Phylogeographic differentiation versus transcriptomic adaptation to warm temperatures in Zostera marina, a globally important seagrass.

Populations distributed across a broad thermal cline are instrumental in addressing adaptation to increasing temperatures under global warming. Using a space-for-time substitution design, we tested for parallel adaptation to warm temperatures along two independent thermal clines in Zostera marina, the most widely distributed seagrass in the temperate Northern Hemisphere. A North-South pair of populations was sampled along the European and North American coasts and exposed to a simulated heatwave in a common-garden mesocosm. Transcriptomic responses under control, heat stress and recovery were recorded in 99 RNAseq libraries with ~13 000 uniquely annotated, expressed genes. We corrected for phylogenetic differentiation among populations to discriminate neutral from adaptive differentiation. The two southern populations recovered faster from heat stress and showed parallel transcriptomic differentiation, as compared with northern populations. Among 2389 differentially expressed genes, 21 exceeded neutral expectations and were likely involved in parallel adaptation to warm temperatures. However, the strongest differentiation following phylogenetic correction was between the three Atlantic populations and the Mediterranean population with 128 of 4711 differentially expressed genes exceeding neutral expectations. Although adaptation to warm temperatures is expected to reduce sensitivity to heatwaves, the continued resistance of seagrass to further anthropogenic stresses may be impaired by heat-induced downregulation of genes related to photosynthesis, pathogen defence and stress tolerance.

Evolution and diversification within the intertidal brown macroalgae Fucus spiralis/F. vesiculosus species complex in the North Atlantic.

We examined 733 individuals of Fucusspiralis from 21 locations and 1093 Fucusvesiculosus individuals from 37 locations throughout their northern hemisphere ranges using nuclear and mitochondrial markers. Three genetic entities of F. spiralis were recovered. In northern and sympatric populations, the presence of "F. spiralis Low" in the mid-intertidal and "F. spiralis High" in the high-intertidal was confirmed and both co-occurred with the sister species F. vesiculosus. The third and newly-discovered entity, "F. spiralis South", was present mainly in the southern range, where it did not co-occur with F. vesiculosus. The South entity diverged early in allopatry, then hybridized with F. vesiculosus in sympatry to produce F. spiralis Low. Ongoing parallel evolution of F. spiralis Low and F. spiralis High is most likely due to habitat preference/local selection and maintained by preferentially selfing reproductive strategies. Contemporary populations of F. spiralis throughout the North Atlantic stem from a glacial refugium around Brittany involving F. spiralis High; F. spiralis South was probably unaffected by glacial episodes. Exponential population expansion for F. vesiculosus began during the Cromer and/Holstein interglacial period (300,000-200,000 yrs BP). Following the last glacial maximum (30,000-22,000 yrs BP), a single mtDNA haplotype from a glacial refugium in SW Ireland colonized Scandinavia, the Central Atlantic islands, and the W Atlantic.

Eelgrass meadows in the California Channel Islands and adjacent coast reveal a mosaic of two species, evidence for introgression and variable clonality.

BACKGROUND AND AIMS: Seagrasses are important facilitator species in shallow, soft-bottom marine environments worldwide and, in many places, are threatened by coastal development and eutrophication. One narrow-leaved species (Zostera marina) and one wide-leaved species, variously designated as Z. marina, Z. pacifica or Z. asiatica, are found off the California Channel Islands and adjacent California-Mexico coast. The aim of the present study was to confirm species identification genetically and to link patterns of genetic diversity, connectivity and hybridization among and within the populations with historical sea levels (Ice Age) or the contemporary environment. METHODS: Samples (n = 11-100) were collected from 28 sites off five California Channel Islands and six sites off the adjacent coast of southern California and Baja California, Mexico. DNA polymorphisms of the rDNA-ITS (internal transcribed spacer) cistron (nuclear), the matK intron (chloroplast) and nine microsatellite loci (nuclear) were examined in a population genetic and phylogeographic context. KEY RESULTS: All wide-leaved individuals were Z. pacifica, whereas narrow-leaved forms were Z. marina. Microsatellite genotypes were consistent with hybridization between the two species in three populations. The present distribution of Z. pacifica follows a glacial age land mass rather than present oceanographic regimes, but no link was observed between the present distribution of Z. marina and past or present environments. Island populations of Z. marina often were clonal and characterized by low genotypic diversity compared with populations along the Baja California coast. The high level of clonal connectivity around Santa Catalina Island indicated the importance of dispersal and subsequent re-establishment of vegetative fragments. CONCLUSIONS: The pristine environmental conditions of offshore islands do not guarantee maximum genetic diversity. Future restoration and transplantation efforts of seagrasses must recognize cryptic species and consider the degree of both genetic and genotypic variation in candidate donor populations.

Pre-zygotic isolation in the macroalgal genus Fucus from four contact zones spanning 100-10 000 years: a tale of reinforcement?

Hybrid zones provide an ideal natural experiment to study the selective forces driving evolution of reproductive barriers and speciation. If hybrid offspring are less fit than the parental species, pre-zygotic isolating barriers can evolve and strengthen in response to selection against the hybrids (reinforcement). Four contact zones between the intertidal macroalgae Fucus serratus (Fs) and Fucus distichus (Fd), characterized by varying times of sympatry and order of species introduction provide an opportunity to investigate reinforcement. We examined patterns of hybridization and reproductive isolation between Fs and Fd in: (i) northern Norway (consisting of two natural sites, 10 000 years old), (ii) the Kattegat near Denmark (Fd introduced, nineteenth century) and (iii) Iceland (Fs introduced, nineteenth century). Using 10 microsatellites and chloroplast DNA, we showed that hybridization and introgression decreased with increasing duration of sympatry. The two younger contact zones revealed 13 and 24% hybrids and several F 1 individuals, in contrast to the older contact zone with 2-3% hybrids and an absence of F 1s. Cross-fertilization experiments revealed that the reduction in hybridization in the oldest zone is consistent with increased gametic incompatibility.

Hybridization of the marine seaweeds, Fucus serratus and Fucus evanescens (Heterokontophyta: Phaeophyceae) in a 100-year-old zone of secondary contact.

Historically, the intertidal seaweeds Fucus serratus (Fs) and Fucus evanescens (Fe) were sympatric only along the western coast of Norway. In the mid-1890s, Fe (monoecious) was accidentally introduced into the Oslofjord. Putative hybridization with the endemic Fs (dioecious) was observed in Oslofjord by 1977 and in the Kattegat and western Baltic Seas by 1998. At Blushøj, Denmark (Kattegat Sea) putative Fs x Fe hybrids were present only when densities of Fe and Fs exceeded 14 and 2 m(-2), respectively. All of the 58 putative hybrids that were collected in 1999 were dioecious and intermediate in morphology. Essentially all (57 out of 58) were reproductively mature, but the oogonia possessed fewer and more variably sized eggs than either parent. Examination of each parental species and putative hybrids with nuclear, mitochondrial and chloroplast molecular markers confirmed the occurrence of hybridization. Furthermore, all of the hybrids possessed Fe-type chloroplasts and mitochondria, indicating that only the Fe egg x Fs sperm pairing was successful in the field. The reciprocal cross of Fs egg x Fe sperm was absent in the field and significantly less successful in laboratory crossings. Asymmetrical hybridization has also been reported for several species of plants and animals.

The foraging ecology of sympatric marine fish in the genus Embiotoca (Embiotocidae): Importance of foraging behavior in prey size selection.

Two locally sympatric temperate marine reef fishes, Embiotoca jacksoni and E. lateralis (Embiotocidae), have high taxonomic similarity in diets. Subdivision of gammarid amphipods, their principal prey, was found. E. jacksoni took more tubicolous gammarid amphipods whereas E. lateralis consumed mostly free-living individuals. The species differed considerably with respect to between-individual variability in taxonomic compositions of their diets. Each E. jacksoni closely resembled other conspecifics in this regard while individual E. lateralis displayed very high between-fish variation. The principal interspecific difference in fish diets concerned the sizes of prey items taken. E. jacksoni ate small but very common items and the mean prey weight in their guts did not differ from random collections of available prey. E. lateralis concentrated on large, rarer sizes such that the average prey weight in their guts was much heavier than available or in the diet of E. jacksoni of the same length. Disparate foraging behaviors was a much better indicator of the relative differences in diets of these two fishes than was external fish morphology. E. jacksoni, which can winnow prey items from unwanted debris, was a relatively indiscriminant forager. E. lateralis did not winnow but actively searched for prey. This species was a much more discriminating forager, but displayed much variability in foraging behavior.

Genomic scans detect signatures of selection along a salinity gradient in populations of the intertidal seaweed Fucus serratus on a 12 km scale.

Detecting natural selection in wild populations is a central challenge in evolutionary biology and genomic scans are an important means of detecting allele frequencies that deviate from neutral expectations among marker loci. We used nine anonymous and 15 EST-linked microsatellites, 362 AFLP loci, and several neutrality tests, to identify outlier loci when comparing four populations of the seaweed Fucus serratus spaced along a 12km intertidal shore with a steep salinity gradient. Under criteria of at least two significant tests in at least two population pairs, three EST-derived and three anonymous loci revealed putative signatures of selection. Anonymous locus FsB113 was a consistent outlier when comparing least saline to fully marine sites. Locus F37 was an outlier when comparing the least saline to more saline areas, and was annotated as a polyol transporter/putative mannitol transporter - an important sugar-alcohol associated with osmoregulation by brown algae. The remaining loci could not be annotated using six different data bases. Exclusion of microsatellite outlier loci did not change either the degree or direction of differentiation among populations. In one outlier test, the number of AFLP outlier loci increased as the salinity differences between population pairs increased (up to 14); only four outliers were detected with the second test and only one was consistent with both tests. Consistency may be improved with a much more rigorous approach to replication and/or may be dependent upon the class of marker used.

Expressed sequence tag-derived polymorphic SSR markers for Fucus serratus and amplification in other species of Fucus.

The seaweed genus Fucus is a dominant component of intertidal shores throughout the North Atlantic and North Pacific and has been the focus of considerable developmental, ecological, and evolutionary research for the past century. Here, we present details of 21 expressed sequence tag-derived simple sequence repeat markers (microsatellites). All 21 were polymorphic for F. serratus, which also display considerable cross-reactivity with the sister species F. distichus (18) and the more distantly related F. vesiculosus (13), and F. spiralis (5).

Glacial refugia and recolonization pathways in the brown seaweed Fucus serratus.

The last glacial maximum (20,000-18,000 years ago) dramatically affected extant distributions of virtually all northern European biota. Locations of refugia and postglacial recolonization pathways were examined in Fucus serratus (Heterokontophyta; Fucaceae) using a highly variable intergenic spacer developed from the complete mitochondrial genome of Fucus vesiculosus. Over 1,500 samples from the entire range of F. serratus were analysed using fluorescent single strand conformation polymorphism. A total of 28 mtDNA haplotypes was identified and sequenced. Three refugia were recognized based on high haplotype diversities and the presence of endemic haplotypes: southwest Ireland, the northern Brittany-Hurd Deep area of the English Channel, and the northwest Iberian Peninsula. The Irish refugium was the source for a recolonization sweep involving a single haplotype via northern Scotland and throughout Scandinavia, whereas recolonization from the Brittany-Hurd Deep refugium was more limited, probably because of unsuitable soft-bottom habitat in the Bay of Biscay and along the Belgian and Dutch coasts. The Iberian populations reflect a remnant refugium at the present-day southern boundary of the species range. A generalized skyline plot suggested exponential population expansion beginning in the mid-Pleistocene with maximal growth during the Eems interglacial 128,000-67,000 years ago, implying that the last glacial maximum mainly shaped population distributions rather than demography.

Hybridization and introgression in a mixed population of the intertidal seaweeds Fucus evanescens and F. serratus.

The introduced Fucus evanescens (hermaphroditic) and the native F. serratus (dioecious) have been in secondary contact along the Danish coast of the Kattegat Sea for 60-100 years and dioecious hybrids have been observed at Blushøj for several years. Hybridization in Fucus is unusual because it appears to always involve a hermaphroditic and a dioecious parental pair. We determined the degree and spatial patterns of introgression for 286 individuals using 10 microsatellite loci and cpDNA. Hybrids accounted for nearly 13% of the population, yet parental species were well differentiated (F(ST) = 0.633). The presence of F. evanescens chloroplasts in 100% of F(1) hybrids revealed asymmetrical hybridization. Fucus evanescens cpDNA was observed in 50% of introgressed and 5.4% of pure F. serratus, but no F. serratus cpDNA was found in F. evanescens. In contrast, nuclear DNA introgression was symmetrical with an equal amount ( approximately 1.5%) of genes introgressed into each parental species. Survivorship and viability data suggest selection against hybrids in the hybrid zone.

Geographically specific heteroplasmy of mitochondrial DNA in the seaweed, Fucus serratus (Heterokontophyta: Phaeophyceae, Fucales).

The presence of more than one type of mitochondrial DNA within the same organism (mtDNA heteroplasmy) has been reported in vertebrates, invertebrates, basidiomycetes and some angiosperms, but never in marine (macro)algae. We examined sequence differences in a 135-base pair (bp) region of the nad11 gene in mitochondria of the intertidal rockweed, Fucus serratus, using single-strand conformation polymorphism (SSCP). Each of 70 and 22 individuals from Blushøj (Denmark) and Oskarshamn (Sweden), respectively, displayed haplotypes 2, 3, and 4 (= mtDNA heteroplasmy), whereas only haplotype 2 was found in each of 24 individuals from locations in Spain, France, Ireland, Iceland and Norway. As Blushøj and Oskarshamn were among the last areas to emerge from ice cover during the Last Glacial Maximum (18000-20000 years BP), the geographically specific heteroplasmy may represent a founder effect and therefore, a valuable marker for understanding the role of post-Ice Age recolonization. Geographically specific heteroplasmy also has important implications in phylogeographical studies based on mtDNA sequences.

Post-ice age recolonization and differentiation of Fucus serratus L. (Phaeophyceae; Fucaceae) populations in Northern Europe.

The seaweed Fucus serratus is hypothesized to have evolved in the North Atlantic and present populations are thought to reflect recolonization from a southern refugium since the last glacial maximum 18 000-20 000 years bp. We examined genetic structure across several spatial scales by analysing seven microsatellite loci in populations collected from 21 localities throughout the species' range. Spatial auto-correlation analysis of seven microsatellite loci revealed no evidence for spatial clustering of alleles on a scale of 100 m despite limited gamete dispersal in F. serratus of approximately 2 m from parental individuals. Pairwise theta analysis suggested that the minimal panmictic unit for F. serratus was between 0.5 and 2 km. Isolation by distance was significant along some contiguous coastlines. Population differentiation was strong within the Skagerrak-Kattegat-Baltic Seas (SKB) (global theta= 0.17) despite a short history of approximately 7500 years. A neighbour-joining tree based on Reynold's distances computed from the microsatellite data revealed a central assemblage of populations on the Brittany Peninsula surrounded by four well-supported clusters consisting of the SKB, the North Sea (Ireland, Helgoland), and two populations from the northern Spanish coast. Samples from Iceland and Nova Scotia were most closely aligned with northwest Sweden and Brittany, respectively. When sample sizes were standardized (N = 41), allelic diversity was twofold higher for Brittany populations than for populations to the north and threefold higher than southern populations. The Brittany region may be a refugium or a recolonized area, whereas the Spanish populations most likely reflect present-day edge populations that have undergone repeated bottlenecks as a consequence of thermally induced cycles of recolonization and extinction.

N2 fixation in marine heterotrophic bacteria: dynamics of environmental and molecular regulation.

Molecular and immunological techniques were used to examine N2 fixation in a ubiquitous heterotrophic marine bacterium, the facultative anaerobic Vibrio natriegens. When batch cultures were shifted from aerobic N-replete to anaerobic N-deplete conditions, transcriptional and post-translational regulation of N2 fixation was observed. Levels of nifHDK mRNA encoding the nitrogenase enzyme were highest at 140 min postshift and undetectable between 6 and 9 h later. Immunologically determined levels of nitrogenase enzyme (Fe protein) were highest between 6 and 15 h postshift, and nitrogenase activity peaked between 6 and 9 h postshift, declining by a factor of 2 after 12-15 h. Unlike their regulation in cyanobacteria, Fe protein and nitrogenase activity were present when nifHDK mRNA was absent in V. natriegens, indicating that nitrogenase is stored and stable under anaerobic conditions. Both nifHDK mRNA and Fe protein disappeared within 40 min after cultures were shifted from N2-fixing conditions (anaerobic, N-deplete) to non- N2-fixing conditions (aerobic, N-enriched) but reappeared when shifted to conditions favoring N2 fixation. Thus, unlike other N2-fixing heterotrophic bacteria, nitrogenase must be resynthesized after aerobic exposure in V. natriegens. Immunological detection based on immunoblot (Western) analysis and immunogold labeling correlated positively with nitrogenase activity; no localization of nitrogenase was observed. Because V. natriegens continues to fix N2 for many hours after anaerobic induction, this species may play an important role in providing "new" nitrogen in marine ecosystems.