomal membrane with large possible oscula openings and desmas immediately beneath, with the ends of the spined globular centre protruding. C, detailed view of the surface, with microstyles protruding from the ectosome around possible oscula openings. D–F, view after treatment with nitric acid. D, outer surface, with dense sphaeroclone desmas surrounding channel openings. E, F, details of sphaeroclone desmas, with several juvenile forms (F) containing a hole in the centre. Figure 3. View largeDownload slide Vetulina stalactites (HBOI 21-V-00-1-004). A–C, view of outer surface before removing the organic material by acid treatment. A, several small and large possible oscula openings. B, detail of the ectosomal membrane with large possible oscula openings and desmas immediately beneath, with the ends of the spined globular centre protruding. C, detailed view of the surface, with microstyles protruding from the ectosome around possible oscula openings. D–F, view after treatment with nitric acid. D, outer surface, with dense sphaeroclone desmas surrounding channel openings. E, F, details of sphaeroclone desmas, with several juvenile forms (F) containing a hole in the centre. Megascleres are sphaeroclone desmas, 100–350 µm in diameter, with an irregularly globular centrum, 0.03–0.06 mm in diameter, with spinose root-like outgrowths emanating from the top of each desma (Fig. 3E). Immature desmas have a hollow centre (hole: ~20–25 µm in diameter; Fig. 3E, F). Three to eight sparsely spinose rays branch from the lower part of the centre (Fig. 3E). Zygomes of the branching rays attach the upper centre part of adjacent desmas to build up the skeleton. The globose centre aligns to the surface, with rays branching and extending towards the choanosome (Fig. 3A–C). Microscleres are microstyles ~50–150 µm long and 4 µm thick, with mucronate pointed tips (Fig. 3C). Microstyles exclusively protrude from the ectosome surface and are sparsely distributed around some ostia (Fig. 3C). DNA barcodes: In the present study, we sequenced partial cox1 (‘Folmer’ fragment) of specimens: HBOI 19-XI-94-1-012, 2-VII-89-2-012, 21-V-00-1-004, 31-VIII-4-004, 12-XI-94-3-005, 14-V-00-1-007, 14-XI-02-3-001, 13-XI-98-3-003, 11-V-003-004, 11-V-00-3-008, 11-V-00-3-006, 31-III-89-1-003 and 14-V-00-1-009. All 13 sequences are identical in all specimens; partial 28S fragment C1–D2 for HBOI 31-VIII-93-4-004, 13-XI-98-3-003, 11-V-00-3-006, 21-V-00-1-004 and 14-V-00-1-007, all of which are identical; ITS for HBOI 11-V-00-3-006, 21-V-00-1-004, 31-VIII-93-4-004, 13-XI-98-3-003 and 11-V-00-3-004, all of which are identical; and partial 18S for HBOI 14-XI-02-3-006, 13-X-03-3-003 and 13-XI-98-3-003, all of which show identical sequences. Additionally, GenBank accession no. KC901963 (Redmond et al., 2013) and AJ224648 (McInerney, Adams & Kelly, 1999) (18S) are added, whereupon sequence AJ224648 has ambiguous characters towards the 3′ end. Remarks: Before this study, V. stalactites was reported only from the type locality of Barbados, between 153 and 601 m (Schmidt, 1879; Van Soest & Stentoft, 1988) and Martinique (229 m; Pomponi et al. 2001). Here, we expand the distribution of this species in the Tropical Western Atlantic to the Bahamas, Turks and Caicos Islands, Jamaica, Curaçao, St Vincent and Bonaire, all collected between depths of 137 and 569 m (see Fig. 1). Our re-examination of V. stalactites, in particular the detailed observation of the ectosomal membrane, clearly produces evidence of the presence of microscleres (styles) throughout the species (Fig. 3C), which were already noticed by Sollas (1888), but considered by Pisera & Lévi (2002b) to be contamination. Finally, spinose microxeas, as observed from the inner surface of the syntype MCZ 6640 by Pisera & Lévi (2002b), could not be detected in any of our examined specimens. Vetulina tholiformis sp. nov. (Figs 2G–J, 4A–F, 5A–C) Diagnosis: Dome-shaped Vetulina with a thick, steep-sided body and circular to oval base and an osculum opening (1–4 mm in diameter) on the apex of the sponge. Microscleres are styles and (sub)tylostyles. Holotype: HBOI 30-X-96-2-003: JSL-I dive 2799, SW Grand Bahama Island, lithoherm (deep-water carbonate mound), Bahamas, 26°37′27.23″N, 78°58′48.83″W, 428 m, October 1996. Paratypes: HBOI 11-XI-02-3-011: JSL-I dive 4500, Eleuthera, Weymyss Bight, Bahamas, 24°45′53.46″N, 76°19′21.58″W, 427 m, November 2002. 19-XI-98-1-005: JSL-I dive 4109, Rum Cay, South Coast of Port Nelson, Bahamas, 23°37′24.42″N, 74°50′4.74″W, 440 m, November 1998. 13-XI-02-1-009: JSL-I dive 4503, Crooked Island, NW Tip, Bahamas, 22°40′0.66″N, 74°21′7.13″W, 367 m, November 2002. Type locality: Northern Caribbean, Bahamas. SW Grand Bahama Island lithoherm (26°37′27.23″N, 78°58′48.83″W), 428 m (Fig. 1A). Distribution: Bahamas (26°37′27.23″N, 78°58′48.83″W), 428 m; Bahamas, 22°49′–26°37′N, 74°21′–78°58′W, 367–440 m. Description: Dome-shaped sponge with a thick, steep-sided body and circular to oval base (Fig. 2G–J). Surface smooth or with several oval depressions (Fig. 2G–J). The entire plain base of the sponge is attached to the hard substratum (Fig. 2G–J). Dimensions of the spherical–bulbous sponge are 0.7 cm × 2.5 cm. Circular osculum, flush with the surface, 1–4 mm in diameter, located apically or laterally on the sponge (Fig. 2G–J). Outer surface smooth; pores not visible. Colour in life is pale yellow, and yellowish brown (Fig. 2G, I) to dark brown in ethanol. Choanosomal skeleton consists of very dense sphaeroclone desmas (150–300 µm in diameter). Megascleres are sphaeroclones; the centre from the outer choanosomal part has spinose root-like outgrowth (Fig. 4E, F), whereas the inner part shows lower tubercles with low, smooth tubercled rays. Three to five main rays branch from the centre and connect with the upper centre part of adjacent desmas, building up the skeleton network (Fig. 4A–D). Rarely, immature desmas are visible, showing a typical hollow of 20–25 µm in diameter instead of a globular centre (Fig. 4E). Figure 4. View largeDownload slide Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, B, dense and irregularly distributed sphaeroclone desmas of the choanosomal skeleton from the outer surface of the sponge. Overview (A) and detailed view (B) showing the spinose root-like outgrowth on top of the sphaeroclone centre. Overview (C) and detailed view (D) of the choanosomal skeleton from the inner surface of the sponge. Note the lower tubercles on the rays and centre of sphaeroclone desmas. E, F, sphaeroclonar desmas from the upper surface. E, immature desma, with the typical hole in the centre (instead of axial canal), attached to mature desmas. F, detailed view of complex interlocking desmas, with spiny branched outgrowths. Figure 4. View largeDownload slide Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, B, dense and irregularly distributed sphaeroclone desmas of the choanosomal skeleton from the outer surface of the sponge. Overview (A) and detailed view (B) showing the spinose root-like outgrowth on top of the sphaeroclone centre. Overview (C) and detailed view (D) of the choanosomal skeleton from the inner surface of the sponge. Note the lower tubercles on the rays and centre of sphaeroclone desmas. E, F, sphaeroclonar desmas from the upper surface. E, immature desma, with the typical hole in the centre (instead of axial canal), attached to mature desmas. F, detailed view of complex interlocking desmas, with spiny branched outgrowths. Microscleres are styles with mucronate tips (Fig. 5A), subtylostyles with a slight swelling on the upper part of the shaft (= polytylote; Fig. 5B) and tylostyles. Both style types are sparsely distributed (perpendicular) in the ectosomal part and concentrated around the osculum openings (not shown). Figure 5. View largeDownload slide Microscleres of Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, mucronate style. B, subtylostyle with a slight swelling (knob) on the upper part of the shaft. C, tylostyle. Figure 5. View largeDownload slide Microscleres of Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, mucronate style. B, subtylostyle with a slight swelling (knob) on the upper part of the shaft. C, tylostyle. Etymology: From Greek tholos, meaning a beehive tomb or domed tombs, which describes the oval dome-shaped gross morphology of the sponge. DNA barcodes: In the present study, we sequenced partial cox1 (‘Folmer’ fragment), ITS and partial 28S (C1–D2 fragment) of the following specimens: HBOI 30-X-96-2-003, 19-XI-98-1-005, 13-XI-02-1-009 and 11-XI-02-3-011. Sequences from each of the gene fragments were identical; partial 18S for HBOI 19-XI-98-1-005, 13-XI-02-1-009 and 11-XI-02-3-011. Remarks: This new species differs from V. stalactites, V. rugosa and V. indica, which form foliose vases and cups (Pisera et al. 2017), by having a dome-shaped external growth form instead of being flabellate vase to cup shaped (Fig. 2). The choanosomal sphaeroclones are very similar to V. rugosa, V. indica and V. stalactites, and differ only in minor details, such as tuberculation and arborescent outgrowth density of the desma centre. In terms of microsclere composition, the new species differs from V. rugosa, V. indica and V. stalactites in that rare stubtylostyles and tylostyles were detected aside from styles. Vetulina tholiformis sp. nov. differs from the new species V. incrustans sp. nov. (for description, see next subsection) by being thinly (vs. thickly) encrusting. Vetulina incrustans sp. nov. (Figs 6A–F, 7A–I) Diagnosis: Thick encrusting, biscuit-shaped Vetulina, with remarkable bright yellow coloration and several large oscula openings (1 mm in diameter) on the surface. Wide and tiny vein-like channels radiate from the osculum. Megascleres are sphaeroclonar desmas. Microscleres are styles and diverse subtylostyles. Holotype: NIWA 109682, BSPG 8020, 0CDN 3443-A, USNM 1470701: Davao, north side of Talikud Island. Deep inside crevices. Local name: ‘Angels Cove’, Philippines, 6°56′43″N, 125°40′56″E, 7–12 m. Collected by Dr Patrick L. Colin, Coral Reef Research Foundation, Republic of Palau, March 1996. Type locality: Talikud Island, Davao, Philippines, 7–12 m (Figs 1, 6A). Distribution: Davao, Philippines. Description: Thick encrusting, biscuit-shaped sponge, ~1 cm thick and up to 20 cm in greatest dimension, with broad hemispherical lobes or domes in the surface (Fig. 6A). Oscules are ~1 mm in diameter, scattered irregularly on the apex of the surface domes, and flush with the surface (Fig. 6B). Aquiferous canals radiate towards the oscules (Fig. 6B). Ostia, 0.1–0.3 mm in diameter, are evenly dispersed over the entire surface (Fig. 6B), forming little pits. Texture is stony but breakable, slightly velvety to the touch owing to a fine plush of microstyle brushes projecting from the surface. Colour in life is a remarkable bright yellow to light orange that extends to ~1 mm deep; the choanosome is beige (Fig. 6A). Beige in ethanol. Figure 6. View largeDownload slide Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A, underwater picture showing the thick (~1 cm) encrusting habit, the bright yellow colour and the evenly scattered oscula (osc) openings over the surface, as well as a transverse section through the choanosome (cho). B, detail of large (1 mm) osculum opening, with several aquiferous channels (ch) radiating towards the osculum. Ostia (ost) dispersed over entire surface. C, overview of choanosomal sphaeroclone desma network, with numerous microscleres dispersed around. D, detailed view of desma network, with immature desma in the centre of the picture indicated by a hollow. E, F, details of spined long root-like outgrowths of the desma centre and rays. Figure 6. View largeDownload slide Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A, underwater picture showing the thick (~1 cm) encrusting habit, the bright yellow colour and the evenly scattered oscula (osc) openings over the surface, as well as a transverse section through the choanosome (cho). B, detail of large (1 mm) osculum opening, with several aquiferous channels (ch) radiating towards the osculum. Ostia (ost) dispersed over entire surface. C, overview of choanosomal sphaeroclone desma network, with numerous microscleres dispersed around. D, detailed view of desma network, with immature desma in the centre of the picture indicated by a hollow. E, F, details of spined long root-like outgrowths of the desma centre and rays. Skeleton is composed of sphaeroclone desmas (Fig. 6C), the ectosome being differentiated by the presence of brushes of microstyles projecting from the surface between the ostial pits (Fig. 6C). Immature desmas are obvious in the surface skeleton. Megascleres are sphaeroclones, with a centrum from which arises an elongated, spined apex, and below which emanate four to five spined rays (Fig. 6E, F) that are joined to other desmas in zygosis. The apex is ornamented with ragged (heavily acanthose) spires (Fig. 6E, F); the branches are more simply spined. Immature desmas are characterized by a hole in the centre (Fig. 6D). In addition, hastate, slightly curved oxea megascleres of various sizes (75–217 µm in length and 3–10 µm thick, N = 15; Fig. 7A–E) were detected but considered here as artefacts of haplosclerid origin. Figure 7. View largeDownload slide Microscleres of Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A–E, hastate oxea megascleres of haplosclerid origin as artefacts. F–H, microsubtylostyles with one (H) or two (F, G) tyles (knobs) towards the tips of the spicule. I, mucronate style. Figure 7. View largeDownload slide Microscleres of Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A–E, hastate oxea megascleres of haplosclerid origin as artefacts. F–H, microsubtylostyles with one (H) or two (F, G) tyles (knobs) towards the tips of the spicule. I, mucronate style. Microscleres are abundant slender, slightly curved, occasionally polytylote microstyles (Fig. 7F–I). Substrate and ecology: Sponges were found deep inside crevices on vertical limestone reef faces with small caves, between 7 and 12 m. Etymology: Named after its unusual encrusting growth form (incrustans = encrusting, Latin). DNA barcodes: In the present study, we sequenced partial cox1 (‘Folmer’ fragment) and 28S (C1–D2 fragment). Remarks: The encrusting domed morphology of V. incrustans sp. nov. is highly distinctive, clearly separating it from all species of Vetulina, particularly V. indica and V. rugosa from Western Australia, which are lamellate. The sphaeroclones are of a similar general structure and diameter, but the ornamentation is characteristic of the new species; the apical spine is covered with ragged spires. Hastate oxeas (Fig. 7A–E), reminiscent of haplosclerid forms, were found to be moderately common in places in the ectosome and shallow choanosome. They represent a variety of sizes and shapes, ranging in length from 75 to 270 µm, and 2–10 µm thick (Fig. 7A–E), suggesting that they are artefacts. We consider the diverse polytylote subtylostyle microscleres to belong to the species owing to their position in the skeleton. Molecular systematics and relaxed molecular clock approach The present study comprises the largest dataset of Vetulina sequences to date, encompassing cox1, 28S, ITS and 18S sequences of all known extant species except for V. incrustans sp. nov., where no ITS and 18S could be amplified (Figs 8, 9). Sequence variation among different Vetulina species is found to be low for ITS (Fig. 8A). Nevertheless, the three Indo-Pacific Vetulina species (V. rugosa, V. indica and V. incrustans sp. nov.) group separately from the Tropical Western Atlantic species (V. tholiformis sp. nov. and V. stalactites; Figs 8, 9). In addition, there is no genetic variation observed between V. rugosa and V. indica from the Indian Ocean. Pairwise sequence differences of these two species to V. incrustans sp. nov. from the Philippines is low in cox1 (0.5%) and 28S (1.6%). There is no variation between V. stalactites and V. tholiformis sp. nov. within cox1, 18S and 28S (C1–D2), but within ITS we observed pairwise sequence difference of 0.8%). GenBank sequence AJ224648 has several ambiguous sites towards the 3′ end of the sequence, which causes the long branch in the 18S gene tree. Figure 8. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial (‘Folmer’) cox1. The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (circles), with colours corresponding to different localities as shown in Figure 1. Figure 8. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial (‘Folmer’) cox1. The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (circles), with colours corresponding to different localities as shown in Figure 1. Figure 9. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial ITS (A), 18S (B) and 28S (C1–D2 region) (C). The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Figure 9. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial ITS (A), 18S (B) and 28S (C1–D2 region) (C). The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Our dated phylogeny based on datasets of cox1 and 28S (C1–D2) calculated in a relaxed molecular clock framework is illustrated in Fig. 10. The Asian Vetulina species form a monophylum, which is sister to the Tropical Western Atlantic Vetulina clade. The origin of all extant Vetulina species is dated to ~43.3 Mya (Eocene, Bartonian), with a credibility interval of 21.1 and 84 Mya, thus before the closure of the Tethyan Seaway, which was dated to 13–11 Mya. The split of V. incrustans sp. nov. from the Philippines and V. indica and V. rugosa from Western Australia is dated to ~22.5 Mya (Early Miocene, Aquitanian), whereas the split of V. tholiformis and V. stalactites is dated earlier, to ~16.1 Mya (Early Miocene, Burdigalian). Furthermore, our relaxed molecular clock approach indicates a possible split of freshwater sponges (Spongillida) and marine Sphaerocladina at ~247.5 Mya (Early Triassic). Figure 10. View largeDownload slide Time-calibrated phylogeny of Sphaerocladina and Spongillida as inferred from BEAST 2 analysis based on cox1 and 28S datasets, plotted on a stratigraphic chart. Newly sequenced species are in bold. Error bars on node ages are in purple. Numbers behind taxon names are voucher numbers (HBOI, NIWA). For references of Spongilla and Xestospongia testudinaria (outgroup) see Material and Methods section. Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Red line indicates the period of the closure of the Tethyan Seaway. Colour code for the geological time scale is according to the Commission for the Geological Map of the World (CGMW), Paris, France. Figure 10. View largeDownload slide Time-calibrated phylogeny of Sphaerocladina and Spongillida as inferred from BEAST 2 analysis based on cox1 and 28S datasets, plotted on a stratigraphic chart. Newly sequenced species are in bold. Error bars on node ages are in purple. Numbers behind taxon names are voucher numbers (HBOI, NIWA). For references of Spongilla and Xestospongia testudinaria (outgroup) see Material and Methods section. Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Red line indicates the period of the closure of the Tethyan Seaway. Colour code for the geological time scale is according to the Commission for the Geological Map of the World (CGMW), Paris, France. DISCUSSION The two recently described Vetulina species from the Indian Ocean, V. incrustans sp. nov. from the Philippines and V. tholiformis sp. nov. from the Bahamas, increase the number of current valid species of the genus to five. Our morphological observations of the choanosomal sphaeroclonar desmas indicate their high similarity among the species, similar to observations by Pisera et al. (2017). However, both new species described in the present study have a unique outer growth form, which is either encrusting or dome shaped. The gross morphological differences and coloration of V. incrustans sp. nov. are striking and certainly add diagnostic characters to separate species in a very difficult group with few morphological characters. Within the fossil record, spherical–bulbous growth forms are known from Cretaceous sphaerocladinid species, e.g. Macrobrochus Schrammen, 1910 and Ozotrachelus Laubenfels, 1955 (Reid, 2004; Pisera, 2006), but no encrusting growth form is reported. Moreover, our morphological re-examination of V. stalactites clearly reveals its unique possession of microstyles, which is a spicule type noticed by Sollas (1885, 1888), but subsequently regarded as contamination by Pisera & Lévi (2002b). Unlike the morphology, difficulties were present in discriminating species based on molecular sequences of cox1 and 18S, which were identical (Figs 8, 9B). Even within the more variable 28S and ITS fragments, only few nucleotide differences were observed, confirming their high genetic similarity. For instance, although V. stalactites and V. tholiformis sp. nov. are both from the Tropical Western Atlantic and both clearly separated on morphological grounds, these two species can be distinguished only by the ITS marker (Fig. 9A). A similar phenomenon is known from the freshwater sponge sister group (Meixner et al., 2007), which suggests the need for comparative analyses on shared factors influencing mutation rates in these two lineages, which is beyond of the scope of the present study. Until now, V. stalactites was known only from Barbados (Schmidt, 1879; Van Soest & Stentoft, 1988) and Martinique (Pomponi et al., 2001). Our study illustrates that V. stalactites is more common in the Tropical Western Atlantic than previously thought (see Fig. 1), but has not yet been found in the Pacific. The disjunct distribution of the genus (Pisera et al., 2017) is not uncommon and has been observed for several other non-‘lithistid’ demosponges (see e.g. Łukowiak et al., 2014; Łukowiak, 2016). However, fossil records of undoubted sphaeroclonar desmas, similar to Recent Sphaerocladina species, are diverse, with ten genera, and known from the Jurassic and Cretaceous (Reid, 2004) as well as from the Eocene (Frisone et al., 2016) Pisera et al. (2017) proposed Vetulina as an example of a relict fauna with a once more widely distributed population, possibly originating in the Tethys Sea. With the most comprehensive dataset available to date, our study first tests this hypothesis by the inclusion of fossils in a relaxed molecular clock framework. The closure of the Tethyan Seaway is dated to the Early Miocene (11–13 Mya, Serravallian; Rögl, 1998) and regarded by Pisera et al. (2017) as the key event causing the isolated and yet rare occurrences of Vetulina species today. With the most comprehensive dataset available to date, our dated phylogeny adds weight to this hypothesis, indicating an origin of all Vetulina species in the Eocene (~43.3 Mya), thus supporting a possible origin of Vetulina in the Tethys Sea. CONCLUSION The original idea of a Tethyan origin for several demosponges was put forward by Reid (1967) and later taken up by several other studies, looking at the diverse modern and Mesozoic sponge fauna and their historical biogeographical distributions (e.g. Wiedenmayer, 1994; Łukowiak et al., 2014; Łukowiak, 2016). However, for the first time in this context, we used an integrative molecular palaeobiological approach to provide supportive information to test the hypothesis that Vetulina is an example of a relict fauna with its origin in the Tethys Sea. This case study demonstrates the advantage of such an approach to address evolutionary and biogeographical hypotheses. [Version of Record, published online 14 March 2018; http://zoobank.org/urn:lsid:zoobank.org:pub:0122DEEF-3F68-4D2F-A119-378D8C4CA5CF] ACKNOWLEDGEMENTS Financial support for this study was provided by the German Science Foundation to D.E. and G.W. (DFG ER 611/3-1 and DFG Wo869/15-1, respectively). LMU Mentoring and the HELGE AX:Son JOHNSON STIFTELSE provided funding for A.S. to visit HBOI (Florida, USA) and NIWA (National Institute of Water and Atmospheric Research, Auckland and Wellington in New Zealand). Financial support for the RV Seward Johnson/Johnson Sea Link I+II expedition was provided by HBOI. Vetulina incrustans sp. nov. was collected by the Coral Reef Research Foundation (CRRF) under contract no. N02-CM-77249 to the US National Cancer Institute. We acknowledge the Government of the Bahamas, Curaçao, Jamaica, Turks and Caicos, Bonaire and St Vincent Islands, and the Philippines, for granting permission to conduct research in their territorial waters. We are grateful to the CRRF for sharing sponge material and pictures of the new species. We thank colleagues Amy Wright, John Reed and Megan Conkling (HBOI-FAU) for their assistance with the HBOI collection of samples and associated data and images and Dr Patrick L. Colin, CRRF, for collecting material attributed to V. incrustans sp. nov. We are also grateful to John Rosser MA (Double Honours), Licentiate Trinity College London, for his assistance with the choice of and correct form of the species name. Gabrielle Büttner and Simone Schätzle (Department of Earth & Environmental Sciences, LMU Munich, Germany) are thanked for sequencing assistance. REFERENCES Bitner MA, Motchurova-Dekova N. 2016. Middle Miocene (Badenian) brachiopods from Yasen, northwestern Bulgaria: taxonomic composition and biogeographical significance. Neues Jahrbuch für Geologie und Paläontologie  279: 7– 22. 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New species and a molecular dating analysis of Vetulina Schmidt, 1879 (Porifera: Demospongiae: Sphaerocladina) reveal an ancient relict fauna with Tethys origin

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Abstract

Abstract Vetulina Schmidt, 1879 (Demospongiae, Sphaerocladina, Vetulinidae) currently constitutes the only living representative of a once diverse Mesozoic group. Molecular data place Vetulina as a sister taxon to freshwater sponges (Spongillida) despite different skeletal composition. To date, only three extant species of this desma-bearing ‘rock sponge’ have been described from the Caribbean and Indian Ocean, all with similar growth forms and spiculation, but different desma and surface details. Comparison of these genetically very similar species was not possible until the present study. The distribution of Vetulina is taken to be a consequence of the closure of the Tethyan Seaway in the Early Miocene, suggesting a more widely distributed population with its origin in the Tethys Sea. To support this hypothesis in a molecular palaeobiological framework, we first increased the taxon sampling by describing and sequencing two new species of Vetulina from the Bahamas and Philippines and report Vetulina stalactites from nine additional locations in the Tropical Western Atlantic. A robust, dated phylogeny was calculated from the combined dataset and amended by five representative fossils. Our results point to an Eocene origin for Vetulina, even before the closure of the Tethyan Seaway in the Miocene, supporting the hypothesis that Vetulina presents a relict fauna with its origin in the Tethys Sea. cox1, DNA barcode, freshwater sponges, molecular clocks, Porifera, relict fauna, Sphaerocladina, Tethys Sea, Vetulina INTRODUCTION Demosponges forming a rock-like skeleton of articulated megascleres (desmas) have historically been called ‘lithistid’ sponges. Morphological and molecular studies assigned 11 out of 13 ‘lithistid’ families to the order Tetractinellida Marshall, 1876 (e.g. Pisera & Lévi, 2002a; Cárdenas et al., 2011; Morrow & Cárdenas, 2015; Schuster et al., 2015). Desmanthidae Topsent, 1893, Vetulinidae von Lendenfeld, 1903 and Crambeidae Lévi, 1963 represent the only desma-bearing group of families outside the Tetractinellida, mostly comprising only one desma-bearing genus (e.g. Schuster et al., 2015): Desmanthidae is allocated to the order Bubarida Morrow and Cárdenas, 2015 (Morrow & Cárdenas, 2015) and Crambeidae to Poecilosclerida Topsent, 1928 (e.g. Van Soest, 2002), whereas Vetulinidae constitutes its own order, Sphaerocladina Schrammen, 1924, which already exists in the fossil classification (Morrow & Cárdenas, 2015; Schuster et al., 2015). Among all extant valid marine demosponges, Sphaerocladina has been recovered as sister taxon to the freshwater sponges (Spongillida; Manconi & Pronzato, 2002) by independent ribosomal (28S and 18S) and mitochondrial (cox1 and complete mitogenomes) markers (Kelly-Borges & Pomponi, 1994; McInerney, Adams, & Kelly, 1999; Redmond et al., 2013; Schuster et al., 2015). However, these two orders clearly differ in their skeleton and spicule composition. Spongillida is mainly characterized by smooth or spined monaxonic megascleres and may possess distinct gemmule microscleres (Manconi & Pronzato, 2002). In contrast, the skeleton of Sphaerocladina is built up by astro- to sphaeroclonar desmas (Pisera & Lévi, 2002b) and various microstyles (Schmidt, 1879; Pisera et al., 2017). Thus, their molecularly close but morphologically and ecologically disparate and enigmatic sister relationship to freshwater sponges remains a matter of further investigation. Within the monogeneric family Vetulinidae, the genus Vetulina Schmidt, 1879 (Pisera & Lévi, 2002b) is described as a flabellate or vase-shaped demosponge, with acrepid polyaxial astro- to sphaeroclonar megascleres (Pisera & Lévi, 2002b) and microscleric styles (Schmidt, 1879; Pisera et al., 2017). Before the recently discovered two new species from the Indian Ocean (Vetulina rugosa Pisera, Łukowiak, Fromont & Schuster, 2017 and Vetulina indica Pisera, Łukowiak, Fromont & Schuster, 2017; Pisera et al., 2017), this genus was monospecific, with Vetulina stalactites Schmidt, 1879 from Barbados (Schmidt, 1879; Van Soest & Stentoft, 1988). All extant Vetulina species were collected from similar rocky substrates and/or sandy slopes at depths between 95 and 220 m (Schmidt, 1879; Pomponi et al., 2001; Pisera et al., 2017). Nevertheless, the extant diversity of Sphaerocladina, with three valid species and one genus, appears considerably low compared with their fossil record, with ten genera known, e.g. from the Jurassic, Cretaceous and Eocene of Europe (Reid, 2004; Frisone, Pisera & Preto, 2016). The known Mesozoic Sphaerocladina fauna and their Recent disjunct occurrences in the Caribbean and Southwest Pacific led Pisera et al. (2017) to conclude that Vetulina represents an example of a relict fauna with a possible origin in the Tethys Sea, which was a large ocean during the Mesozoic Era that separated the supercontinent of Laurasia in the north from Gondwana in the south until its closure in the Miocene (Serravallian; 13–11 Mya; Rögl, 1998). The closure of the Tethyan Seaway in the Miocene is considered to be a key factor for the Recent rare occurrences and disjunct distribution of extant Vetulina species (Pisera et al., 2017). A correlation between the biogeographical distribution of modern and Mesozoic ‘lithistid’ demosponges to the Tethys Sea was already noticed and discussed by Reid (1967). He recognized that the ‘lithistid’ sponge fauna from the Upper Cretaceous of Northern Europe, which, at that time, represented a subtropical/warm-temperate region, included several representatives from the western end of the Tethys Sea, which was by that time a tropical/subtropical region still connected with the Atlantic Ocean. Thus, the Tethys Sea was climatically very similar to the present known regions of the Tropical Western Atlantic and the Pacific Ocean. Both of these regions are known for their rich extant ‘lithistid’ fauna (see e.g. Pomponi et al., 2001; Kelly, 2007). Further Recent ‘lithistid’ species, similar to Vetulina, also appear to have a disjunct distribution. The genus Isabella Schlacher-Hoenlinger, Pisera and Hooper, 2005 (Family Corallistidae Sollas, 1888) was first described in the southwest Pacific and recently discovered in the north-east Atlantic (Carvalho, Pomponi & Xavier, 2015). Corallistidae has a diverse (11 genera) and well-known Mesozoic fossil record, and was recently discovered from the Eocene of Europe (e.g. Pisera, 2002; Świerczewska-Gładysz, 2017). Likewise, Boury-Esnault, Pansini & Uriz (1992) noticed the similarity between the Recent Mediterranean Discorhabdella hindei Boury-Esnault, Pansini & Uriz, 1992 species and the fossil pre-Messinian Indo-Pacific species, but clearly disclosed any affinity to the Atlantic Discorhabdella species. Further support for a Tethyan origin is provided by the study of Maldonado et al. (2001), who reported the first occurrences of Discorhabdella Dendy, 1924 and Crambe Vosmaer, 1880 (desma bearing) from the eastern Pacific, which until then were known only from the Mediterranean Sea and the Atlantic. Further examples of non-‘lithistid’ demosponges and their hypothesized Tethyan origin were discussed and proposed in the studies of Łukowiak, Pisera & Schlögl (2014) and Łukowiak (2016), who compared the late Early Miocene fossil sponges from the central Paratethys in Slovakia as well as the Eocene fossil and modern sponge fauna of southern Australia. Although several examples of relict faunas with a possible origin in the Tethys Sea are predicted, not only among sponges (see e.g. Bitner & Motchurova-Dekova, 2016 for brachiopods; and Obura, 2016 for scleractinian corals), no attempt has yet been made to test this hypothesis for any of these groups in a molecular palaeobiological approach using fossil data. Its application requires not only a well-studied fossil record, but also a comprehensive molecular dataset. The availability of suitable Sphaerocladina sequences in GenBank and other sources is currently poor. At present, no common gene regions of the Caribbean and the Indian Ocean Vetulina species have been sequenced. Only one 28S (D3–D5) and three 18S sequences of V. stalactites from the Caribbean and one cox1 sequence of V. rugosa and V. indica, both from the Indian Ocean (Redmond et al., 2013; Schuster et al., 2015; Pisera et al., 2017; McInerney et al., 1999; Kelly-Borges & Pomponi, 1994) are available, hampering an integrative molecular palaeobiological approach. As a result, all hypotheses of a Tethys origin for Vetulina and other sponges (desma and non-desma bearing), are currently based only on empirical data from distribution patterns and fossils (see examples above), but in-depth molecular dating analyses are needed to corroborate current hypotheses about these processes. The aims of the present study, therefore, were as follows: (1) to provide a morphological description and illustrate V. stalactites from the Tropical Western Atlantic and to describe two new species, Vetulina incrustans sp. nov. from the Philippines and Vetulina tholiformis sp. nov. from the Bahamas; (2) to discriminate all valid Vetulina species with genetic markers and to calculate a robust phylogenetic tree that can be used as a basis for dating analyses; and (3) to combine the phylogenies of two independent markers (28S and cox1) with selected representative fossils in a relaxed molecular clock analysis, with the objective of testing the hypothesis of whether Vetulina constitutes another example of a relict fauna with its origin in the Tethys Sea. MATERIAL AND METHODS Specimen collection and morphological investigations Vetulina stalactites (13 specimens) and V. tholiformis sp. nov. (four specimens) were collected by the Harbor Branch Oceanographic Institution (HBOI) during expeditions to the Bahamas, Curaçao, Turks and Caicos, Jamaica, Bonaire and St Vincent Island from 1989 to 2003. Samples were collected at 12 stations from water depths ranging from 115 to 569 m (Fig. 1A, Table 1). Vetulina incrustans sp. nov. was collected from the Philippines during a Coral Reef Research Foundation (CRRF; Republic of Palau) expedition in 1996, by scuba diving, from 7 m (Fig. 1B). Upon collection, specimens were preserved in 70–80% ethanol. All samples collected from the Tropical Western Atlantic regions are accessioned within the Harbor Branch Oceanographic Museum (HBOM), Fort Pierce, FL, USA, although microscope slides and scanning electron microscopy (SEM) stubs of the skeleton and a small piece of the holotype and paratypes of V. tholiformis sp. nov. are stored at the Bavarian State Collection for Palaeontology and Geology (BSPG) in Munich, Germany. The holotype of V. incrustans sp. nov. is accessioned within the collections of the National Institute of Water & Atmospheric Research (NIWA) Invertebrate collection (NIC), and a small piece of the holotype is archived at the BSPG with stubs and slides. A small piece of the holotype of V. incrustans is also accessioned into the collections of the National Museum of Natural History (USNM) Smithsonian Institute, Washington, DC, USA. A detailed list of all Vetulina specimens from this study with their corresponding museum numbers, locations and accession numbers, is given in Table 1. Table 1. Species list, accession numbers and Sponge Barcoding Project (SBP) numbers of Vetulina specimens used in this study Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  View Large Table 1. Species list, accession numbers and Sponge Barcoding Project (SBP) numbers of Vetulina specimens used in this study Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  Species  Museum identification  Sponge Barcoding Project numbers  cox1  28S  18S  ITS  Location  Depth (m)  Vetulina incrustans sp. nov.  NIWA 109682  1734  LT960515  LT960539      Philippines  7  Vetulina stalactites  HBOI 2-VII-89-2-12  1735  LT960516            Vetulina stalactites  HBOI 21-V-00-1-004  1736  LT960517  LT960540    LT960566  Curaçao  212  Vetulina stalactites  HBOI 31-VIII-93-4-004  1737  LT960518  LT960541    LT960567  Jamaica  350  Vetulina stalactites  HBOI 12-XI-94-3-005  1738  LT960519        Turks and Caicos  550  Vetulina stalactites  HBOI 14-V-00-1-007  1739  LT960520  LT960542      Bonaire  256  Vetulina stalactites  HBOI 14-XI-02-3-001  1740  LT960521        Bahamas  569  Vetulina stalactites  HBOI 13-XI-98-3-003  1741  LT960522  LT960543  LT960538  LT960568  Bahamas  498  Vetulina stalactites  HBOI 11-V-00-3-004  1742  LT960523      LT960569  Curaçao  219  Vetulina stalactites  HBOI 11-V-00-3-008  1743  LT960524        Curaçao  137  Vetulina stalactites  HBOI 11-V-00-3-006  1744  LT960525  LT960544    LT960570  Curaçao  180  Vetulina stalactites  HBOI 31-III-89-1-003  1745  LT960526        St Vincent, York Bay  247  Vetulina stalactites  HBOI 14-XI-02-3-006  1746      LT960531    Bahamas  569  Vetulina stalactites  HBOI 13-X-03-3-003  1747      LT960532    Bahamas  909  Vetulina stalactites  HBOM 003:01011        KC901963    Curaçao  212  Vetulina stalactites          AJ224648        Vetulina tholiformis sp. nov.  HBOI 13-XI-02-1-009  1748  LT960527  LT960545  LT960533  LT960571  Bahamas  367  Vetulina tholiformis sp. nov.  HBOI 19-XI-98-1-005  1749  LT960528  LT960546  LT960534  LT960572  Bahamas  440  Vetulina tholiformis sp. nov.  HBOI 30-X-96-2-003  1750  LT960529  LT960547    LT960573  Bahamas  428  Vetulina tholiformis sp. nov.  HBOI 11-XI-02-3-011  1751  LT960530  LT960548  LT960535  LT960574  Bahamas  427  Vetulina indica  WAM Z35842  1752  LN624211  LT960549  LT960536  LT960575  Western Australia, Ashmore Reef  95  Vetulina rugosa  WAM Z36103  1753  LN624212  LT960550  LT960537  LT960576  Western Australia, Broome  100–108  View Large Figure 1. View largeDownload slide Maps illustrating the disjunct occurrences of currently valid Vetulina species. A, distribution of Vetulina stalactites (red) and Vetulina tholiformis sp. nov. (blue) in the Tropical Western Atlantic. B, distribution of Vetulina indica (pink) and Vetulina rugosa (brown) from the Indian Ocean, and Vetulina incrustans sp. nov. (yellow) from the Philippine Sea. Holotype locations are indicated as stars and paratypes as circles. The map was made with GeoMapApp 3.6.3 http://www.geomapapp.org (Ryan et al. 2009). Figure 1. View largeDownload slide Maps illustrating the disjunct occurrences of currently valid Vetulina species. A, distribution of Vetulina stalactites (red) and Vetulina tholiformis sp. nov. (blue) in the Tropical Western Atlantic. B, distribution of Vetulina indica (pink) and Vetulina rugosa (brown) from the Indian Ocean, and Vetulina incrustans sp. nov. (yellow) from the Philippine Sea. Holotype locations are indicated as stars and paratypes as circles. The map was made with GeoMapApp 3.6.3 http://www.geomapapp.org (Ryan et al. 2009). A vertically oriented piece of sponge tissue from the surface to the choanosome was cut, dried and prepared for SEM as outlined by Pisera & Pomponi (2015). Additionally, for V. stalactites, a small piece of the surface was cut and mounted on the stub without chemical (70% nitric acid) treatment to check for rare microscleres (styles), which are supposed to occur around pores (Pisera et al., 2017). All stubs were sputter-coated with gold or platinum and examined with a Hitachi SU 5000 SEM at the Department for Earth and Environmental Sciences (Munich, Germany) and on a Philips L-20 SEM at the Institute of Paleobiology (Warsaw, Poland). Taxonomic authority is limited to Schuster, Pisera and Kelly. Molecular systematics A small piece of sponge tissue from the choanosome of each specimen was cut, and genomic DNA was isolated using the NucleoSpin (Machery-Nagel) or the DNeasy (Quiagen) Blood and Tissue Kit according to the manufacturer’s protocol. An additional centrifugation step, immediately before transferring the lysate to the spin column, was inserted to avoid clogging of the membrane by sponge spicules. The isolated genomic DNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Amplifications of independent mitochondrial (cox1) and nuclear markers [28S, 18S and internal transcribed spacer (ITS)] were obtained by using different primers and PCR conditions. The mitochondrial cox1 gene (‘Folmer’ fragment, 659 bp) was amplified using the primers dgLCO1490 and dgHCO2198 (Meyer, Geller & Paulay, 2005). The ribosomal 28S gene (C1–D2 fragment, 802 bp) was amplified using the primers C1’ASTR (Cárdenas et al., 2010) and D2 (Le, Lecointre & Perasso, 1993). The PCR reagents and settings for cox1 and 28S were the same as those described by Schuster et al. (2015). Primers SP18aF and SP18gR (Lavrov, Wang & Kelly, 2008) were used to amplify a nearly complete fragment of 18S (~1800 bp) using the following PCR settings: 95 °C for 3 min; (95 °C for 30 s; 50°C for 40 s; 72 °C for 2 min 50 s) × 35–40 cycles; and 72 °C for 10 min. Additionally, full-length ITS 1 and 2, 5.8S and partial 28S rDNA were amplified with primers ITS-RA2-fwd and ITS2.2-rvse (Wörheide, 1998) and the following PCR settings: 95 °C for 3 min; (95 °C for 30 s; 50 °C for 30 s; 72 °C for 20 s) × 35 cycles; and 72 °C for 3 min. The success of the PCR was checked visually on a 1% agarose gel. A PCR dilution of 1:10 together with BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA) chemicals and the same PCR primers was mixed for cycle sequencing, except for 18S, for which multiple primers (400F18S, 1200F18S, 600R18S and 1350R18S; Lavrov et al., 2008) were needed. Sequencing was performed on an ABI 3730 Genetic Analyzer at the Sequencing Service of the Department of Biology (LMU München, Germany). Sponge origin of newly generated Vetulina spp. sequences were assessed by BLAST searches against NCBI GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Raw trace files were base-called using CodonCode Aligner v.3.7.1.1 (CodonCode Corporation). The assembly of forward and reverse reads was performed using Geneious v.8.1.8 (http://www.geneious.com;Kearse et al., 2012). Owing to heterogeneous taxa sets and sequences available in GenBank for cox1, 28S, 18S and ITS, in particular for freshwater sponges and outgroup taxa, single-gene alignments were generated and analysed separately. Sequences of the 28S fragment C1–D2 for Spongilla lacustris Linnaeus, 1759 and Ephydatia muelleri Lieberkühn, 1856 were obtained from transcriptomic data by BLASTN search [S. lacustris (Riesgo et al., 2014; Ludeman et al., 2014); E. muelleri: Compagen http://www.compagen.org/datasets.html]. For Xestospongia testudinaria Lamarck, 1815, raw reads were downloaded from the Sequence Read Archive (SRA) of NCBI and assembled using the software Trinity v2.0.6 before BLAST search was undertaken (Eitel M., personal communication). Further sequences of freshwater sponges and Vetulina specimens (not sequenced in the present study), as well as all outgroup taxa were downloaded from GenBank and aligned to the new sequences from this study using the implemented Muscle v.3.6 (Edgar, 2004) aligner in AliView v1.18 (Larsson, 2014). A statistical summary of all four alignments, comprising the total number of sequences, new sequences, alignment length, constant and parsimony uninformative/informative characters, is given in Supporting Information, Table 1. The alignments from the present study are freely available at OpenDataLMU (https://doi.org/10.5282/ubm/data.111). Bayesian inference (BI) of phylogenies was executed separately for each dataset on a parallel version of MrBayes v.3.2.4 (Ronquist et al., 2012) on a Linux cluster. The generalised time-reversible + Gamma + Invariant sites (GTR + G + I) evolutionary model was chosen as calculated from jModelTest v.2.1.7 (Darriba et al., 2012). Analyses were run in two concurrent runs of four Metropolis-coupled Markov chains (MCMC) for 100000000 generations and stopped when the average standard deviation of split frequencies reached 0.01. For further analysis, 25% (burn-in) of the sampled trees were removed. Maximum likelihood (ML) and bootstrap analyses (1000 replicates) were performed under the gernalised time-reversible + Gamma (GTR + G) model as results from jModelTest v.2.1.7 (Darriba et al., 2012) using RAxML v8.0.26 (Stamatakis, 2014) on a Linux cluster. Tree topologies resulting from BI and ML analyses were compared and visualized using Figtree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). Molecular clock approach In this study, we considered Mesozoic fossils only within Sphaerocladina and freshwater sponges (see Table 2). Palaeozoic sponges with sphaeroclonar desma skeletons are known from the Ordovician to Permian (Astylospongiidae Zittel, 1877), but their relationship to younger Mesozoic and Recent taxa is debated, owing to their more massively formed skeleton and the large (~200 Myr) stratigraphic gap (Mostler & Balogh, 1994; Pisera, 2002). Owing to these rather doubtful fossils, we considered Jumarella astrorhiza Mehl & Fürsich, 1997 (Bathonian) as the oldest reliable fossil representing Recent Sphearocladina. The currently oldest freshwater sponge spicules were described from the Permo-Carboniferous by Schindler & Wuttke (2008); however, this record is doubted by Schultze (2009), who interprets the stratigraphic level including these sponge spicules as marine or marine influenced. Therefore, in the present study, Eospongilla morrisonensis Dunagan, 1999 from the Upper Jurassic is considered as the oldest and most reliable fossil representing Recent freshwater sponges (see Table 2). Table 2. Fossil species used to calibrate divergence times under the fossilized birth–death (FBD) model in BEAST v2.4.3 Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  View Large Table 2. Fossil species used to calibrate divergence times under the fossilized birth–death (FBD) model in BEAST v2.4.3 Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  Species  Age in BEAST  Stratigraphic range  Citation  Sphaerocladina  Jumarella astrorhiza Mehl & Fürsich, 1997   166.1  Bathonian  Mehl & Fürsich (1997)  Pachytrachelus conicus Schrammen, 1910  86.3  Coniacian/Santonian  Schrammen (1910)  Ozotrachelus conicus Roemer, 1841  41.2  Early Lutetian, Eocene  Frisone et al. (2016)  Spongillida  Eospongilla morrisonensis Dunagan, 1999  145.0  Upper Jurassic  Dunagan (1999)  Ephydatia chileana Piera & Sáez, 2003  5.8  Late Miocene  Pisera & Sáez (2003)  View Large In order to use the advantage of having several representative fossil taxa available, hence not only the oldest fossils, we applied a Bayesian relaxed molecular clock analysis under the fossilized birth–death (FBD) model (Heath, Huelsenbeck & Stadler, 2014) using BEAST v2.4.3 (Bouckaert et al., 2014). The FBD model allows the assignment of multiple fossils of different ages to a clade without requiring morphological information in the analysis (Heath et al., 2014). For the performance of this method, we took two datasets comprising the unlinked mitochondrial cox1 and ribosomal 28S (C1–D2) genes. Owing to heterogeneous datasets available, chimeric sequences were built for E. muelleri, S. lacustris (freshwater sponges) and X. testudinaria (outgroup taxa). The final dataset was complemented by fossil taxa (Table 2) and their ages. As the FBD model requires the specification of point fossil ages (Heath et al., 2014), the youngest stratigraphic age for each of the five fossils was taken (see Table 2). The fossils used were either considered ancestor or extinct sister taxa and associated with their appropriate subclades, based on results from BI and ML trees (see Results Figs 8, 9). The software BEAUti v.2.4.3 (integrated in the BEAST 2 package) was used to configure the FBD model parameters and set up the XML file for the analysis. We used an uncorrelated relaxed clock model. An earlier study has shown that partitioning on data matrices had no influence on the divergence time estimates; therefore, no partitioning of our data matrix was applied (Ma & Yang, 2016). For the molecular sequence data, the GTR substitution model was specified with the ‘shape’ and ‘substitution rate’ parameters set to ‘estimate’ for both genes. A gamma prior for rates (rateAC.s; rateAG.s; rateAT.s; rateCG.s; rateCT.s; rateGT.s) on both genes (cox1 and 28S) was parameterized by setting α = 2 and β = 0.25. The root of the tree was set to 1000 Myr, because previous molecular clock analyses (Sperling et al., 2010; Gold et al., 2016; Schuster et al. 2017) suggested a deep origin of demosponges. A lognormal prior was induced on this root (mean = 241 Myr, SD minimum = 168 Myr, maximum = 796 Myr) with two hyperparameters, ucldMean.c (set to exponential < 1) and ucldStdev.c (set to exponential 0.333). A ‘uniform’ (0,1) distribution was chosen for the sampling proportion and turnover of the FBD. Sampling proportion was set to a β distribution with Alpha 2.0. Two independent Markov chain analyses were run for 400 million generations, sampling every 5000th generation. Runs were evaluated using Tracer v.1.6 (Rambaut et al., 2014) to assure stationarity of each Markov chain, an effective sample size (EES) for all parameters > 200, and convergence of the independent runs. The first 25% of the sampled tree topologies from both analyses were discarded as burn-in, and the remaining trees were combined in LogCombiner and summarized in TreeAnnotator (both programs were implemented in the BEAST 2 package) with mean divergence times and 95% highest posterior density (HPD). Before this, all fossils were removed from the tree using the FullToExtantTreeConverter tool (a tool implemented in BEAUti). Possible prior influences on the posterior distribution estimates were checked by specifying the sampling from the prior only, followed by re-running of the analysis. RESULTS Species descriptions Class Demospongiae Sollas, 1885 Subclass Heteroscleromorpha Cárdenas, Pérez & Boury-Esnault, 2012 Order Sphaerocladina Schrammen, 1924 Family Vetulinidae Lendenfeld, 1903 Genus Vetulina Schmidt, 1879 Vetulina Schmidt, 1879: 19. Vetulina—Sollas, 1885: 486; Sollas, 1888: 354; de Laubenfels, 1955: E63; Van Soest & Stentoft, 1988 : 71; Gruber, 1993: 49, pl. 14: 4–8. Type species: Vetulina stalactites Schmidt, 1879 (by monotypy). Emended diagnosis: Polymorphic lithistid Demospongiae: encrusting to hemispherical, lamellate or vase shaped; acrepid polyaxial desmas (sphaeroclone to astroclone) as megascleres; microscleres if present are styles, (sub)tylostyles and strongyles (modified from Pisera et al., 2017). Remarks: Only lamellate or vase-shaped growth forms are published in the diagnosis of the genus (Pisera & Lévi, 2002b; Pisera et al., 2017). However, in the present study we describe two new species, the first with a thick encrusting, spreading morphology, and the second forming a thick hemispherical encrustation with a spherical outline, requiring amendment to the diagnosis of the genus. In addition, (sub)tylostyles and strongyles of various sizes (75–125 µm) are discovered and considered here as microstyles, despite their size, following the same argument as Pisera et al. (2017), who considered the location/function of these styles in the skeleton as being microscleres. Vetulina stalactites Schmidt, 1879 (Figs 2A–F, 3A–F) Vetulina stalactites Schmidt, 1879: 19, pl.1: 1, pl. 2: 9. Vetulina stalactites—Sollas, 1885: 486: pls 3, 4; Sollas, 1888: 454. von Lendenfeld, 1903: 150; de Laubenfels, 1955: E63, fig. 46: 4; Van Soest & Stentoft, 1988: 71, text-fig. P.43; Gruber, 1993: 49, pl. 14: 4–8; McInerney et al., 1999: p. 346. Vetulina sp.,— Redmond et al., 2013: p. 400. Material examined: HBOM 003:01011 (HBOI 21-V-00-1-004): Johnson Sea-Link II (JSL-II) dive 3226, South Central Coast, off Piscadera Bay, Curaçao, 12°6′38.77″N, 68°58′43.25″W, 212 m, May 2000. HBOI 11-V-00-3-006: JSL-II dive 3210, South Central Coast, Seamount off Porto Mari Bay, Curaçao, 12°12′51.19″N, 69°5′50.21″W, 180 m, May 2000. HBOI 11-V-00-3-004: JSL-II dive 3210, South Central Coast, Seamount off Porto Mari Bay, Curaçao, 12°12′51.19″N, 69°5′50.21″W, 219 m, May 2000. HBOI 31-VIII-93-4-004: JSL-I dive 3601, North Coast, 4 Nautical miles (NM) SE of Galina Port, Jamaica, 18.355°N, 76.833°W, 350 m, August 1993. HBOI 14-V-00-1-007: JSL-II dive 3214, West Coast, Wecua Port, Bonaire, 12°13′20.35″N, 68°25′2.93″W, 256 m, May 2000. HBOI 13-XI-98-3-003: JSL-I dive 4098, Andros Island, East of Stafford Creek, Bahamas, 24°54′49.79″N, 77°52′12.61″W, 498 m, June 1998. Type locality: Eastern Caribbean, Barbados (13°10′0″N, 59°40′0″W), 183–329 m, RV Hassler Expedition (Fig. 1A). Distribution: Barbados (13°10′0″N, 59°40′0″W), 135–601 m (Schmidt, 1879); Curaçao, 12°6′–12°12′N, 68°58′–69°05′W, 137–219 m (present study); Jamaica, 18°21′18.07″N, 76°49′59.41″W, 350 m (present study); Turks and Caicos, 21°31′– 21°52′N, 71°8′–72°20′W, 520–550 m (present study); Bahamas, 23°38′–24°54′N, 74°56′–77°52′W, 498–569 m (present study); St Vincent Island, 13°9′38.99″N, 61°16′31.19″W, 247 m (present study); Bonaire, 12°13′20.35″N, 68°25′2.93″W, 256 m (present study); Martinique 14°30′18″N, 61°6′12″W, 220 m (Pomponi et al., 2001). Description: The gross morphology is irregularly lamellate to undulating and foliose, occasionally with tubular outgrowth (Fig. 2A–E) or cup to vase shaped (Fig. 2F); the base is attached to hard substratum. Dimensions of the cups are ~5–20 cm in diameter and ~7–26 cm high, with walls of 0.6–1 cm thickness and round, flattened margins. Outer surfaces contain well-developed, regularly concentric growth lines (Fig. 2A–F) and irregularly distributed pores of ~0.1 mm in diameter (Fig. 3A, B), while the inner surface is rather smooth and finely porous (Fig. 2E). Texture stony. Colour in life is pale yellow, yellowish brown to tan in ethanol. Figure 2. View largeDownload slide Deck photographs of Vetulina stalactites (A–F) and Vetulina tholiformis sp. nov. (G–J). Abbreviations behind the (HBOI) voucher number: BAH, Bahamas; bas, base; BON, Bonaire; CUR, Curaçao; dep, depression; JAM, Jamaica; osc, osculum. Figure 2. View largeDownload slide Deck photographs of Vetulina stalactites (A–F) and Vetulina tholiformis sp. nov. (G–J). Abbreviations behind the (HBOI) voucher number: BAH, Bahamas; bas, base; BON, Bonaire; CUR, Curaçao; dep, depression; JAM, Jamaica; osc, osculum. The skeleton is composed of a dense and regular network of sphaeroclone desmas (Fig. 3D–F) stacked upon one another (Fig. 3D). Figure 3. View largeDownload slide Vetulina stalactites (HBOI 21-V-00-1-004). A–C, view of outer surface before removing the organic material by acid treatment. A, several small and large possible oscula openings. B, detail of the ectosomal membrane with large possible oscula openings and desmas immediately beneath, with the ends of the spined globular centre protruding. C, detailed view of the surface, with microstyles protruding from the ectosome around possible oscula openings. D–F, view after treatment with nitric acid. D, outer surface, with dense sphaeroclone desmas surrounding channel openings. E, F, details of sphaeroclone desmas, with several juvenile forms (F) containing a hole in the centre. Figure 3. View largeDownload slide Vetulina stalactites (HBOI 21-V-00-1-004). A–C, view of outer surface before removing the organic material by acid treatment. A, several small and large possible oscula openings. B, detail of the ectosomal membrane with large possible oscula openings and desmas immediately beneath, with the ends of the spined globular centre protruding. C, detailed view of the surface, with microstyles protruding from the ectosome around possible oscula openings. D–F, view after treatment with nitric acid. D, outer surface, with dense sphaeroclone desmas surrounding channel openings. E, F, details of sphaeroclone desmas, with several juvenile forms (F) containing a hole in the centre. Megascleres are sphaeroclone desmas, 100–350 µm in diameter, with an irregularly globular centrum, 0.03–0.06 mm in diameter, with spinose root-like outgrowths emanating from the top of each desma (Fig. 3E). Immature desmas have a hollow centre (hole: ~20–25 µm in diameter; Fig. 3E, F). Three to eight sparsely spinose rays branch from the lower part of the centre (Fig. 3E). Zygomes of the branching rays attach the upper centre part of adjacent desmas to build up the skeleton. The globose centre aligns to the surface, with rays branching and extending towards the choanosome (Fig. 3A–C). Microscleres are microstyles ~50–150 µm long and 4 µm thick, with mucronate pointed tips (Fig. 3C). Microstyles exclusively protrude from the ectosome surface and are sparsely distributed around some ostia (Fig. 3C). DNA barcodes: In the present study, we sequenced partial cox1 (‘Folmer’ fragment) of specimens: HBOI 19-XI-94-1-012, 2-VII-89-2-012, 21-V-00-1-004, 31-VIII-4-004, 12-XI-94-3-005, 14-V-00-1-007, 14-XI-02-3-001, 13-XI-98-3-003, 11-V-003-004, 11-V-00-3-008, 11-V-00-3-006, 31-III-89-1-003 and 14-V-00-1-009. All 13 sequences are identical in all specimens; partial 28S fragment C1–D2 for HBOI 31-VIII-93-4-004, 13-XI-98-3-003, 11-V-00-3-006, 21-V-00-1-004 and 14-V-00-1-007, all of which are identical; ITS for HBOI 11-V-00-3-006, 21-V-00-1-004, 31-VIII-93-4-004, 13-XI-98-3-003 and 11-V-00-3-004, all of which are identical; and partial 18S for HBOI 14-XI-02-3-006, 13-X-03-3-003 and 13-XI-98-3-003, all of which show identical sequences. Additionally, GenBank accession no. KC901963 (Redmond et al., 2013) and AJ224648 (McInerney, Adams & Kelly, 1999) (18S) are added, whereupon sequence AJ224648 has ambiguous characters towards the 3′ end. Remarks: Before this study, V. stalactites was reported only from the type locality of Barbados, between 153 and 601 m (Schmidt, 1879; Van Soest & Stentoft, 1988) and Martinique (229 m; Pomponi et al. 2001). Here, we expand the distribution of this species in the Tropical Western Atlantic to the Bahamas, Turks and Caicos Islands, Jamaica, Curaçao, St Vincent and Bonaire, all collected between depths of 137 and 569 m (see Fig. 1). Our re-examination of V. stalactites, in particular the detailed observation of the ectosomal membrane, clearly produces evidence of the presence of microscleres (styles) throughout the species (Fig. 3C), which were already noticed by Sollas (1888), but considered by Pisera & Lévi (2002b) to be contamination. Finally, spinose microxeas, as observed from the inner surface of the syntype MCZ 6640 by Pisera & Lévi (2002b), could not be detected in any of our examined specimens. Vetulina tholiformis sp. nov. (Figs 2G–J, 4A–F, 5A–C) Diagnosis: Dome-shaped Vetulina with a thick, steep-sided body and circular to oval base and an osculum opening (1–4 mm in diameter) on the apex of the sponge. Microscleres are styles and (sub)tylostyles. Holotype: HBOI 30-X-96-2-003: JSL-I dive 2799, SW Grand Bahama Island, lithoherm (deep-water carbonate mound), Bahamas, 26°37′27.23″N, 78°58′48.83″W, 428 m, October 1996. Paratypes: HBOI 11-XI-02-3-011: JSL-I dive 4500, Eleuthera, Weymyss Bight, Bahamas, 24°45′53.46″N, 76°19′21.58″W, 427 m, November 2002. 19-XI-98-1-005: JSL-I dive 4109, Rum Cay, South Coast of Port Nelson, Bahamas, 23°37′24.42″N, 74°50′4.74″W, 440 m, November 1998. 13-XI-02-1-009: JSL-I dive 4503, Crooked Island, NW Tip, Bahamas, 22°40′0.66″N, 74°21′7.13″W, 367 m, November 2002. Type locality: Northern Caribbean, Bahamas. SW Grand Bahama Island lithoherm (26°37′27.23″N, 78°58′48.83″W), 428 m (Fig. 1A). Distribution: Bahamas (26°37′27.23″N, 78°58′48.83″W), 428 m; Bahamas, 22°49′–26°37′N, 74°21′–78°58′W, 367–440 m. Description: Dome-shaped sponge with a thick, steep-sided body and circular to oval base (Fig. 2G–J). Surface smooth or with several oval depressions (Fig. 2G–J). The entire plain base of the sponge is attached to the hard substratum (Fig. 2G–J). Dimensions of the spherical–bulbous sponge are 0.7 cm × 2.5 cm. Circular osculum, flush with the surface, 1–4 mm in diameter, located apically or laterally on the sponge (Fig. 2G–J). Outer surface smooth; pores not visible. Colour in life is pale yellow, and yellowish brown (Fig. 2G, I) to dark brown in ethanol. Choanosomal skeleton consists of very dense sphaeroclone desmas (150–300 µm in diameter). Megascleres are sphaeroclones; the centre from the outer choanosomal part has spinose root-like outgrowth (Fig. 4E, F), whereas the inner part shows lower tubercles with low, smooth tubercled rays. Three to five main rays branch from the centre and connect with the upper centre part of adjacent desmas, building up the skeleton network (Fig. 4A–D). Rarely, immature desmas are visible, showing a typical hollow of 20–25 µm in diameter instead of a globular centre (Fig. 4E). Figure 4. View largeDownload slide Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, B, dense and irregularly distributed sphaeroclone desmas of the choanosomal skeleton from the outer surface of the sponge. Overview (A) and detailed view (B) showing the spinose root-like outgrowth on top of the sphaeroclone centre. Overview (C) and detailed view (D) of the choanosomal skeleton from the inner surface of the sponge. Note the lower tubercles on the rays and centre of sphaeroclone desmas. E, F, sphaeroclonar desmas from the upper surface. E, immature desma, with the typical hole in the centre (instead of axial canal), attached to mature desmas. F, detailed view of complex interlocking desmas, with spiny branched outgrowths. Figure 4. View largeDownload slide Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, B, dense and irregularly distributed sphaeroclone desmas of the choanosomal skeleton from the outer surface of the sponge. Overview (A) and detailed view (B) showing the spinose root-like outgrowth on top of the sphaeroclone centre. Overview (C) and detailed view (D) of the choanosomal skeleton from the inner surface of the sponge. Note the lower tubercles on the rays and centre of sphaeroclone desmas. E, F, sphaeroclonar desmas from the upper surface. E, immature desma, with the typical hole in the centre (instead of axial canal), attached to mature desmas. F, detailed view of complex interlocking desmas, with spiny branched outgrowths. Microscleres are styles with mucronate tips (Fig. 5A), subtylostyles with a slight swelling on the upper part of the shaft (= polytylote; Fig. 5B) and tylostyles. Both style types are sparsely distributed (perpendicular) in the ectosomal part and concentrated around the osculum openings (not shown). Figure 5. View largeDownload slide Microscleres of Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, mucronate style. B, subtylostyle with a slight swelling (knob) on the upper part of the shaft. C, tylostyle. Figure 5. View largeDownload slide Microscleres of Vetulina tholiformis sp. nov. (HBOI 30-X-96-2-003). A, mucronate style. B, subtylostyle with a slight swelling (knob) on the upper part of the shaft. C, tylostyle. Etymology: From Greek tholos, meaning a beehive tomb or domed tombs, which describes the oval dome-shaped gross morphology of the sponge. DNA barcodes: In the present study, we sequenced partial cox1 (‘Folmer’ fragment), ITS and partial 28S (C1–D2 fragment) of the following specimens: HBOI 30-X-96-2-003, 19-XI-98-1-005, 13-XI-02-1-009 and 11-XI-02-3-011. Sequences from each of the gene fragments were identical; partial 18S for HBOI 19-XI-98-1-005, 13-XI-02-1-009 and 11-XI-02-3-011. Remarks: This new species differs from V. stalactites, V. rugosa and V. indica, which form foliose vases and cups (Pisera et al. 2017), by having a dome-shaped external growth form instead of being flabellate vase to cup shaped (Fig. 2). The choanosomal sphaeroclones are very similar to V. rugosa, V. indica and V. stalactites, and differ only in minor details, such as tuberculation and arborescent outgrowth density of the desma centre. In terms of microsclere composition, the new species differs from V. rugosa, V. indica and V. stalactites in that rare stubtylostyles and tylostyles were detected aside from styles. Vetulina tholiformis sp. nov. differs from the new species V. incrustans sp. nov. (for description, see next subsection) by being thinly (vs. thickly) encrusting. Vetulina incrustans sp. nov. (Figs 6A–F, 7A–I) Diagnosis: Thick encrusting, biscuit-shaped Vetulina, with remarkable bright yellow coloration and several large oscula openings (1 mm in diameter) on the surface. Wide and tiny vein-like channels radiate from the osculum. Megascleres are sphaeroclonar desmas. Microscleres are styles and diverse subtylostyles. Holotype: NIWA 109682, BSPG 8020, 0CDN 3443-A, USNM 1470701: Davao, north side of Talikud Island. Deep inside crevices. Local name: ‘Angels Cove’, Philippines, 6°56′43″N, 125°40′56″E, 7–12 m. Collected by Dr Patrick L. Colin, Coral Reef Research Foundation, Republic of Palau, March 1996. Type locality: Talikud Island, Davao, Philippines, 7–12 m (Figs 1, 6A). Distribution: Davao, Philippines. Description: Thick encrusting, biscuit-shaped sponge, ~1 cm thick and up to 20 cm in greatest dimension, with broad hemispherical lobes or domes in the surface (Fig. 6A). Oscules are ~1 mm in diameter, scattered irregularly on the apex of the surface domes, and flush with the surface (Fig. 6B). Aquiferous canals radiate towards the oscules (Fig. 6B). Ostia, 0.1–0.3 mm in diameter, are evenly dispersed over the entire surface (Fig. 6B), forming little pits. Texture is stony but breakable, slightly velvety to the touch owing to a fine plush of microstyle brushes projecting from the surface. Colour in life is a remarkable bright yellow to light orange that extends to ~1 mm deep; the choanosome is beige (Fig. 6A). Beige in ethanol. Figure 6. View largeDownload slide Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A, underwater picture showing the thick (~1 cm) encrusting habit, the bright yellow colour and the evenly scattered oscula (osc) openings over the surface, as well as a transverse section through the choanosome (cho). B, detail of large (1 mm) osculum opening, with several aquiferous channels (ch) radiating towards the osculum. Ostia (ost) dispersed over entire surface. C, overview of choanosomal sphaeroclone desma network, with numerous microscleres dispersed around. D, detailed view of desma network, with immature desma in the centre of the picture indicated by a hollow. E, F, details of spined long root-like outgrowths of the desma centre and rays. Figure 6. View largeDownload slide Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A, underwater picture showing the thick (~1 cm) encrusting habit, the bright yellow colour and the evenly scattered oscula (osc) openings over the surface, as well as a transverse section through the choanosome (cho). B, detail of large (1 mm) osculum opening, with several aquiferous channels (ch) radiating towards the osculum. Ostia (ost) dispersed over entire surface. C, overview of choanosomal sphaeroclone desma network, with numerous microscleres dispersed around. D, detailed view of desma network, with immature desma in the centre of the picture indicated by a hollow. E, F, details of spined long root-like outgrowths of the desma centre and rays. Skeleton is composed of sphaeroclone desmas (Fig. 6C), the ectosome being differentiated by the presence of brushes of microstyles projecting from the surface between the ostial pits (Fig. 6C). Immature desmas are obvious in the surface skeleton. Megascleres are sphaeroclones, with a centrum from which arises an elongated, spined apex, and below which emanate four to five spined rays (Fig. 6E, F) that are joined to other desmas in zygosis. The apex is ornamented with ragged (heavily acanthose) spires (Fig. 6E, F); the branches are more simply spined. Immature desmas are characterized by a hole in the centre (Fig. 6D). In addition, hastate, slightly curved oxea megascleres of various sizes (75–217 µm in length and 3–10 µm thick, N = 15; Fig. 7A–E) were detected but considered here as artefacts of haplosclerid origin. Figure 7. View largeDownload slide Microscleres of Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A–E, hastate oxea megascleres of haplosclerid origin as artefacts. F–H, microsubtylostyles with one (H) or two (F, G) tyles (knobs) towards the tips of the spicule. I, mucronate style. Figure 7. View largeDownload slide Microscleres of Vetulina incrustans sp. nov. (NIWA 109682, USNM 1470701). A–E, hastate oxea megascleres of haplosclerid origin as artefacts. F–H, microsubtylostyles with one (H) or two (F, G) tyles (knobs) towards the tips of the spicule. I, mucronate style. Microscleres are abundant slender, slightly curved, occasionally polytylote microstyles (Fig. 7F–I). Substrate and ecology: Sponges were found deep inside crevices on vertical limestone reef faces with small caves, between 7 and 12 m. Etymology: Named after its unusual encrusting growth form (incrustans = encrusting, Latin). DNA barcodes: In the present study, we sequenced partial cox1 (‘Folmer’ fragment) and 28S (C1–D2 fragment). Remarks: The encrusting domed morphology of V. incrustans sp. nov. is highly distinctive, clearly separating it from all species of Vetulina, particularly V. indica and V. rugosa from Western Australia, which are lamellate. The sphaeroclones are of a similar general structure and diameter, but the ornamentation is characteristic of the new species; the apical spine is covered with ragged spires. Hastate oxeas (Fig. 7A–E), reminiscent of haplosclerid forms, were found to be moderately common in places in the ectosome and shallow choanosome. They represent a variety of sizes and shapes, ranging in length from 75 to 270 µm, and 2–10 µm thick (Fig. 7A–E), suggesting that they are artefacts. We consider the diverse polytylote subtylostyle microscleres to belong to the species owing to their position in the skeleton. Molecular systematics and relaxed molecular clock approach The present study comprises the largest dataset of Vetulina sequences to date, encompassing cox1, 28S, ITS and 18S sequences of all known extant species except for V. incrustans sp. nov., where no ITS and 18S could be amplified (Figs 8, 9). Sequence variation among different Vetulina species is found to be low for ITS (Fig. 8A). Nevertheless, the three Indo-Pacific Vetulina species (V. rugosa, V. indica and V. incrustans sp. nov.) group separately from the Tropical Western Atlantic species (V. tholiformis sp. nov. and V. stalactites; Figs 8, 9). In addition, there is no genetic variation observed between V. rugosa and V. indica from the Indian Ocean. Pairwise sequence differences of these two species to V. incrustans sp. nov. from the Philippines is low in cox1 (0.5%) and 28S (1.6%). There is no variation between V. stalactites and V. tholiformis sp. nov. within cox1, 18S and 28S (C1–D2), but within ITS we observed pairwise sequence difference of 0.8%). GenBank sequence AJ224648 has several ambiguous sites towards the 3′ end of the sequence, which causes the long branch in the 18S gene tree. Figure 8. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial (‘Folmer’) cox1. The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (circles), with colours corresponding to different localities as shown in Figure 1. Figure 8. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial (‘Folmer’) cox1. The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (circles), with colours corresponding to different localities as shown in Figure 1. Figure 9. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial ITS (A), 18S (B) and 28S (C1–D2 region) (C). The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Figure 9. View largeDownload slide Bayesian inference (MrBayes, GTR + G + I model) phylogeny of Sphaerocladina and Spongillida based on partial ITS (A), 18S (B) and 28S (C1–D2 region) (C). The maximum likelihood (RaxML) tree is congruent, otherwise marked with a dash at the support values. Numbers above or close to the branch length are bootstrap supports (left) and posterior probabilities (right). Specimens representing Vetulina are shaded light grey. Numbers behind taxon names are either voucher numbers (HBOI, NIWA) or GenBank accession numbers. New sequences from this study are in bold. Abbreviations behind the numbers correspond to different localities in the Pacific and Tropical Western Atlantic (BAH, Bahamas; BON, Bonaire; CAR, Caribbean; CUR, Curaçao; JAM, Jamaica; PHI, Philippines; TUR, Turks and Caicos; W-AUS, Western Australia). Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Our dated phylogeny based on datasets of cox1 and 28S (C1–D2) calculated in a relaxed molecular clock framework is illustrated in Fig. 10. The Asian Vetulina species form a monophylum, which is sister to the Tropical Western Atlantic Vetulina clade. The origin of all extant Vetulina species is dated to ~43.3 Mya (Eocene, Bartonian), with a credibility interval of 21.1 and 84 Mya, thus before the closure of the Tethyan Seaway, which was dated to 13–11 Mya. The split of V. incrustans sp. nov. from the Philippines and V. indica and V. rugosa from Western Australia is dated to ~22.5 Mya (Early Miocene, Aquitanian), whereas the split of V. tholiformis and V. stalactites is dated earlier, to ~16.1 Mya (Early Miocene, Burdigalian). Furthermore, our relaxed molecular clock approach indicates a possible split of freshwater sponges (Spongillida) and marine Sphaerocladina at ~247.5 Mya (Early Triassic). Figure 10. View largeDownload slide Time-calibrated phylogeny of Sphaerocladina and Spongillida as inferred from BEAST 2 analysis based on cox1 and 28S datasets, plotted on a stratigraphic chart. Newly sequenced species are in bold. Error bars on node ages are in purple. Numbers behind taxon names are voucher numbers (HBOI, NIWA). For references of Spongilla and Xestospongia testudinaria (outgroup) see Material and Methods section. Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Red line indicates the period of the closure of the Tethyan Seaway. Colour code for the geological time scale is according to the Commission for the Geological Map of the World (CGMW), Paris, France. Figure 10. View largeDownload slide Time-calibrated phylogeny of Sphaerocladina and Spongillida as inferred from BEAST 2 analysis based on cox1 and 28S datasets, plotted on a stratigraphic chart. Newly sequenced species are in bold. Error bars on node ages are in purple. Numbers behind taxon names are voucher numbers (HBOI, NIWA). For references of Spongilla and Xestospongia testudinaria (outgroup) see Material and Methods section. Coloured symbols after the taxon names indicate holotypes (stars) and paratypes (dots), with colours corresponding to different localities as shown in Figure 1. Red line indicates the period of the closure of the Tethyan Seaway. Colour code for the geological time scale is according to the Commission for the Geological Map of the World (CGMW), Paris, France. DISCUSSION The two recently described Vetulina species from the Indian Ocean, V. incrustans sp. nov. from the Philippines and V. tholiformis sp. nov. from the Bahamas, increase the number of current valid species of the genus to five. Our morphological observations of the choanosomal sphaeroclonar desmas indicate their high similarity among the species, similar to observations by Pisera et al. (2017). However, both new species described in the present study have a unique outer growth form, which is either encrusting or dome shaped. The gross morphological differences and coloration of V. incrustans sp. nov. are striking and certainly add diagnostic characters to separate species in a very difficult group with few morphological characters. Within the fossil record, spherical–bulbous growth forms are known from Cretaceous sphaerocladinid species, e.g. Macrobrochus Schrammen, 1910 and Ozotrachelus Laubenfels, 1955 (Reid, 2004; Pisera, 2006), but no encrusting growth form is reported. Moreover, our morphological re-examination of V. stalactites clearly reveals its unique possession of microstyles, which is a spicule type noticed by Sollas (1885, 1888), but subsequently regarded as contamination by Pisera & Lévi (2002b). Unlike the morphology, difficulties were present in discriminating species based on molecular sequences of cox1 and 18S, which were identical (Figs 8, 9B). Even within the more variable 28S and ITS fragments, only few nucleotide differences were observed, confirming their high genetic similarity. For instance, although V. stalactites and V. tholiformis sp. nov. are both from the Tropical Western Atlantic and both clearly separated on morphological grounds, these two species can be distinguished only by the ITS marker (Fig. 9A). A similar phenomenon is known from the freshwater sponge sister group (Meixner et al., 2007), which suggests the need for comparative analyses on shared factors influencing mutation rates in these two lineages, which is beyond of the scope of the present study. Until now, V. stalactites was known only from Barbados (Schmidt, 1879; Van Soest & Stentoft, 1988) and Martinique (Pomponi et al., 2001). Our study illustrates that V. stalactites is more common in the Tropical Western Atlantic than previously thought (see Fig. 1), but has not yet been found in the Pacific. The disjunct distribution of the genus (Pisera et al., 2017) is not uncommon and has been observed for several other non-‘lithistid’ demosponges (see e.g. Łukowiak et al., 2014; Łukowiak, 2016). However, fossil records of undoubted sphaeroclonar desmas, similar to Recent Sphaerocladina species, are diverse, with ten genera, and known from the Jurassic and Cretaceous (Reid, 2004) as well as from the Eocene (Frisone et al., 2016) Pisera et al. (2017) proposed Vetulina as an example of a relict fauna with a once more widely distributed population, possibly originating in the Tethys Sea. With the most comprehensive dataset available to date, our study first tests this hypothesis by the inclusion of fossils in a relaxed molecular clock framework. The closure of the Tethyan Seaway is dated to the Early Miocene (11–13 Mya, Serravallian; Rögl, 1998) and regarded by Pisera et al. (2017) as the key event causing the isolated and yet rare occurrences of Vetulina species today. With the most comprehensive dataset available to date, our dated phylogeny adds weight to this hypothesis, indicating an origin of all Vetulina species in the Eocene (~43.3 Mya), thus supporting a possible origin of Vetulina in the Tethys Sea. CONCLUSION The original idea of a Tethyan origin for several demosponges was put forward by Reid (1967) and later taken up by several other studies, looking at the diverse modern and Mesozoic sponge fauna and their historical biogeographical distributions (e.g. Wiedenmayer, 1994; Łukowiak et al., 2014; Łukowiak, 2016). However, for the first time in this context, we used an integrative molecular palaeobiological approach to provide supportive information to test the hypothesis that Vetulina is an example of a relict fauna with its origin in the Tethys Sea. This case study demonstrates the advantage of such an approach to address evolutionary and biogeographical hypotheses. [Version of Record, published online 14 March 2018; http://zoobank.org/urn:lsid:zoobank.org:pub:0122DEEF-3F68-4D2F-A119-378D8C4CA5CF] ACKNOWLEDGEMENTS Financial support for this study was provided by the German Science Foundation to D.E. and G.W. (DFG ER 611/3-1 and DFG Wo869/15-1, respectively). LMU Mentoring and the HELGE AX:Son JOHNSON STIFTELSE provided funding for A.S. to visit HBOI (Florida, USA) and NIWA (National Institute of Water and Atmospheric Research, Auckland and Wellington in New Zealand). Financial support for the RV Seward Johnson/Johnson Sea Link I+II expedition was provided by HBOI. Vetulina incrustans sp. nov. was collected by the Coral Reef Research Foundation (CRRF) under contract no. N02-CM-77249 to the US National Cancer Institute. We acknowledge the Government of the Bahamas, Curaçao, Jamaica, Turks and Caicos, Bonaire and St Vincent Islands, and the Philippines, for granting permission to conduct research in their territorial waters. 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