Circadian clock-controlled gene expression in co-cultured, mat-forming cyanobacteria.

Natural coastal microbial mat communities are multi-species assemblages that experience fluctuating environmental conditions and are shaped by resource competition as well as by cooperation. Laboratory studies rarely address the natural complexity of microbial communities but are usually limited to homogeneous mono-cultures of key species grown in liquid media. The mat-forming filamentous cyanobacteria Lyngbya aestuarii and Coleofasciculus chthonoplastes were cultured under different conditions to investigate the expression of circadian clock genes and genes that are under their control. The cyanobacteria were grown in liquid medium or on a solid substrate (glass beads) as mono- or as co-cultures under a light-dark regime and subsequently transferred to continuous light. TaqMan-probe based qPCR assays were used to quantify the expression of the circadian clock genes kaiA, kaiB, and kaiC, and of four genes that are under control of the circadian clock: psbA, nifH, ftsZ, and prx. Expression of kaiABC was influenced by co-culturing the cyanobacteria and whether grown in liquid media or on a solid substrate. Free-running (i.e. under continuous light) expression cycle of the circadian clock genes was observed in L. aestuarii but not in C. chthonoplastes. In the former organism, maximum expression of psbA and nifH occurred temporally separated and independent of the light regime, although the peak shifted in time when the culture was transferred to continuous illumination. Although functionally similar, both species of cyanobacteria displayed different 24-h transcriptional patterns in response to the experimental treatments, suggesting that their circadian clocks have adapted to different life strategies adopted by these mat-forming cyanobacteria.

Daily rhythmicity in coastal microbial mats.

Cyanobacteria are major primary producers in coastal microbial mats and provide biochemical energy, organic carbon, and bound nitrogen to the mat community through oxygenic photosynthesis and dinitrogen fixation. In order to anticipate the specific requirements to optimize their metabolism and growth during a day-and-night cycle, Cyanobacteria possess a unique molecular timing mechanism known as the circadian clock that is well-studied under laboratory conditions but little is known about its function in a natural complex community. Here, we investigated daily rhythmicity of gene expression in a coastal microbial mat community sampled at 6 time points during a 24-h period. In order to identify diel expressed genes, meta-transcriptome data was fitted to periodic functions. Out of 24,035 conserved gene transcript clusters, approximately 7% revealed a significant rhythmic expression pattern. These rhythmic genes were assigned to phototrophic micro-eukaryotes, Cyanobacteria but also to Proteobacteria and Bacteroidetes. Analysis of MG-RAST annotated genes and mRNA recruitment analysis of two cyanobacterial and three proteobacterial microbial mat members confirmed that homologs of the cyanobacterial circadian clock genes were also found in other bacterial members of the microbial mat community. These results suggest that various microbial mat members other than Cyanobacteria have their own molecular clock, which can be entrained by a cocktail of Zeitgebers such as light, temperature or metabolites from neighboring species. Hence, microbial mats can be compared to a complex organism consisting of multiple sub-systems that have to be entrained in a cooperative way such that the corpus functions optimally.

Time-calibrated molecular phylogeny of pteropods.

Pteropods are a widespread group of holoplanktonic gastropod molluscs and are uniquely suitable for study of long-term evolutionary processes in the open ocean because they are the only living metazoan plankton with a good fossil record. Pteropods have been proposed as bioindicators to monitor the impacts of ocean acidification and in consequence have attracted considerable research interest, however, a robust evolutionary framework for the group is still lacking. Here we reconstruct their phylogenetic relationships and examine the evolutionary history of pteropods based on combined analyses of Cytochrome Oxidase I, 28S, and 18S ribosomal rRNA sequences and a molecular clock calibrated using fossils and the estimated timing of the formation of the Isthmus of Panama. Euthecosomes with uncoiled shells were monophyletic with Creseis as the earliest diverging lineage, estimated at 41-38 million years ago (mya). The coiled euthecosomes (Limacina, Heliconoides, Thielea) were not monophyletic contrary to the accepted morphology-based taxonomy; however, due to their high rate heterogeneity no firm conclusions can be drawn. We found strong support for monophyly of most euthecosome genera, but Clio appeared as a polyphyletic group, and Diacavolinia grouped within Cavolinia, making the latter genus paraphyletic. The highest evolutionary rates were observed in Heliconoides inflatus and Limacina bulimoides for both 28S and 18S partitions. Using a fossil-calibrated phylogeny that sets the first occurrence of coiled euthecosomes at 79-66 mya, we estimate that uncoiled euthecosomes evolved 51-42 mya and that most extant uncoiled genera originated 40-15 mya. These findings are congruent with a molecular clock analysis using the Isthmus of Panama formation as an independent calibration. Although not all phylogenetic relationships could be resolved based on three molecular markers, this study provides a useful resource to study pteropod diversity and provides general insight into the processes that generate and maintain their diversity in the open ocean.

Lock, stock and two different barrels: comparing the genetic composition of morphotypes of the indo-pacific sponge Xestospongia testudinaria.

The giant barrel sponge Xestospongiatestudinaria is an ecologically important species that is widely distributed across the Indo-Pacific. Little is known, however, about the precise biogeographic distribution and the amount of morphological and genetic variation in this species. Here we provide the first detailed, fine-scaled (<200 km(2)) study of the morphological and genetic composition of X. testudinaria around Lembeh Island, Indonesia. Two mitochondrial (CO1 and ATP6 genes) and one nuclear (ATP synthase ß intron) DNA markers were used to assess genetic variation. We identified four distinct morphotypes of X. testudinaria around Lembeh Island. These morphotypes were genetically differentiated with both mitochondrial and nuclear markers. Our results indicate that giant barrel sponges around Lembeh Island, which were all morphologically identified as X. testudinaria, consist of at least two different lineages that appear to be reproductively isolated. The first lineage is represented by individuals with a digitate surface area, CO1 haplotype C5, and is most abundant around the harbor area of Bitung city. The second lineage is represented by individuals with a predominantly smooth surface area, CO1 haplotype C1 and can be found all around Lembeh Island, though to a lesser extent around the harbor of Bitung city. Our findings of two additional unique genetic lineages suggests the presence of an even broader species complex possibly containing more than two reproductively isolated species. The existence of X. testudinaria as a species complex is a surprising result given the size, abundance and conspicuousness of the sponge.

The effects of nutrient enrichment and herbivore abundance on the ability of turf algae to overgrow coral in the Caribbean.

Turf algae are multispecies communities of small marine macrophytes that are becoming a dominant component of coral reef communities around the world. To assess the impact of turf algae on corals, we investigated the effects of increased nutrients (eutrophication) on the interaction between the Caribbean coral Montastraea annularis and turf algae at their growth boundary. We also assessed whether herbivores are capable of reducing the abundance of turf algae at coral-algae boundaries. We found that turf algae cause visible (overgrowth) and invisible negative effects (reduced fitness) on neighbouring corals. Corals can overgrow neighbouring turf algae very slowly (at a rate of 0.12 mm 3 wk(-1)) at ambient nutrient concentrations, but turf algae overgrew corals (at a rate of 0.34 mm 3 wk(-1)) when nutrients were experimentally increased. Exclusion of herbivores had no measurable effect on the rate turf algae overgrew corals. We also used PAM fluorometry (a common approach for measuring of a colony's "fitness") to detect the effects of turf algae on the photophysiology of neighboring corals. Turf algae always reduced the effective photochemical efficiency of neighbouring corals, regardless of nutrient and/or herbivore conditions. The findings that herbivores are not capable of controlling the abundance of turf algae and that nutrient enrichment gives turf algae an overall competitive advantage over corals together have serious implications for the health of Caribbean coral reef systems. At ambient nutrient levels, traditional conservation measures aimed at reversing coral-to-algae phase shifts by reducing algal abundance (i.e., increasing herbivore populations by establishing Marine Protected Areas or tightening fishing regulations) will not necessarily reduce the negative impact of turf algae on local coral communities. Because turf algae have become the most abundant benthic group on Curaçao (and likely elsewhere in the Caribbean), new conservation strategies are required to mitigate their negative impact on coral communities.