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In many freshwater habitats, green algae form intracellular symbioses with a variety of heterotrophic host taxa including several species of freshwater sponge. These sponges perform important ecological roles in their habitats, and the poriferan:green algae partnerships offers unique opportunities to study the evolutionary origins and ecological persistence of endosymbioses. We examined the association between Ephydatia muelleri and its chlorophyte partner to identify features of host cellular and genetic responses to the presence of intracellular algal partners. Chlorella-like green algal symbionts were isolated from field-collected adult E. muelleri tissue harboring algae. The sponge-derived algae were successfully cultured and subsequently used to reinfect aposymbiotic E. muelleri tissue. We used confocal microscopy to follow the fate of the sponge-derived algae after inoculating algae-free E. muelleri grown from gemmules to show temporal patterns of symbiont location within host tissue. We also infected aposymbiotic E. muelleri with sponge-derived algae, and performed RNASeq to study differential expression patterns in the host relative to symbiotic states. We compare and contrast our findings with work in other systems (e.g., endosymbiotic Hydra) to explore possible conserved evolutionary pathways that may lead to stable mutualistic endosymbioses. Our work demonstrates that freshwater sponges offer many tractable qualities to study features of intracellular occupancy and thus meet criteria desired for a model system.

Intracellular habitats have been invaded by a remarkable diversity of organisms, and strategies employed to successfully reside in another species’ cellular space are varied. Common selective pressures may be experienced in symbioses involving phototrophic symbionts and heterotrophic hosts. Here I refine and elaborate the Arrested Phagosome Hypothesis that proposes a mechanism that phototrophs use to gain access to their host’s intracellular habitat. I employ the economic concept of production possibility frontiers (PPF) as a useful heuristic to clearly define the trade-offs that an intracellular phototroph is likely to face as it allocates photosynthetically-derived pools of energy. Fixed carbon can fuel basic metabolism/respiration, it can support mitotic division, or it can be translocated to the host. Excess photosynthate can be stored for future use. Thus, gross photosynthetic productivity can be divided among these four general categories, and natural selection will favor phenotypes that best match the demands presented to the symbiont by the host cellular habitat. The PPF highlights trade-offs that exist between investment in growth (i.e., mitosis) or residency (i.e., translocating material to the host). Insights gained from this perspective might help explain phenomena such as coral bleaching because deficits in photosynthetic production are likely to diminish a symbiont’s ability to afford the costs of intracellular residency. I highlight deficits in our current understanding of host:symbiont interactions at the molecular, genetic, and cellular level, and I also discuss how semantic differences among scientists working with different symbiont systems may diminish the rate of increase in our understanding of phototrophic-based associations. I argue that adopting interdisciplinary (in this case, inter-symbiont-system) perspectives will lead to advances in our general understanding of the phototrophic symbiont’s intracellular niche.

Canonical and non-canonical Wnt signaling, as well as the Pax/Six gene network, are involved in patterning the freshwater sponge aquiferous system. Using computational approaches to identify transcription factor binding motifs in a freshwater sponge genome, we located putative PaxB binding sites near a Secreted Frizzled Related Protein (SFRP) gene in Ephydatia muelleri. EmSFRP is expressed throughout development, but with highest levels in juvenile sponges. In situ hybridization and antibody staining show EmSFRP expression throughout the pinacoderm and choanoderm in a subpopulation of amoeboid cells that may be differentiating archeocytes. Knockdown of EmSFRP leads to ectopic oscula formation during development, suggesting that EmSFRP acts as an antagonist of Wnt signaling in E. muelleri. Our findings support a hypothesis that regulation of the Wnt pathway by the Pax/Six network as well as the role of Wnt signaling in body plan morphogenesis was established before sponges diverged from the rest of the metazoans.

Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges.We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosa(p), Myxospongiae(p), Spongillida(p), Haploscleromorpha(p) (the marine haplosclerids) and Democlavia(p). We found conflicting results concerning the relationships of Keratosa(p) and Myxospongiae(p) to the remaining demosponges, but our results strongly supported a clade of Haploscleromorpha(p)+Spongillida(p)+Democlavia(p). In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillida(p)) are sister to Haploscleromorpha(p) rather than part of Democlavia(p). Within Keratosa(p), we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiae(p), Chondrosida and Verongida were monophyletic. A well-supported clade within Democlavia(p), Tetractinellida(p), composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlavia(p). Within Tetractinellida(p), we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida.These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets.