Sponge species from pool-cave-canyon systems
Above, we have provided a non-exhaustive description of several sponge species from pool-cave-canyon systems of the Taiwanese island of Lanyu, in addition to two common species (X. testudinaria and S. carteri) sampled from adjacent coral reef habitat. Both X. testudinaria and S. carteri have been extensively studied in previous publications as pertains to their microbial symbionts (Cleary et al. 2015, 2018, 2020, 2021, 2022; de Voogd et al. 2015; 2019; Polónia et al. 2015, 2016, 2017, 2018, 2021). Below we will go into more detail with respect to specific groups.
Sclerosponges
In the present study, we sampled two sclerosponge species, namely, A. willeyana (order Agelasida) and A. wellsi (order Clionaida). Sclerosponges form a hypercalcified coral-like skeleton surrounded by a very thin layer of living tissue embedded with siliceous spicules and are superficially similar albeit in distantly related orders. Modern sclerosponges, relicts of a bygone age, are morphologically similar to now extinct stromatoporids, which once formed great reefs prior to and concurrent with the first coral reefs (Basile et al. 1984). They prevailed as the main reef-building marine organisms throughout the Phanerozoic, but can still be found in modern marine environments (Vacelet 1985, Asami et al., 2021 and references therein). They are long-lived, slow growing, often mushroom-shaped, and are mainly found in caves and at greater depths; in certain locations, they contribute to reef-building (Lang et al. 1975; Asami et al., 2020; Macartney et al., 2020). These characteristics, together with the fact that their exoskeleton is formed by the slow deposition of calcium carbonate in sequential layers through time makes them excellent paleo-proxy recorders of environmental conditions at depths where photosynthetic scleractinian corals are not present (Asami et al., 2020; Grottoli et al., 2020).
Acanthochaetetes wellsi (Fig. 10) belongs to the Acanthochaetetidae family, which is an ancient family of now, largely extinct species (Rützler and Vacelet 2002). Only two species remain, namely, A. wellsi and Willardia caicosensis. The genus Acanthochaetetes was based on the extinct fossil species A. seunesi Fischer, 1970 with A. wellsi being a living fossil. It was originally discovered in shallow-water caves of Guam, but has since been recorded in other Indo-Pacific locations in similar, cave habitats. In addition to A. wellsi another agelasid, the non-sclerosponge Acanthostylotella cornuta (Topsent, 1897), was also observed. This mono-specific poorly-known species was originally described from Indonesia and was recently placed in the Agelasida (Morrow and Cardenas, 2015.
Our results showed that, despite their hypercalcified skeletal similarity, that the sclerosponge species housed distinct prokaryotic microbiomes. Richness and evenness were higher in A. willeyana than in A. wellsi. Chloroflexi were, however, relatively abundant in both taxa. Acidobacteriota, in turn, were more abundant in A. willeyana than in A. wellsi. In terms of composition, A. willeyana clustered closer to the known high microbial abundance (HMA) sponge species X. testudinaria. Our results for A. willeyana align with those of Karlińska-Batres and Wörheide (2013a, 2015) for A. willeyana sampled from the Great Barrier Reef and from sites across the Indo-Pacific region, respectively, namely, a prokaryotic community dominated by Chloroflexi with major Actinobacteriota, Acidobacteriota and Proteobacteria components and minor Gemmatimonadota and Poribacteria components. In a previous study, the coralline Ceratoporella nicholsoni (order Agelasida) maintained a stable microbiome through a depth gradient (37 to 97m) consisting mainly of Chloroflexi, Thaumarcheaota, Proteobacteria, Acidobacteria and Actinobacteria (Macartney et al., 2020). Similar results were obtained for the coralline sponge Vaceletia crypta (order Dictyoceratida) with Chloroflexi being the most abundant phyla (35%) (Karlińska-Batres & Wörheide 2013b). Some studies have suggested that the symbiotic microbial communities of coralline members may be involved in calcification (Jackson et al. 2011; Jackson & Wörheide 2014). Karlińska-Batres and Wörheide (2013a) also noted that their samples separated according to sampling location suggesting an important role of geographic distance in structuring prokaryotic community composition in line with our study for two HMA sponge species, X. testudinaria and Hyrtios erectus (Cleary et al. 2022).
Acanthochaetetes wellsi was the most compositionally distinct of the two coralline sponges sampled, had the lowest richness of all sponge species and housed the most taxon-restricted OTUs. These OTUs, furthermore, had relatively low sequence similarities to organisms in GenBank. Moreover, the Chloroflexi in A. wellsi were exclusively assigned to the Dehalococcoidia with no OTUs assigned to the Anaerolineae, in contrast to the other, presumably, HMA species in the present study. To the best of our knowledge, this is the first next-generation assessment of the prokaryotic community of the sclerosponge Acanthochaetetes wellsi.
Lithistid sponges
In addition to sclerosponges, we observed numerous other sponge species of which the lithistids were particularly diverse. The lithistids, also referred to as Desma-bearing or rock sponges, are characterised by a particularly dense skeletal structure (Pisera and Gerovasileiou 2021). They are also an ancient group. Upper Cambrian reefs consisted of lithistid sponges, which together with microbial biofilms formed columnar ‘stromatolites' (Coulson and Brand 2016). Lithistid sponges are noted for the sheer diversity of pharmaceutically interesting compounds, which have been isolated. Valeria D'Auria et al. (2002) identified them as a ‘spectacular source of new metabolites’. These included alkaloids, pigments, novel sterols, cyclic and linear peptides, polyketides and macrolides. Valeria D'Auria et al. (2002) noted that the structural similarity of many of these compounds to compounds produced by microorganisms suggests that they may be synthesised by microbial symbionts of the sponges.
Lithistids are a polyphyletic group of ancient (Cambrian) Demospongiae with hypersilicified and articulated skeletons of desma megascleres. Modern Lithistids can be found in cryptic habitats (caves and at greater depths) (Manconi et al., 2006 and references therein; Xavier et al., 2021). Although some species occur in shallow caves, most of the species live at greater depths (e.g., seamounts or along rocky slopes) (Maldonado et al. 2015). Based on molecular systematics, most of the lithistid sponges have been placed in the Tetractinellida order (Schuster et al, 2021). Sponges of this order have a subradial or radial skeletal configuration, both monactine and triaene megascleres, aster, sigma, microxea, raphide and microrhabd microscleres and desmas sometimes present (Morrow & Cárdenas 2015). They can form sponge grounds i.e., reef-like aggregations on coarse sand and gravel at greater depths (Hourigan et al., 2017). Although poorly studied, like coral reefs, these sponge grounds are structural habitats that provide habitat for a variety of vertebrate and invertebrate species and play a role in nutrient recycling and bentho-pelagic coupling (Hawkes et al., 2019, Cathalot et al., 2015; Maldonado et al., 2020; Rooks et al., 2020, Xavier et al., 2021).
The bright yellow Vetulina incrustans (Vetulinidae: Sphaerocladina), recently described from deep crevices of small caves in the Philippines (Schuster et al., 2018) was observed and collected from one location. This group of sponges is closely related to freshwater sponges of the order Spongillida. Lithistids previously had their own order, the Lithistida. The shift of lithistids to different orders was based on molecular and morphological data, which indicated polyphyly (Morrow and Cardenas, 2015). The tetracladinid and dicranocladinid lithistids, however, remained monophyletic and branched with the choristid demosponges. Rhizomorinid families, in contrast, were identified as polyphyletic (Kelly-Borges and Pomponi 1994). The Tetractinellida presently includes three suborders, the Astrophorina, Spirophorina and Thoosina.
The tetractinellid species in the present study formed two distinct clusters in the PCO analysis with one cluster including both species in the Sclerotidermidae family (Aciculites ciliata and Scleritoderma novo spec.) whereas the other cluster contained all remaining species in the families Ancorinidae, Corallistidae, Geodiidae, and Theonellidae. The tetractinellid species, in general, were characterised by having high evenness and richness and relatively high abundances of HMA indictator taxa, such as Chloroflexi, Actinobacteriota, and Acidobacteriota (Moitinho-Silva et al. 2017) although there was considerable variation among species. The microbial community of the tetractinellid cold water Geodia barretti (Tetractinellida, Demospongiae) was shown to be dominated by Chloroflexi (SAR202), Poribacteria and Acidobacteria (Radax et. al., 2012). Proteobacteria and more specifically Gammaproteobacteria were the most numerous taxa followed by Chloroflexi, Firmicutes and Alphaproteobacteria in the lithistid sponge Discodermia spp. collected at different depths (24–161 m) in the Bahamas (Brück & McCarthy 2012).
The tetractinellid species in the present study shared a large number of dominant OTUs, many of which were not found in other taxa with the exception of the species Polymastia sp. (Order: Polymastiida), which clustered between the tetractinellid species and the other sponge species. Unfortunately, we were only able to sample a single specimen of this species. Despite their highly distinct composition, the dominant OTUs recorded in the tetractinellids had relatively high sequence similarities to organisms in GenBank, in contrast to the sclerosponge A. wellsi.
Petrosiidae
Sponges of the family Petrosiidae have a broad geographical distribution and inhabit shallow to deep (4–280 m) and cold to warm waters. They are massive, bulbous (but sometimes branching or encrusting), have a stony and brittle texture, a smooth surface and can have a vase shape resembling a volcano crater. The family is divided into four genera and two subgenera. The genus Petrosia is defined by having a network of free spicules with oxea (of 3 different size classes; subgenus Petrosia) and strongyle (subgenus Strongylophora) megascleres. They can be found in North, central and South Pacific Oceans, central Atlantic and Indian Oceans (Desqueyroux-Faúndez & Valentine, 2002).
Three host sponge species sampled in the present study belonged to the family Petrosiidae including the known HMA species, X. testudinaria. in addition to Xestospongia novo spec. and P. corticata (Fig. 11). Previous studies have identified various species of the Petrosiidae family as HMA species (Gloeckner et al. 2014; Moitinho-Silva et al. 2017). For example, X. testudinaria has consistently been shown to harbour a HMA-type prokaryotic community characterised by an abundance of HMA-indicator taxa (Cleary et al. 2018, 2020, Cleary, Polónia, Huang et al. 2019; Cleary, Swierts, Coelho et al. 2019). Likewise, in a compositional analysis, Petrosia elephantotus sampled from the Red Sea clustered together with other known HMA species such as X. testudinaria, and had a relatively high abundance of Poribacteria compared to non-HMA species sampled from the same area (Cleary et al. 2020). In Mayotte, a species identified as Petrosia aff. spheroida clustered together with the known HMA species H. erectus and X. testudinaria (de Voogd et al. 2019). The Mediterranean Petrosia ficiformis was also identified as a HMA species (Ribes et al. 2015). Interestingly, P. ficiformis occurs in two distinct colour morphs. The white/pink morph is found in dark/shady environments, such as inside or at the entrances of caves, without phototrophic symbionts, as opposed to a violet morph inhabiting illuminated habitats, such as rocky cliffs, with a vast community of intracellular cyanobacteria. The presence of the cyanobacterium Synechococcus feldmannii has, furthermore, been confirmed (with the aid of TEM observation and Chlorophyll a measurements) in the pink and violet morphs, but not in the white morphs (Burgsdorf et al., 2014). A 16S rRNA pyrosequencing-based assessment of these two different colour morphs also showed a stable bacterial community dominated by Chloroflexi, Gammaproteobacteria, and Acidobacteria in all colour morphs. Geographical variation was also shown to be a more important structural component of variation in microbial diversity than host-genetic variability (Burgsdorf et al., 2014).
Phototrophic sponges
Another characteristic component of the sponge fauna in the present study were the phototrophic sponges, which are characterized by their reliance on photosynthetic symbionts to attain most of their energy requirements. Previous studies have reported different distributions of photo- and heterotrophic sponges with phototrophic sponges more abundant in the Indo-Pacific region (e.g., Great barrier reef) and mostly in outer reefs (more distant from shore) and heterotrophic sponges relatively more abundant in the Caribbean (Erwin & Thacker 2007; Bell et al., 2018, 2020). Phototrophic sponges are also primarily found in clearer, oligotrophic waters.
Cyanobacteria are one of the most abundant photosynthetic organisms found in symbiotic relationship with phototrophic sponges. Members of the genus Lamellodysidea, for example, have been consistently shown to host the cyanobacterial symbiont, Hormoscilla spongeliae (previously known as Oscillatoria spongeliae), and appear unable to survive without the species. This dependence has also made it hitherto impossible to culture H. spongeliae given that the symbiont appears unable to survive without its host (Usher, 2008; Schorn et al., 2019). Hormoscilla spongeliae is known to produce high amounts of several halogenated compounds and polybrominated diphenyl ethers (PBDEs), which are chemically identical to anthropogenic pollutants, but with potential antimicrobial and antipredator properties (Flatt et al. 2005; Agarwal et al. 2017). In addition to Cyanobacteria, the microbial community of Lamellodysidea members and more specifically L. herbacea also consisted of Bacteroidetes, Alphaproteobacteria, Gammaproteobacteria, and Oligoflexia members (Podell et al., 2020). In the present study, both phototrophic sponges were characterised by low evenness and richness. In contrast to many of the other sponge species in the present study, both sponge species were only found in the high light intensity pool environments growing over rock surfaces. In addition to L. herbacea, H. spongeliae, recorded as Oscillatoria spongeliae, has also been found to be a prominent member of the microbiomes of the phototrophic sponges Phyllospongia papyracea and Lendenfeldia chondrodes (Ridley et al. 2005).
SAR202 were enriched in cave sponges
In the present study, Chloroflexi were the most abundant overall bacterial phylum and were only rare in species from the high light intensity pools or adjacent coral reef habitat, namely, the phototrophic species or S. carteri, a known LMA sponge species. A number of studies have found Chloroflexi members to be particularly abundant in HMA species (Schmitt et al. 2011; Moitinho-Silva et al. 2017; Swierts et al. 2018; Cleary, Polónia, Huang et al. 2019; Cleary et al. 2020, 2021). Although Chloroflexi members are absent or rare in many LMA species, they can be abundant in others. For example, we previously observed Chloroflexi members in the species Paratetilla bacca from Mayotte, Acanthella cavernosa from Taiwan, and Ectyoplasia coccinea, Cinachyrella sp. and Topsentia aqabaensis from the Red Sea, all of which were otherwise compositionally similar to samples from known LMA species from the same locations (Cleary, Polónia, Huang et al. 2019; Cleary et al. 2020; de Voogd et al. 2019). In the present study, Chloroflexi abundance was high in both the agelasid A. cornuta, and the sclerosponge A. wellsi. In A. wellsi, this was mainly due to high levels of Dehalococcoidia and in A. cornuta high levels of Dehalococcoidia (mainly SAR202) and TK17. In the Silva 138 database the SAR202 clade is an order of the Dehalococcoidia. In both species, levels of Anaerolineae were very low in contrast to the known HMA species X. testudinaria sampled from coral reef habitat, which had the highest Anaerolineae levels. SAR202 members have been shown to be abundant in bathypelagic waters and Anaerolinae members in a wide range of habitats varying from arctic permafrost to the mammalian gastrointestinal tract (Varela et al. 2008; Campbell et al. 2014; Hug et al. 2013). In humans, Anaerolinae members scavenge material from human tissue and lysed bacterial cells and may perform a similar function in sponges given their rapid cell turnover.
Bayer et al. (2018) noted that in sponges, Chloroflexi members appeared to be aerobic and heterotrophic. The energy-producing pathways (partially) identified involved glycolysis, the tricarboxylic acid cycle, pentose phosphate pathway and the respiratory chain in addition to the reductive citrate acid cycle and Wood-Ljungdahl pathway (Bayer et al. 2018). The latter of these entail autotrophic carbon fixation with the suggestion that they may function under anoxic conditions when the sponge stops pumping (Bayer et al. 2018). Bayer et al. (2018) found that all the Chloroflexi genomes studied contained genes encoding for ammonia import and assimilation, nitrite transport, and sulfur incorporation into S-containing amino acids. There were, however, also pronounced differences among genomes from different clades. SAR202 genomes, but not Anaerolinae, contained genes encoding for glutamate synthesis from glutamine and ammonia, reduction to ammonia, biosynthesis of thiamine diphosphate, and riboflavin biosynthesis. The SAR202 genomes also contained clustered, regularly interspaced, short, palindromic repeats (CRISPR)-Cas systems, indicative of high exposure to waterborne phages, which were lacking in the studied Anaerolinae genomes, polyketide synthase gene clusters and appeared to have the requisite genomic repertoire for chemical defense in the sponge.
Anaerolinae, in turn, but not SAR2020, encoded for genes involved in assimilatory reduction of sulfate, monosaccharide and oligosaccharide transport (ABC), uronic acid degradation, thiamine transport and conversion to thiamine diphosphate. In contrast to the SAR202 genomes, the Anaerolineae genomes lacked gene clusters involved in the biosynthesis of secondary metabolites. The ABC transporters identified in Anaerolinae genomes transported a range of monosaccharides including inositol. Bayer et al. (2018) noted that in a previous study (Kamke et al. 2013) it was postulated that Poribacteria use myo-inositol as a carbon source and potentially a regulatory agent and went on to remark that Anaerolineae (and Caldilineae) genomes contained all the components involved in the degradation of inositol. Inositol, itself, is involved in a wide range of functions in the sponge host and its resident microbiome including being a precursor to lipid molecules, ‘stress-protective solutes’ of eukaryotes, potential signal transduction and a component of the cell walls of eukaryotes and Archaea (Bayer et al. 2018 and references therein). In the present study, all the sponge hosts, in which Poribacteria were absent also had very low levels of Anaerolinae (data not shown) suggestive of a possible link between both taxa. This did not appear the case between Poribacteria and Dehalococcoidia.
The SAR202 and Anaerolineae genomes are also complementary to a certain extent. Anaerolineae, for example, contain genes involved in carbohydrate degradation while SAR202 lack genes for carbohydrate transport and degradation, but instead potentially use amino and fatty acid degradation to fulfill energy requirements. Bayer et al. (2018) also noted that Anaerolineae take up cofactors while SAR202 genomes contained genes for their synthesis.
Previous studies have suggested that HMA sponges are specialised for uptake of dissolved organic matter (DOM), wheras LMA speonges are specialised for the uptake of particulate organic matter (Bayer et al. 2018; McMurray et al. 2018). Chloroflexi, in particular, may play a key role in DOM dynamics. SAR202, for example, have also been implicated in the degradation of recalcitrant or refractory DOM (Landry et al. 2017; Colatriano et al. 2018). In the genomes of deep sea Chloroflexi members, Landry et al. (2017) identified enzymes involved in recalcitrant compound oxidation, some of which were found in SAR202 genomes of sponge symbionts (Bayer et al. 2018). Given the above, it is possible that SAR202 members in sponges play a role in the degradation of DOM, particularly in environments with high DOM content such as areas close to rivers or submarine groundwater discharge (Kim and Kim 2017). The sheer preponderance of Chloroflexi in cave-dwelling sponges whether in tetractinellids, other presumably HMA species, or species such as A. cornuta and A. wellsi hints that DOM-rich groundwater discharge from Lanyu island may be the main environmental component structuring the cave sponge communities. This, however, remains to be tested. The implications are, however, interesting and suggest that the sponge-filled cave systems surrounding Lanyu and other similar areas may act as giant DOM filters with important repercussions for the surrounding marine environment.
In summary, the present study identifies a potentially novel ecosystem with a number of conspicuous species new to science. The species described in the present study also only represent a fraction of the total sponge fauna. In particular, there was a rich calcareous sponge fauna, which we sampled, but have not yet had time to identify to species. The new species in the present study also await formal description. In addition to sponges, other groups of organisms including corals and algae were observed. Surveys should also be undertaken to discover potentially similar systems in the Philippines and Japan. In the Mediterranean, caves support a large part of total poriferan diversity (Gerovasileiou and Voultsiadou 2012), but remain understudied elsewhere. With respect to the prokaryotic communities, we identified marked enrichment of all cave sponge species with Dehalococcoidia (mainly SAR202) members. The implications hereof for nutrient dynamics across the land-sea interface warrant further study. Finally, it should be noted that although in apparent good condition that the pool-cave-canyon systems receive no formal protection. We, however, observed frequent visits by groups of tourists. Perhaps, more preoccupying, is the matter of the main pool-cave-canyon systems being located next to a nuclear waste storage facility.