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Quantitative gene expression analysis is an important tool in the scientist's belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter's unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage., Competing Interests: The authors have declared that no competing interests exist.

Closely related taxa provide significant case studies for understanding evolution of new species but may simultaneously challenge species identification and definition. In the Baltic Sea, two dominant and perennial brown algae share a very recent ancestry. Fucus vesiculosus invaded this recently formed postglacial sea 8000 years ago and shortly thereafter Fucus radicans diverged from this lineage as an endemic species. In the Baltic Sea both species reproduce sexually but also recruit fully fertile new individuals by asexual fragmentation. Earlier studies have shown local differences in morphology and genetics between the two taxa in the northern and western Bothnian Sea, and around the island of Saaremaa in Estonia, but geographic patterns seem in conflict with a single origin of F. radicans. To investigate the relationship between northern and Estonian distributions, we analysed the genetic variation using 9 microsatellite loci in populations from eastern Bothnian Sea, Archipelago Sea and the Gulf of Finland. These populations are located in between earlier studied populations. However, instead of bridging the disparate genetic gap between N-W Bothnian Sea and Estonia, as expected from a simple isolation-by-distance model, the new populations substantially increased overall genetic diversity and showed to be strongly divergent from the two earlier analysed regions, showing signs of additional distinct populations. Contrasting earlier findings of increased asexual recruitment in low salinity in the Bothnian Sea, we found high levels of sexual reproduction in some of the Gulf of Finland populations that inhabit extremely low salinity. The new data generated in this study supports the earlier conclusion of two reproductively isolated but very closely related species. However, the new results also add considerable genetic and morphological complexity within species. This makes species separation at geographic scales more demanding and suggests a need for more comprehensive approaches to further disentangle the intriguing relationship and history of the Baltic Sea fucoids.

Northern hemisphere rockweeds (Fucus) are thought to have evolved in the North Pacific and then spread to the North Atlantic following the opening of the Bering Strait. They have dispersed and widely speciated in the North Atlantic and its tributary seas. Fucus distichus is likely near the ancestral member of this genus, and studies have shown that there are several species/subspecies in this complex (i.e. F. evanescens and F. gardneri). We used phylogenetic and haplotype analyses to test the phylogenetic relationships and biogeography of F. distichus. Our data and subsequent analyses demonstrate that, unlike previous studies that lacked samples from an extensive geographical area of the Arctic and Subarctic, there is a distinct Arctic haplotype that is the source of subspecies in both the North Pacific and North Atlantic. Fucus distichus occupies a low tide zone habitat, and in Arctic/Subarctic regions it is adapted to the severe stress of sea ice coverage and disturbance during many months per year. We hypothesize that the very large geographic area of Arctic and Subarctic rocky shores available to this species during interglacials, supported by large Arctic/Subarctic fringe areas as well as unglaciated refugia during glacial cycles, provided a robust population and gene pool (described by the Thermogeographic Model). This gene pool dilutes that of the more fragmented and area-limited Temperate/Boreal area populations when they are brought together during glacial cycles. We suggest that similar subspecies complexes for a variety of Arctic/Subarctic shore biota should be examined further in this context, rather than arbitrarily being split up into numerous species.

A role as UV sunscreens has been suggested for phlorotannins, the phenolic compounds that accumulate in brown algae in response to a number of external stimuli and take part in cell wall structure. After exposure of the intertidal brown alga Fucus vesiculosus to artificial UV-B radiation, we examined its physiological responses by following the transcript level of the pksIII gene encoding a phloroglucinol synthase, likely to be involved in the first step of phlorotannins biosynthesis. We also monitored the expression of three targeted genes, encoding a heat shock protein (hsp70), which is involved in global stress responses, an aryl sulfotransferase (ast), which could be involved in the sulfation of phlorotannins, and a vanadium bromoperoxidase (vbpo), which can potentially participate in the scavenging of Reactive Oxygen Species (ROS) and in the cross-linking and condensation of phlorotannins. We investigated whether transcriptional regulation of these genes is correlated with an induction of phlorotannin accumulation by establishing metabolite profiling of purified fractions of low molecular weight phlorotannins. Our findings demonstrated that a high dose of UV-B radiation induced a significant overexpression of hsp70 after 12 and 24 hours following the exposure to the UV-B treatment, compared to control treatment. The physiological performance of algae quantified by the photosynthetic efficiency (Fv/Fm) was slightly reduced. However UV-B treatment did not induce the accumulation of soluble phlorotannins in F. vesiculosus during the kinetics of four weeks, a result that may be related to the lack of induction of the pksIII gene expression. Taken together these results suggest a constitutive accumulation of phlorotannins occurring during the development of F.vesiculosus, rather than inducible processes. Gene expression studies and phlorotannin profiling provide here complementary approaches to global quantifications currently used in studies of phenolic compounds in brown algae.

Sympatric assemblages of congeners with incomplete reproductive barriers offer the opportunity to study the roles that ecological and non-ecological factors play in reproductive isolation. While interspecific asynchrony in gamete release and gametic incompatibility are known prezygotic barriers to hybridization, the role of mating system variation has been emphasized in plants. Reproductive isolation between the sibling brown algal species Fucus spiralis, Fucus guiryi (selfing hermaphrodite) and Fucus vesiculosus (dioecious) was studied because they form hybrids in parapatry in the rocky intertidal zone, maintain species integrity over a broad geographic range, and have contrasting mating systems. We compared reproductive synchrony (spawning overlap) between the three species at several temporal scales (yearly/seasonal, semilunar/tidal, and hourly during single tides). Interspecific patterns of egg release were coincident at seasonal (single peak in spring to early summer) to semilunar timescales. Synthesis of available data indicated that spawning is controlled by semidiurnal tidal and daily light-dark cues, and not directly by semilunar cycles. Importantly, interspecific shifts in timing detected at the hourly scale during single tides were consistent with a partial ecological prezygotic hybridization barrier. The species displayed patterns of gamete release consistent with a power law distribution, indicating a high degree of reproductive synchrony, while the hypothesis of weaker selective constraints for synchrony in selfing versus outcrossing species was supported by observed spawning in hermaphrodites over a broader range of tidal phase than in outcrossers. Synchronous gamete release is critical to the success of external fertilization, while high-energy intertidal environments may offer only limited windows of reproductive opportunity. Within these windows, however, subtle variations in reproductive timing have evolved with the potential to form ecological barriers to hybridization.

Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency. Congeneric, externally-fertilizing fucoid brown algae occur as distinct morphotypes along intertidal exposure gradients despite gene flow. Combining analyses of genetic and phenotypic traits, we investigate the potential for physiological resilience to emersion stressors to act as an isolating mechanism in the face of gene flow. Along vertical exposure gradients in the intertidal zone of Northern Portugal and Northwest France, the mid-low shore species Fucus vesiculosus, the upper shore species Fucus spiralis, and an intermediate distinctive morphotype of F. spiralis var. platycarpus were morphologically characterized. Two diagnostic microsatellite loci recovered 3 genetic clusters consistent with prior morphological assignment. Phylogenetic analysis based on single nucleotide polymorphisms in 14 protein coding regions unambiguously resolved 3 clades; sympatric F. vesiculosus, F. spiralis, and the allopatric (in southern Iberia) population of F. spiralis var. platycarpus. In contrast, the sympatric F. spiralis var. platycarpus (from Northern Portugal) was distributed across the 3 clades, strongly suggesting hybridization/introgression with both other entities. Common garden experiments showed that physiological resilience following exposure to desiccation/heat stress differed significantly between the 3 sympatric genetic taxa; consistent with their respective vertical distribution on steep environmental clines in exposure time. Phylogenetic analyses indicate that F. spiralis var. platycarpus is a distinct entity in allopatry, but that extensive gene flow occurs with both higher and lower shore species in sympatry. Experimental results suggest that strong selection on physiological traits across steep intertidal exposure gradients acts to maintain the 3 distinct genetic and morphological taxa within their preferred vertical distribution ranges. On the strength of distributional, genetic, physiological and morphological differences, we propose elevation of F. spiralis var. platycarpus from variety to species level, as F. guiryi.