Systematics of ‘lithistid’ tetractinellid demosponges from the Tropical Western Atlantic—implications for phylodiversity and bathymetric distribution

Higher-taxa relationships of desma-bearing tetractinellids

The 296 lithistid sequences of at least 88 species from 27 genera (35 known) constitute the largest and most comprehensive taxon set on desma-bearing tetractinellids to date. Our phylogenies (overview in Fig. 2, see Figs. 3–8 (28S) and Figs. 9–16 (cox1) for details) corroborate the monophyly of Tetractinellida, currently including the suborders Astrophorina and Spirophorina (Morrow & Cárdenas, 2015) (Fig. 2C). In addition, the affinity of eight desma-bearing families to the suborder Astrophorina (Cárdenas et al., 2011; Morrow & Cárdenas, 2015; Schuster et al., 2015) is confirmed (Figs. 2A–2C). The 28S phylogeny (Fig. 2A) indicates a sister relationship of Astrophorina and Spirophorina. In both gene trees (Figs. 2A–2B) desma-bearing tetractinellids do not group with the Spirophorina (only represented by the Tetillidae in our sampling). In both gene trees the rhizomorine-bearing families Scleritodermidae, Siphonidiidae and Azoricidae form a clade (Fig. 2). However, Gastrophanella (Siphonidiidae) is distinct and sister (1.0 Posterior Probability (PP)) to Scleritodermidae/Siphonidiidae/Azoricidae in the 28S phylogeny (Fig. 2A). This sister-group relationship could not be corroborated by cox1 analysis as no sequence of Gastrophanella could be generated. We suspected an intron insertion within cox1 due to the discovery of these in closely related rhizomorine-bearing genera (Setidium Schmidt, 1879, Microscleroderma Kirkpatrick, 1903, Aciculites Schmidt, 1879, Scleritoderma Sollas, 1888) (Schuster et al., 2017). Based on this, various primer sets suggested by Schuster et al. (2017) were tested, however, without success. We suspect that Gastrophanella has one or several intron insertions in the cox1 gene in a yet unknown position. By including several additional rhizomorine-bearing genera such as Gastrophanella, Leiodermatium, Siphonidium and Amphibleptula in our datasets, the family Thrombidae could not be recovered within Astrophorina as hypothesized by the Systema Porifera (Hooper & Van Soest, 2002). The 28S gene tree recovers Thrombidae as sister to all rhizomorine-bearing tetractinellids, but this relationship is not supported (0.72 PP) (Figs. 2 and 8) and needs further investigation, including also 28S for Stupendidae Kelly & Cárdenas, 2016, a recently established new family (Kelly & Cárdenas, 2016). In the cox1 phylogeny (Fig. 2B) Stupendidae is a highly supported sister taxon to Scleritodermidae/Siphonidiidae/Azoricidae. It should be noted that Thrombidae and Gastrophanella are missing in the cox1 phylogeny (Fig. 2B).

Intra-subordinal relationships of astrophorine ‘lithistids’

The majority (15 out of 23) of the currently known tetractinellid families are located within the Astrophorina (Morrow & Cárdenas, 2015). This includes eight desma-bearing families (Corallistidae, Isoraphiniidae Schrammen, 1924, Macandrewiidae Schrammen, 1924, Neopeltidae, Pleromidae, Phymaraphiniidae Schrammen, 1924, Phymatellidae and Theonellidae) and seven non-desma bearing families (Cárdenas et al., 2011; Morrow & Cárdenas, 2015; Schuster et al., 2015) (see also Figs. 2A–2C). Thus, the present study supports earlier findings, which were based on lower taxon sampling and additionally provides deeper insights into the intraspecific relationship of desma-bearing astrophorids.

The family Theonellidae consists of the genera Discodermia Du Bocage, 1869, Manihinea Pulitzer-Finali, 1993, Racodiscula Zittel, 1878, Siliquariaspongia Hoshino, 1981 and Theonella Gray, 1868. Theonellidae possesses tetraclone desmas and phyllotriaenes to discotriaenes as characteristic megascleres. Typical microscleres are acanthorhabds, spirasters and amphiasters (Pisera & Lévi, 2002a). Until now, only Theonella and Discodermia species as well as one Manihinea sp. were sequenced in different phylogenetic studies using 18S, 28S and cox1 (see e.g., Redmond et al., 2013; Hall, Ekins & Hooper, 2014; Schuster et al., 2015). By providing sequences for all known genera, our 28S phylogeny (Fig. 3) recovers Theonellidae as monophyletic (PP = 1.0), thus conclusively support earlier findings of Schuster et al. (2015), while the cox1 phylogeny (Fig. 13) lacks support in this respect. The 28S phylogeny indicates the monophyly of the genera Discodermia, Manihinea, Racodiscula and a potential new taxon, here denoted as Theonellidae gen. sp., a potential new genus mainly distinct by the layered network of tetraclone desmas with smooth rays and strongly tuberculated tips and the less abundant microscleres on the ectosome (SBD#2102–2106). The sister relationship of Manihinea conferta to Theonella sp. is highly supported (PP = 0.99) by 28S (Fig. 3), whereas it is not supported by cox1 (Fig. 13). A close relationship of Theonella and Manihinea was observed in an earlier study by Redmond et al. (2013) using a nearly complete 18S gene fragment, but unsupported. The genus Discodermia is sister to a clade consisting of Manihinea +Theonella +Siliquariaspongia, which is sister to Racodiscula +Theonellidae gen. sp.

The genus Racodiscula is highly supported (PP=1.0) as sister to Theonellidae gen. sp. Although the outer morphology of Theonellidae gen. sp. (SBD#2106 A–D) is very similar to that of Racodiscula, it differs in spicule composition, desma and skeleton structure: the usually abundant spinose microacanthorhabds, covering the surface of Racodiscula species (SBD#2065) building a dense crust on the surface, are rarer or even absent in Theonellidae gen. sp. Instead of microacanthorabds, phyllo- to discotriaenes are the main components of the dense surface crust. In addition, Theonellidae gen. sp. possesses desmas with smooth rays and strongly tuberculated tips (SBD#2105 and SBD#2106) building a layered network (SBD#2102), which clearly differs from Racodiscula (Schuster et al., 2018).

Theonella mirabilis (De Laubenfels, 1954) was first named within the homosclerophorid genus Placinolopha (Class Homoscleromorpha, Order Homosclerophorida, Family Plakinidae) on the possession of what de Laubenfels described as ’lophotetractines’. The key size and shape differences between the ’tetralophs’ of T. mirabilis and other Placinolopha species were noted by Muricy & Diaz (2002), who suggested that the species mirabilis had a more likely affinity with species in family Theonellidae. Sequences of 28S (Fig. 3) unite specimens identified as Theonella mirabilis in a single clade with a specimen identified as T. conica (Kieschnick, 1896) which also has tetraloph-like desmas, suggesting that species with non-articulated ’tetraloph’ desmas may be monophyletic and separate from other Theonella spp. However, cox1 sequences (Fig. 13) separate T. mirabilis into two groups, nesting them within diverse species of Theonella. Theonella mirabilis is very similar in spicule complement to the type species of the genus Siliquariaspongia, S. japonica Hoshino, 1981 (Family Theonellidae), although the latter lacks the strongyles and possesses frilly discotrianes, the latter occasionally recorded in T. mirabilis. Our phylogenies clearly place all of the sequenced Theonella mirabilis species within the Theonella +Manihinea clade (Figs. 3 and 13), confirming that this species belongs to the family Theonellidae. This result is supported by the discovery of potent new depsipeptides mirabamides A-D, that inhibit HIV-1 infection, adding to a small class first exemplified by the papuamides from various Theonella spp. (Plaza et al., 2007).

The family Macandrewiidae is monogeneric with currently seven valid species (Van Soest et al., 2018a). Until know, only Macandrewia rigida Lévi & Lévi, 1989 from the Solomon Islands has been sequenced (28S C1-D2 region, LN624160, G317931) (Schuster et al., 2015). The present study includes a further sequence of an undescribed Macandrewia sp. from the Bahamas (909 m depth), which clearly differs from M. rigida (Fig. 4). Morphological differences in desmas (SBD#2004) corroborate the genetic difference to M. rigida and provide further evidence of a possible new species, which would be the first record in the TWA. Nevertheless, further morphological observations and comparison with the type material of M. rigida as well as its sequences are needed to conclusively describe and distinguish this potential new species. Both Macandrewia species group within the Geodiidae, close to the Erylinae Sollas, 1888, within a clade of non-desma bearing astrophorins (Calthropella Sollas, 1888, Caminella Von Lendenfeld, 1894) (Fig. 4). This relationship is currently not supported by morphology (Cárdenas et al., 2018) and in any case suggests a distinct evolutionary history of Macandrewia to other ‘lithistid’ families Corallistidae and Neopeltidae (Schuster et al., 2015), where Macandrewia was previously allocated (Kelly, 2000; Pomponi et al., 2001).

The family Phymatellidae currently includes three genera: Neoaulaxinia, Neosiphonia and Reidispongia. A highly supported sister group relationship of Neoaulaxinia Pisera & Lévi, 2002e to the genus Gignouxia Moret, 1926 is observed and Reidispongia coerulea Lévi & Lévi, 1988 is sister to this clade (Fig. 4). Morphological characters of both sequenced Gignouxia species were further investigated and illustrated (SBD#1894). Both species are potentially new to science. Gignouxia sp. 1 (SBD#1894) possesses the characteristic pseudophyllotriaenes (SBD#1894) known from the Neopeltidae, while Gignouxia sp. 2 (SBD#1895) possesses dichotriaenes characteristic for the Phymatellidae. These dichotriaenes, however, have a unique shape with indented cladomes (SBD#1895). A spicule drawing of Corallistes tubulatus Van Soest & Stentoft, 1988 from Barbados, now Neophrissospongia tubulata, resembles those unique dichotriaenes implying that N. tubulata may need to be reallocated to Gignouxia. Interestingly, dichotriaenes of Gignouxia sp. 2 resemble those of the fossil Gignouxia niciensis Moret, 1926 (Corallistidae) from the Late Cretaceous (Pl. XVIII, Fig. 2, 2′fig-txt 37). We suggest to allocate Gignouxia to the family Phymatellidae. Gignouxia will include Gignouxia sp. 2 as well as N. tubulata comb. nov.

Neopeltidae polyphyly is given by the highly supported (PP = 1.0) sister relationship of the newly sequenced species Neopelta sp. to the non-desma bearing astrophorid Neamphius huxley Sollas, 1888 (Fig. 4). Morphologically, these two species only share choanosomal amphiasters with spiny rays and microxeas. Thus, monocrepid desmas and pseudodiscotriaenes characterizing Neopelta were lost in Neamphius. Spicule losses and gains are not uncommon within tetractinellids and have frequently been shown (Chombard, Boury-Esnault & Tillier, 1998; Cárdenas et al., 2011; Schuster et al., 2015). Nevertheless, these two genera form a robust sister clade to a non-desma bearing Ancorinidae clade consisting of Stelletta Schmidt, 1862, Ancorina Schmidt, 1862, Asteropus Sollas, 1888 and Stryphnus Sollas, 1886 (see Fig. 4).

Corallistidae is another major family dominating the HBOI collection and subsequently our phylogenies (Figs. 5 and 9). These taxa were the focus of biomedical investigations (e.g., Haar et al., 1996; Wright, 2010), thus targeted during the HBOI expeditions. Even though Corallistes were frequently sampled in the past and 15 species are described to date (Van Soest et al., 2018b), only five sequences are published (Kelly Borges & Pomponi, 1994; Chombard, Boury-Esnault & Tillier, 1998; McInerney, Adams & Kelly, 1999). With 52 Corallistes specimens sequenced, this study presents the largest data set to date and reveals the monophyly of this genus (28S, PP = 1.0, Fig. 5). Corallistes typus (Schmidt, 1870) specimens, type species of the genus, were examined (SBD#1888, 1889, 1892) and sequenced (Figs. 5 and 9). In addition, a Corallistes isabela Desqueyroux-Faúndez & Van Soest, 1997 sample from Honduras was sequenced. Until this study Corallistes isabela was only known from the Eastern Pacific (Galápagos) and discussed as endemic to the Galápagos (Desqueyroux-Faúndez & Van Soest, 1997 and Schuster et al., 2018). In addition, six Corallistes (C. sp. 2 to C. sp. 7, see Fig. 5) differ by 1–3 bp in the 28S fragment, while no differences were found in cox1 (Fig. 9). Morphological differences are observed between C. sp. 2 (SBD#1872) and C. sp. 4 (SBD#1879). For example, C. sp. 2 has long (700 µm) thin ectosomal oxeas (SBD#1872 A), while C. sp. 4 has ectosomal styles (SBD#1879 D) and subectosomal microxea with spined surfaces (SBD#1879 C). Morphological identifications are in progress and necessary to discriminate the remaining Corallistes species.

The polyphyletic corallistid genus Herengeria Lévi & Lévi, 1988, only known from the Pacific Norfolk Ridge, New Caledonia, and New Zealand (Schlacher-Hoenlinger, Pisera & Hooper, 2005; Kelly et al., 2009), was sequenced (Fig. 5) and morphologically illustrated (SBD#1949) in the present study for the first time from the Bahamas and Honduras, representing a new genus for the Atlantic. Only two species of Herengeria are described (H. auriculata Lévi & Lévi, 1988) and H. vasiformis Schlacher-Hoenlinger, Pisera and Hooper, 2005). The morphological observations delimit the new TWA species from H. auriculata (Schlacher-Hoenlinger, Pisera & Hooper, 2005) due to a lack of subectosomal rhabd-like spirasters. With respect to molecular markers, Herengeria spp. from the TWA are distinct from H. vasiformis/Herengeria sp. from the Pacific (Fig. 5). Our 28S (Fig. 5) and cox1 (Fig. 10) phylogenies strongly support Neoschrammeniella Pisera & Lévi, 2002d as sister to the Herengeria/Isabella clade. Currently, seven valid species of Neophrissospongia (Pisera & Lévi, 2002d) are described (Van Soest et al., 2018c). Only a few Neophrissospongia sequences (two 28S and one 18S) from the Pacific Ocean are currently published. However, in order to gain a better understanding of their geographical distribution and genetic differences, additional material from the Caribbean were sequenced in this study: Their resulting 28S phylogeny clearly separates Neophrissospongia from the Pacific and the Caribbean Islands. Neophrissospongia sp. 1 from different Caribbean Islands is sister (PP=1.0) to N. microstylifera and Neophrissospongia sp. 3, from the Pacific (Fig. 6). A further Caribbean Neophrissospongia species (sp. 2) forms a robust sister clade to Neophrissospongia from the Eastern Pacific (Galápagos, Panama) (Fig. 6).

A clade of six as yet unidentified specimens from the HBOI collection (SBD#1814) is sister to the monophyletic Neophrissospongia (PP = 1.0, Fig. 6). We assume that this clade consists of species from the as yet unsequenced genus Awhiowhio Kelly, 2007 from the Pacific based on morphological evidence. These show similar mega- and microsclere types to Awhiowhio such as dicranoclone desmas and smooth dichotriaenes in Awhiowhio sp. 1 from the Bahamas (SBD#1815), most similar to the Awhiowhio osheai from New Zealand (Kelly, 2007), but slightly different in terms of desma ornamentation. Streptaster microscleres and acanthose microrhabds in Awhiowhio sp. 1 (SBD#1814, 1815) differ from those in Awhiowhio osheai in sizes and shapes (SBD#1814). The cox1 phylogeny (Fig. 9) indicates the sister group relationship of Awhiowhio osheai Kelly, 2007 to Neophrissospongia. A close relationship of Awhiowhio to Herengeria as suggested by Kelly (2007) based on morphological features is not supported by any of our phylogenies. Instead, both markers independently suggest a close relationship (strongly supported by PP = 1.0) to Neophrissospongia. The genus Pleroma Sollas, 1888 (family Pleromidae) is recovered as paraphyletic in both phylogenies (Figs. 5, 6, 9 and 13). Pleroma menoui (Sollas, 1888) is distant to other Pleroma spp. (including the type species P. turbinatum) in a close relationship to Corallistidae (Figs. 5 and 9).

Intra-subordinal relationships of spirophorine ‘lithistids’

The suborder Spirophorina is characterized by sigmaspire microscleres and its members share triaene spicules with Astrophorina. Currently, three families are known: Samidae Sollas, 1888, Spirasigmidae Hallmann, 1912 and Tetillidae Sollas, 1886, whereas the latter is the largest in terms of genera and species (e.g., Van Soest & Hooper, 2002). The relationships of major clades within our cox1 and 28S phylogenies (Figs. 3–16) were in concordance with the findings of Carella et al. (2016) and Schuster et al. (2017). The latest revised classification of Morrow & Cárdenas (2015) included the desma-bearing families Azoricidae, Scleritodermidae and Siphonidiidae within Spirophorina. Since then, several studies (Schuster et al., 2015; Schuster et al., 2017; Kelly & Cárdenas, 2016) using cox1, 18S and 28S markers showed the separation of all rhizomorine-bearing sponges from Spirophorina (=Tetillidae). The present enlarged dataset corroborates again the absence of desma-bearing sponges in Spirophorina and their grouping in a well supported clade along with the Stupendidae and the Thrombidae (Fig. 2). In order to establish this clade as a new taxa, we await further molecular data from the latter two families (work in progress, MK and PC).

Subordinal structure of Tetractinellida

Kelly & Cárdenas (2016) provided strong support for families Azoricidae, Scleritodermidae and Siphonidiidae within a proposed suborder, supported in part by the common possession of rhizomorine desmas.

Regarding Azoricidae, Maldonado et al. (2015) discovered a dense and large aggregation of Leiodermatium pfeifferae Carter, 1873 on seamounts in the Mediterranean building complex reef-forming structures. Even though Leiodermatium Schmidt, 1870 has very few diagnostic characters (no microscleres) to discriminate between species (Pisera, 2002; Pisera & Lévi, 2002b), 11 species are valid to date (WPD access Jan. 2020). In the present study we sequenced the 28S C1-D2 fragment for 52 Leiodermatium specimens from several regions in the TWA (Fig. 8) representing at least 8 species and the largest sequenced dataset for this genus to date. The monophyly of Leiodermatium is highly supported by our 28S phylogeny (PP = 1.0; Fig. 8). The amplification of cox1 unfortunately failed, most likely due to the presence of introns similar to other rhizomorine-bearing genera like e.g., Microscleroderma and Scleritoderma (Schuster et al., 2017). Preliminary morphological investigations (SBD# 1966, 1967, 1959, 1965, 1956, 1955, 1975, 1977, 1976, 1994, 2000, 2001, 2003) adumbrate detailed differences of Leiodermatium spp., in particular their surfaces (oscules and ostia sizes), diactines and desma morphology. For instance Leiodermatium sp. 1 (SBD#1955) has large and marginate oscules, while Leiodermatium sp. 6 (SBD#1994) and Leiodermatium sp. 8 (SBD#2003) have large but elevated oscules on exterior margins, in contrast to Leiodermatium sp. 7, whose oscules are small and closely distributed. Based on molecular and morphological data we propose eight different species of Leiodermatium (Fig. 8) in the TWA, however further morphological investigations are needed to corroborate this assumption. Leiodermatium is unsupported (PP = 0.65) sister to a clade of Siphonidium spp. (Siphonidiidae); the same relationship was revealed with small fragment of the 18S gene (482 bp) for Leiodermatium sp. (Kelly Borges & Pomponi, 1994; Kelly & Cárdenas, 2016). Further investigation and a review of all extant and fossil Leiodermatium species is suggested to better understand the geographical distribution and recent diversification of this paleontological important group.

Within the polyphyletic rhizomorine family Scleritodermidae, its genera Aciculites is polyphyletic and Scleritoderma is paraphyletic, while Microscleroderma, Amphibleptula Schmidt, 1879 and Setidium Schmidt, 1879 are monophyletic (Fig. 8). The genus Amphibleptula is currently monospecific with A. madrepora Schmidt, 1879 from the Caribbean (Pisera & Lévi, 2002c). Morphologically, A. madrepora is very similar and easy to confuse with Microscleroderma spirophora Lévi, 1960 as discussed in Van Soest & Stentoft (1988). Amphibleptula is here sequenced for the first time and our 28S phylogeny shows Microscleroderma and Amphibleptula sp. 1 as sister groups, although unsupported in the 28S phylogeny (PP = 0.63). Morphological observations (SBD#1802,1803) provide conclusive evidence that our three samples are Amphibleptula species, due to their dense tuberculated/blunt spinose rhizoclones, the protruding bundles of oxeas in the oscula area (SBD#1802,1803) as well as the presence of sigmaspires (SBD#1802). Differences to A. madrepora are the diactine spicules present in all three Amphibleptula sp. 1. In addition, fusiform spined microxeas and acanthorhabds are found in the specimen from Jamaica (HBOI 1-IX-93-1-006, SBD#1802). To conclude, we sequenced two potential new species of Amphibleptula with clear unique morphological characters, different from A. madrepora.