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Sponges are among the earliest branching extant animals. As such, genetic data from this group are valuable for understanding the evolution of various traits and processes in other animals. However, like many marine organisms, they are notoriously difficult to sequence, and hence, genomic data are scarce. Here, we present the draft genome assembly for the North Atlantic deep-sea high microbial abundance species Geodia barretti Bowerbank 1858, from a single individual collected on the West Coast of Sweden. The nuclear genome assembly has 4,535 scaffolds, an N50 of 48,447 bp and a total length of 144 Mb; the mitochondrial genome is 17,996 bp long. BUSCO completeness was 71.5%. The genome was annotated using a combination of ab initio and evidence-based methods finding 31,884 protein-coding genes.

Sponge microbiomes contribute to host health, nutrition, and defense through the production of secondary metabolites. Chlamydiae, a phylum of obligate intracellular bacteria ranging from animal pathogens to endosymbionts of microbial eukaryotes, are frequently found associated with sponges. However, sponge-associated chlamydial diversity has not yet been investigated at the genomic level and host interactions thus far remain unexplored. Here, we sequenced the microbiomes of three sponge species and found high, though variable, Chlamydiae relative abundances of up to 18.7% of bacteria. Using genome-resolved metagenomics 18 high-quality sponge-associated chlamydial genomes were reconstructed, covering four chlamydial families. Among these, Candidatus Sororchlamydiaceae shares a common ancestor with Chlamydiaceae animal pathogens, suggesting long-term co-evolution with animals. Based on gene content, sponge-associated chlamydiae resemble members from the same family more than sponge-associated chlamydiae of other families, and have greater metabolic versatility than known chlamydial animal pathogens. Sponge-associated chlamydiae are also enriched in genes for degrading diverse compounds found in sponges. Unexpectedly, we identified widespread genetic potential for secondary metabolite biosynthesis across Chlamydiae, which may represent an unexplored source of novel natural products. This finding suggests that Chlamydiae members may partake in defensive symbioses and that secondary metabolites play a wider role in mediating intracellular interactions. Furthermore, sponge-associated chlamydiae relatives were found in other marine invertebrates, pointing towards wider impacts of the Chlamydiae phylum on marine ecosystems.

Sponges are among the earliest diverging taxa in the animal tree of life. They are sessile, filter-feeding animals found in marine and freshwater habitats. Many species are characterized by a close, specific and consistent association with microbes, mainly Bacteria and Archaea. This feature has been known for a long time and is suggested to be a factor contributing to the rich and diverse chemical output of the sponges. This thesis explored the effect of the habitat, specifically water mass or depth on sponges, their associated microbes, and their combined chemical output. The focal species of this thesis was the North Atlantic deep-sea high microbial abundance (HMA) demosponge Geodia barretti.

In Paper I, 16S rRNA gene amplicon sequencing and untargeted metabolomics were used to quantify variation in prokaryotic community composition and chemical output in three sponge species. Water masses structured the prokaryotic community composition in the HMA species G. barretti and Stryphnus fortis. The community composition of the low microbial abundance (LMA) sponge Weberella bursa was unaffected by depth. Untargeted metabolomic data was modelled by depth. This allowed for identification of individual compounds varying with depth. Among those compounds were many putative osmolytes as well as diketopiperazines. Bioactive peptides and brominated tryptophan derivatives were unaffected by depth.

In Paper II the diversity of the barrettide peptide family was explored in DNA sequencing data and chemical profiles across a wide selection of sponge species and G. barretti in particular. Five new barrettides were predicted and one sequence, barrettide C, was confirmed by solid phase peptide synthesis and co-elution with a native extract, antifouling bioassays and NMR structure elucidation. The confidence gained from sequence analysis and validating predictions lead us to suggest barrettides are a family of antifouling peptides in G. barretti.

In Paper III, a reduced representation sequencing approach was used to evaluate the Stacks de novo pipeline in HMA sponges with the help of a whole genome assembled for this purpose. With this data, gene flow and connectivity were investigated in G. barretti populations sampled across the North Atlantic. The de novo pipeline was found to assemble and retain many putatively microbial loci and should thus only be used with reservations in HMA sponges. However, regarding biological inferences, strong population structure was recovered despite the apparent contamination.

Marine sponges (phylum Porifera) are leading organisms for the discovery of bioactive compounds from nature. Their often rich and species-specific microbiota is hypothesised to be producing many of these compounds. Yet, environmental influences on the sponge-associated microbiota and bioactive compound production remain elusive. Here, we investigated the changes of microbiota and metabolomes in sponges along a depth range of 1232 m. Using 16S rRNA gene amplicon sequencing and untargeted metabolomics, we assessed prokaryotic and chemical diversities in three deep-sea sponge species: Geodia barretti, Stryphnus fortis, and Weberella bursa. Both prokaryotic communities and metabolome varied significantly with depth, which we hypothesized to be the effect of different water masses. Up to 35.5 % of microbial ASVs (amplicon sequence variants) showed significant changes with depth while phylum-level composition of host microbiome remained unchanged. The metabolome varied with depth, with relative quantities of known bioactive compounds increasing or decreasing strongly. Other metabolites varying with depth were compatible solutes regulating osmolarity of the cells. Correlations between prokaryotic community and the bioactive compounds in G. barretti suggested members of Acidobacteria, Proteobacteria, Chloroflexi, or an unclassified prokaryote as potential producers.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC₅₀ 0.64 μM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC–MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC–MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 μg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.

The tropical ginger genus Amomum (Zingiberaceae) has always posed challenges for classification based on morphological characters. Previous molecular phylogenetic studies showed Amomum to be paraphyletic but limited sampling and absence of the data of the type Amomum subulatum made it impossible to resolve the paraphyly and make nomenclatural changes. Here, Amomum is further investigated in a multi-marker phylogenetic framework using matK and nrITS including multiple accessions of the type, the genus Elettaria and additional accessions of Amomum, Alpinia, Elettariopsis, Geocharis, Geostachys and Hornstedtia. Amomum is shown to consist of nine clades and Alpinia of six. The genera Elettaria, Elettariopsis, Plagiostachys, and species in Hornstedtia are nested within these clades. Morphological studies of species previously subsumed in Amomum support recognition of new genera that correspond to well-delimited clades in the phylogenetic framework presented here. Recircumscription of the paraphyletic genus Amomum facilitates identification and creates nomenclatural stability. Three genera, Conamomum, Meistera and Wurfbainia, are resurrected, and three new genera Epiamomum, Lanxangia and Sundamomum are described, together with a key to the genera and a nomenclatural synopsis placing 384 specific names (incl. all synonyms) into the new generic framework. Of these 129 represent new combinations and 3 are replacement names. Types of Geocharis and Geostachys are designated. Further studies and specific sampling will be needed to resolve other branches of Alpinioideae containing other polyphyletic genera.

AimThe Cucurbitaceae genus Trichosanthes is widespread in Asia and Australia, and previous studies have shown that the genus originated in Asia, and that three independent lineages dispersed through the Sunda archipelago to Australasia. The timing and routes of these three dispersals, as well as the dispersal of two widespread species found in Australia and New Guinea, were investigated. LocationSunda-Sahul dispersals with a focus on New Guinea and Australia. MethodsA combined dataset of nuclear ribosomal (ITS1-5.8S-ITS2) and plastid DNA (matK, ndhF, rpl20-rps12, rps16) was used for maximum likelihood and Bayesian phylogenetic analysis to infer the relationships of the studied taxa. A fossil-calibrated molecular dating was used to time the dispersal events, and a biogeographical analysis was used to study the origin and dispersal of the genus. ResultsThe two widespread species, T. pilosa and T. cucumerina, form monophyletic groups in Australia, suggesting single dispersals. Molecular dating analysis dates four of the dispersal events to the Miocene, and two to the Oligo-Miocene boundary and the initial Sahul shelf collision with the Philippine plate. Most known Sahul-Philippine dispersals concern species that migrated from the Sahul shelf, whereas dispersals south are fewer. Southward Miocene dispersals include species that were present on the Sunda shelf before the tectonic formation of the Makassar Straits. All lineages that dispersed to Australasia have undergone extensive diversification following dispersal. Lineages adapted to wet tropical climates have speciated mostly in New Guinea, and lineages adapted to monsoon tropical climates have speciated mostly in northern Australia. Main conclusionsDispersals in Trichosanthes pre-date human colonization of Australasia, suggesting natural long-distance dispersal and establishment of all lineages. Diversification within lineages found in both Australia and New Guinea is limited, corroborating frequent land connections between these areas during the Pleistocene. Sunda-Sahul dispersals are likely to have been more common through time than previously suggested, especially in genera with floating fruit such as Trichosanthes.

Reduced representation sequencing appraches such as ddRADseq allow to assess population connectivity and infer population summary statistics, both with and without a reference genome. However, as ddRADseq employs total DNA indiscriminate of the origin, the method warrants validation prior to application in microbial rich systems. One example of a complex system are sponges such as the North Atlantic high microbial abundance sponge Geodia barretti. This species is known to maintain large, putatively disjoint populations across the deep-sea, but its dispersal capabilities remain unclear as larvae have never been observed. To study the effect of microbial contamination on data processing and population genetic inference in ddRADseq, we produced a reference genome of G. barretti and collected 163 individuals across its habitat range and bathymetry (35–1560 m) in the North Atlantic. We processed the data with Stacks2 both with and without a reference genome (de novo and hybrid/‘reference-integrated’ approach). We found that strong population structures are recovered by both approaches and across different population genetic analyses (fastStructure, PCA, F_ST). Compared to previous work using microsatellites in shallow populations, we found only very weak population structure across large geographic stretches (>1000 km). However, over a third  (34%) of the final loci produced by the de novo pipeline did not map to the reference genome indicating that these might be of microbial origin. For comparably complex systems this means that de novo RRS genotyping approaches may contain a considerable amount of off-target loci potentially biasing the results.