Genomics of Terpene Biosynthesis in Dictyoceratid Sponges (Porifera) - What Do We (Not) Know?

Sponges are recognized as promising sources for novel bioactive metabolites. Among them are terpenoid metabolites that constitute key biochemical defense mechanisms in several sponge taxa. Despite their significance, the genetic basis for terpenoid biosynthesis in sponges remains poorly understood. Dictyoceratida comprise demosponges well-known for their bioactive terpenoids. In this study, we explored the currently available genomic data for insights into the metabolic pathways of dictyoceratid terpenoids. We first identified prenyltransferase (PT) and terpene cyclase (TC) enzymes essential for the terpenoid biosynthetic processes in the terrestrial realm by analyzing available transcriptomic and genomic data of Dictyoceratida sponges and 10 other sponge species. All Dictyoceratida sponges displayed various PTs involved in either sesqui- or diterpene, steroid and carotenoid production. Additionally, it was possible to identify a potential candidate for a dictyoceratid sesterterpene PT. However, analogs of common terrestrial TCs were absent, suggesting the existence of a distinct or convergently evolved sponge-specific TC. Our study aims to contribute to the foundational understanding of terpene biosynthesis in sponges, unveiling the currently evident genetic components for terpenoid production in species not previously studied. Simultaneously, it aims to identify the known and unknown factors, as a starting point for biochemical and genetic investigations in sponge terpenoid production.

A Soft Spot for Chemistry-Current Taxonomic and Evolutionary Implications of Sponge Secondary Metabolite Distribution.

Marine sponges are the most prolific marine sources for discovery of novel bioactive compounds. Sponge secondary metabolites are sought-after for their potential in pharmaceutical applications, and in the past, they were also used as taxonomic markers alongside the difficult and homoplasy-prone sponge morphology for species delineation (chemotaxonomy). The understanding of phylogenetic distribution and distinctiveness of metabolites to sponge lineages is pivotal to reveal pathways and evolution of compound production in sponges. This benefits the discovery rate and yield of bioprospecting for novel marine natural products by identifying lineages with high potential of being new sources of valuable sponge compounds. In this review, we summarize the current biochemical data on sponges and compare the metabolite distribution against a sponge phylogeny. We assess compound specificity to lineages, potential convergences, and suitability as diagnostic phylogenetic markers. Our study finds compound distribution corroborating current (molecular) phylogenetic hypotheses, which include yet unaccepted polyphyly of several demosponge orders and families. Likewise, several compounds and compound groups display a high degree of lineage specificity, which suggests homologous biosynthetic pathways among their taxa, which identifies yet unstudied species of this lineage as promising bioprospecting targets.

Antibacterial scalarane from Doriprismatica stellata nudibranchs (Gastropoda, Nudibranchia), egg ribbons, and their dietary sponge Spongia cf. agaricina (Demospongiae, Dictyoceratida).

Investigations on the biochemical relationship between Doriprismatica stellata (Chromodorididae, Doridoidea) nudibranchs, their egg ribbons, and the associated dietary sponge Spongia cf. agaricina (Demospongiae, Porifera) led to the isolation of the structurally new scalarane-type sesterterpene 12-deacetoxy-4-demethyl-11,24-diacetoxy-3,4-methylenedeoxoscalarin, with an unprecedented position of the cyclopropane ring annelated to the ring A. Unlike other scalaranes, which are most often functionalized at C-12 of ring C, it bears two acetoxy groups at C-11 and C-24 instead. The compound was present in all three samples, supporting the dietary relationship between chromodorid nudibranchs of the genus Doriprismatica and scalarane-containing dictyoceratid sponges of the Spongiidae family. The results also indicate that D. stellata passes the scalarane metabolite on to its egg ribbons, most likely for protective purposes. The scalarane showed antibacterial activity against the Gram-positive bacteria Arthrobacter crystallopoietes (DSM 20117) and Bacillus megaterium (DSM 32).

Minimalist barcodes for sponges: a case study classifying African freshwater Spongillida.

African sponges, particularly freshwater sponges, are understudied relative to demosponges in most other geographical regions. Freshwater sponges (Spongillida) likely share a common ancestor; however, their evolutionary history, particularly during their radiation into endemic and allegedly cosmopolitan groups, is unclear. Freshwater sponges of at least 58 species of 17 genera and four families are described from Central and Eastern Africa, but the diversity is underestimated due to limited distinguishable morphological features. The discovery of additional cryptic species is very likely with the use of molecular techniques such as DNA barcoding. The Royal Museum of Central Africa (MRAC, Tervuren, Belgium) hosts one of the largest collections of (Central) African freshwater sponge type material. Type specimens in theory constitute ideal targets for molecular taxonomy; however, the success is frequently hampered by DNA degradation and deamination, which are a consequence of suboptimal preservation techniques. Therefore, we genotyped African demosponge holotype material of the MRAC with specific short primers suitable for degenerated tissue and compare the results with the current, morphology-based classification. Our results demonstrate the utility of minimalistic barcodes for identification of sponges, potentially enabling efficient identification of individuals in taxonomic or metabarcoding studies, and highlight inconsistencies in the current freshwater sponge classification.

Naturally Drug-Loaded Chitin: Isolation and Applications.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis. A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Naturally Prefabricated Marine Biomaterials: Isolation and Applications of Flat Chitinous 3D Scaffolds from Ianthella labyrinthus (Demospongiae: Verongiida).

Marine sponges remain representative of a unique source of renewable biological materials. The demosponges of the family Ianthellidae possess chitin-based skeletons with high biomimetic potential. These three-dimensional (3D) constructs can potentially be used in tissue engineering and regenerative medicine. In this study, we focus our attention, for the first time, on the marine sponge Ianthella labyrinthus Bergquist & Kelly-Borges, 1995 (Demospongiae: Verongida: Ianthellidae) as a novel potential source of naturally prestructured bandage-like 3D scaffolds which can be isolated simultaneously with biologically active bromotyrosines. Specifically, translucent and elastic flat chitinous scaffolds have been obtained after bromotyrosine extraction and chemical treatments of the sponge skeleton with alternate alkaline and acidic solutions. For the first time, cardiomyocytes differentiated from human induced pluripotent stem cells (iPSC-CMs) have been used to test the suitability of I. labyrinthus chitinous skeleton as ready-to-use scaffold for their cell culture. Results reveal a comparable attachment and growth on isolated chitin-skeleton, compared to scaffolds coated with extracellular matrix mimetic Geltrex®. Thus, the natural, unmodified I. labyrinthus cleaned sponge skeleton can be used to culture iPSC-CMs and 3D tissue engineering. In addition, I. labyrinthus chitin-based scaffolds demonstrate strong and efficient capability to absorb blood deep into the microtubes due to their excellent capillary effect. These findings are suggestive of the future development of new sponge chitin-based absorbable hemostats as alternatives to already well recognized cellulose-based fabrics.

Divergence times in demosponges (Porifera): first insights from new mitogenomes and the inclusion of fossils in a birth-death clock model.

BACKGROUND: Approximately 80% of all described extant sponge species belong to the class Demospongiae. Yet, despite their diversity and importance, accurate divergence times are still unknown for most demosponge clades. The estimation of demosponge divergence time is key to answering fundamental questions on the origin of Demospongiae, their diversification and historical biogeography. Molecular sequence data alone is not informative on an absolute time scale, and therefore needs to be "calibrated" with additional data such as fossils. Here, we calibrate the molecular data with the fossilized birth-death model, which compared to strict node dating, allows for the inclusion of young and old fossils in the analysis of divergence time. We use desma-bearing sponges, a diverse group of demosponges that form rigid skeletons and have a rich and continuous fossil record dating back to the Cambrian (~500 Ma), to date the demosponge radiation and constrain the timing of key evolutionary events, like the transition from marine to freshwater habitats. To infer a dated phylogeny of Demospongiae we assembled the mitochondrial genomes of six desma-bearing demosponges from reduced-representation genomic libraries. The total dataset included 33 complete demosponge mitochondrial genomes and 30 fossils. RESULTS: Our study supports a Neoproterozoic origin of Demospongiae. Novel age estimates for the split of freshwater and marine sponges dating back to the Carboniferous and the previously assumed recent (~18 Ma) diversification of freshwater sponges is supported. Moreover, we provide detailed age estimates for a possible diversification of Tetractinellidae (~315 Ma), the Astrophorina (~240 Ma), the Spirophorina (~120 Ma) and the family Corallistidae (~188 Ma) all of which are considered as key groups for dating the Demospongiae due to their extraordinary rich and continuous fossil history. CONCLUSION: This study provides novel insights into the evolution of Demospongiae. Observed discrepancies of our dated phylogeny with their putative first fossil appearance dates are discussed for selected sponge groups. For instance, a Carboniferous origin of the order Tetractinellida seems to be too late, compared to their first appearance in the fossil record in the Middle Cambrian. This would imply that Paleozoic spicule forms are not homologous to post-Paleozoic forms.

Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding.

BACKGROUND: Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns. RESULTS: For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. CONCLUSION: This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested.

Antiprotozoal Linear Furanosesterterpenoids from the Marine Sponge Ircinia oros.

Chemical investigation of the marine sponge Ircinia oros yielded four linear furanosesterterpenoids, including the known metabolites ircinin-1 (1) and ircinin-2 (2) and two new compounds, ircinialactam E (3) and ircinialactam F (4). Their chemical structures were elucidated by using a combination of [α]D, NMR, HRMS, and FT-IR spectroscopy. The absolute configuration of C-18 in compounds 1-3 was identified as R by electronic circular dichroism (ECD) spectroscopy coupled with time-dependent density functional theory calculations. Compounds 1-4 showed moderate leishmanicidal, trypanocidal, and antiplasmodial activities (IC50 values 28-130 µM). This is the second report of rare glycinyl lactam derivatives 3 and 4 from the genus Ircinia.

A mitochondrial intron in a verongid sponge.

We discovered for the first time a mitochondrial intron in a non-tetillid demosponge, which sheds new light on the interpretation of mitochondrial intron evolution among non-bilaterian animals and has consequences for phylogenetic and DNA barcoding studies. The newly discovered class 1 intron of Aplysinella rhax (Verongida) CO1 has an ORF for a putative LAGLIDADG-type and resembles other sponge and cnidarian mitochondrial introns. Our analysis of the Aplysinella rhax intron underlines that the patchy distribution of introns in sponges is caused by a combination of horizontal and vertical transmission. Further implications for CO1 phylogenetic and barcoding projects are discussed.

First record of a living species of the genus Janulum (Class Demospongiae) in the Southern Hemisphere.

A new species of the enigmatic sponge genus Janulum de Laubenfels, 1936 was discovered recently on the Louisville Seamount Chain, in International Waters to the east of New Zealand; two small specimens were found encrusting the interstices of the stony coral Solenosmilia variabilis Duncan at a depth of 1200-1600 m. Janulum imago sp. nov., is described and compared with the genus type J. spinispiculum (Carter, 1876) from the North Atlantic. Janulum was also recorded from the Late Eocene Oamaru Diatomite of southern New Zealand in 1892, but was misidentified as genus Plocamia Schmidt (Order Poecilosclerida Topsent, Family Microcionidae Carter). Fossil species Janulum princeps sp. nov. is also described herein and represents the first record of this North Atlantic-Arctic Ocean genus in the Southern Hemisphere. The validity of J. filholi (Topsent, 1890), the second and only other North Atlantic species currently assigned to Janulum, is considered in the context of J. spinispiculum and the new species J. imago sp. nov.

The genomes of the aquarium sponges Tethya wilhelma and Tethya minuta (Porifera: Demospongiae).

Sponges (Phylum Porifera) are aquatic sessile metazoans found worldwide in marine and freshwater environments. They are significant in the animal tree of life as one of the earliest-branching metazoan lineages and as filter feeders play crucial ecological roles, particularly in coral reefs, but are susceptible to the effects of climate change. In the face of the current biodiversity crisis, genomic data is crucial for species conservation efforts and predicting their evolutionary potential in response to environmental changes. However, there is a limited availability of culturable sponge species with annotated high-quality genomes to further comprehensive insights into animal evolution, function, and their response to the ongoing global change. Despite the publication of a few high-quality annotated sponge genomes, there remains a gap in resources for culturable sponge species. To address this gap, we provide high quality draft genomes of the two congeneric aquarium species Tethya wilhelma and Tethya minuta, small ball-shaped demosponges that are easily maintained long-term in ex situ culture. As such, they offer promising opportunities as laboratory models to contribute to advancing our understanding of sponge biology and provide valuable resources for studying animal evolution, function, and responses to environmental challenges.

A novel target-enriched multilocus assay for sponges (Porifera): Red Sea Haplosclerida (Demospongiae) as a test case.

With declining biodiversity worldwide, a better understanding of species diversity and their relationships is imperative for conservation and management efforts. Marine sponges are species-rich ecological key players on coral reefs, but their species diversity is still poorly understood. This is particularly true for the demosponge order Haplosclerida, whose systematic relationships are contentious due to the incongruencies between morphological and molecular phylogenetic hypotheses. The single gene markers applied in previous studies did not resolve these discrepancies. Hence, there is a high need for a genome-wide approach to derive a phylogenetically robust classification and understand this group's evolutionary relationships. To this end, we developed a target enrichment-based multilocus probe assay for the order Haplosclerida using transcriptomic data. This probe assay consists of 20,000 enrichment probes targeting 2956 ultraconserved elements in coding (i.e. exon) regions across the genome and was tested on 26 haplosclerid specimens from the Red Sea. Our target-enrichment approach correctly placed our samples in a well-supported phylogeny, in agreement with previous haplosclerid molecular phylogenies. Our results demonstrate the applicability of high-resolution genomic methods in a systematically complex marine invertebrate group and provide a promising approach for robust phylogenies of Haplosclerida. Subsequently, this will lead to biologically unambiguous taxonomic revisions, better interpretations of biological and ecological observations and new avenues for applied research, conservation and managing declining marine diversity.

Genetic data confirms the enigmatic demosponge Janulum as haplosclerid.

Historically, sponge classification is based on the interpretation of morphological characters, whose phylogenetic information content is frequently limited, subject to homoplasies, or prone to environmental plasticity (e.g., Chombard et al. 1998). Therefore, the currently accepted order-level classification of its largest class, Demospongiae, has been largely revised with molecular phylogenetic data (Morrow & Cárdenas 2015). Nevertheless, numerous sponge genera with ambiguous or provisoric phylogenetic placement still await definite classification.

Deep Water Polymastiidae (Porifera, Polymastiida) from the South West Pacific.

This study reports on some deep water sponges in the family Polymastiidae collected during the 2017 Abyss Cruise off the East Coast of Australia and the 2003 NORFANZ Expedition to the Lord Howe and Norfolk Ridges in the Tasman Sea, Southwest Pacific Ocean. Species of Radiella, Spinularia, Ridleia, Tentorium and Polymastia were collected from abyssal and bathyal depths. From these collections, seven new species were discovered: Radiella nidula sp. nov., Radiella pumix sp. nov., Radiella sclera sp. nov., Spinularia flagellata sp. nov., Tentorium labium sp. nov., Ridleia echidna sp. nov. and Polymastia norfanzii sp. nov. In addition, two species previously known are redescribed based on the new collections: viz. Polymastia zitteli and Polymastia invaginata.

Revision of the genus Fascaplysinopsis, the type species Fascaplysinopsis reticulata (Hentschel, 1912) (Porifera, Dictyoceratida, Thorectidae) and descriptions of two new genera and seven new species.

The present study examines the taxonomy of sponge specimens with unique chemistry collectively known as Fascaplysinopsis reticulata (Hentschel, 1912). Examination of Hentschels original species upon which the genus Fascaplysinopsis Bergquist, 1980 was based in conjunction with a comparison with recent Indo-west Pacific collections, using morphological and molecular analyses (ITS and 28S rDNA), revealed extensive variation. Fascaplysinopsis reticulata was found to be a species complex comprising the genus Fascaplysinopsis, as well as two new genera: Skolosachlys gen. nov. and Rubrafasciculus gen. nov. The new species of Fascaplysinopsis described are F. palauensis sp. nov., F. klobos sp. nov. and F. ronquinni sp. nov. The new species of Skolosachlys gen. nov. described herein are: S. enlutea sp. nov. and S. nidus sp. nov. The new species described of Rubrafasciculus gen. nov. includes: R. cerasus sp. nov. and R. fijiensis sp. nov..

Horny sponges and their affairs: on the phylogenetic relationships of keratose sponges.

The demosponge orders Dictyoceratida and Dendroceratida are historically assigned to the keratose (or "horny") sponges, which are mostly devoid of primary skeletal elements, but possess an elaborate skeleton of organic fibres instead. This paucity of complex mineral skeletal elements makes their unambiguous classification and phylogenetic reconstruction based on morphological features difficult. Here we present the most comprehensive molecular phylogeny to date for the Dendroceratida, Dictyoceratida, and also other sponge orders that largely lack a mineral skeleton or skeletal elements at all (i.e. Verongida, Halisarcida, Chondrosida), based on independent mitochondrial and nuclear markers. We used molecular data to validate the coherence of all recognised orders, families and subfamilies that are currently defined using morphological characteristics. We discussed the significance of morphological and chemotaxonomic characters for keratose sponges, and suggested adapted definitions for the classification of dendroceratid, dictyoceratid, and verongid higher taxa. Also, we found that chondrosid sponges are non-monophyletic with respect to Halisarcida. Verongida and Dendroceratida were monophyletic, however most of their classically recognised families were not recovered. This indicated that the current distinction between dendritic and mesh-like fibre skeletons is not significant at this level of classification. Dysideidae were found to be the sister-group to the remaining Dictyoceratida. Irciniidae formed a distinct clade, however Thorectidae and Spongiidae could not be separated with the molecular markers used. Finally, we are establishing the name Verongimorpha for the clade combining verongid, chondrosid and halisarcid taxa and readjust the content of its sister-clade Keratosa.

New antiplasmodial bromotyrosine derivatives from Suberea ianthelliformis Lendenfeld, 1888.

Four samples of Suberea ianthelliformis were investigated and furnished five new and 13 known brominated tyrosine-derived compounds. Two of the new compounds were identified as araplysillin N20-formamide and its N-oxide derivative. Three other new compounds, araplysillins IV, V, and VI, were isolated and identified as analogs of araplysillin II. Most of these compounds exhibit moderate inhibitory activities against chloroquine-resistant and -sensitive strains of Plasmodium falciparum, and were investigated for their PFTase inhibitory properties. The chemical content of the investigated sponges is correlated with their molecular phylogeny.

Insights into the evolution of freshwater sponges (Porifera: Demospongiae: Spongillina): Barcoding and phylogenetic data from Lake Tanganyika endemics indicate multiple invasions and unsettle existing taxonomy.

Sponges are a conspicuous element in many benthic habitats including in Africa's oldest, deepest lake, Lake Tanganyika. Despite their prevalence and pivotal ecological role as filter feeders, knowledge of the evolutionary history of sponges is in its infancy. Here, we provide the first molecular analysis targeting the evolution of sponges from Lake Tanganyika. Independent markers indicate the occurrence of several colonisation events which have shaped the current Tanganyikan lacustrine sponge biodiversity. This is in contrast to a range of previously studied organisms that have diversified within the lake from single lineages. Our tree reconstructions indicate the presence of two genera, Oncosclera and Eunapius, which are globally distributed. Therefore, we reject the hypothesis of monophyly for the sponges from Lake Tanganyika and challenge existing higher taxonomic structure for freshwater sponges.

Systematics of 'lithistid' tetractinellid demosponges from the Tropical Western Atlantic-implications for phylodiversity and bathymetric distribution.

BACKGROUND: Among all present demosponges, lithistids represent a polyphyletic group with exceptionally well-preserved fossils dating back to the Cambrian. Knowledge of their recent diversity, particularly in the Tropical Western Atlantic Ocean (TWA) where they are common in deep waters, is scarce making any comparison between present and past major 'lithistid' faunas difficult. In addition, the lack of sufficient molecular and morphological data hamper any predictions on phylogenetic relationships or phylodiversity from this region. The Harbor Branch Oceanographic Institute (HBOI, Fort Pierce, Florida) holds the largest collection of TWA lithistid sponges worldwide, however, the majority remain to be taxonomically identified and revised. PRINCIPAL FINDINGS: In this study we provide sequences of 249 lithistid demosponges using two independent molecular markers (28S rDNA (C1-D2) and cox1 mtDNA). In addition, a morphological documentation of 70 lithistid specimens is provided in the database of the Sponge Barcoding Project (SBP). This integrated dataset represents the largest and most comprehensive of the TWA lithistids to date. The phylogenetic diversity of 'lithistid' demosponges in the Bahamas and Jamaica are high in comparison to other TWA regions; Theonellidae and Corallistidae dominate the fauna, while Neopeltidae and Macandrewiidae are rare. A proposed tetractinellid suborder, one undescribed genus and several undescribed species are recognized and the Pacific 'lithistid' genera, Herengeria and Awhiowhio, are reported from the TWA for the first time. The higher-taxa relationships of desma-bearing tetractinellids are discussed and topics for revision suggested. CONCLUSION: This first integrative approach of TWA 'lithistid' demosponges contributes to a better understanding of their phylogenetic affinities, diversity and bathymetric distribution patterns within the TWA. As in the Pacific, the TWA 'lithistid' demosponges dominate deep-water habitats. Deeper taxonomic investigations will undoubtedly contribute to a better comparison between present major 'lithistid' faunas and their fossil record in the Mesozoic.