The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea.

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

An evaluation of small-scale genetic diversity and the mating system in Zostera noltii on an intertidal sandflat in the Wadden Sea.

BACKGROUND AND AIMS: The dwarf eelgrass, Zostera noltii, is a predominant inhabitant of soft-bottom intertidal regions along the coasts of northern Europe. It is a monoecious, protogynous angiosperm in which the potential for self-fertilization and inbreeding are high, especially if clone sizes exceed pollen dispersal distances. The aim of the present study was to determine the relationship between mating system and clonal structure, examine the relative roles of geitonogamous selfing and biparental inbreeding, measure pollen availability (multiple paternities) and estimate pollen dispersal. METHODS: A 100-m(2) plot was established in a large, intertidal Z. noltii meadow on the island of Sylt in the German Wadden Sea. A total of 256 adult shoots was sampled: one from the centre of 100 fixed 1-m(2) quadrats (large scale resolution) and an additional 156 from within eight randomly selected 1-m(2) sub-quadrats (small-scale resolution). DNA was extracted from seeds and leaf tissue of all samples and genotyped with nine microsatellite loci. KEY RESULTS: Mating system analysis revealed high multilocus and single locus outcrossing rates. Average pollen dispersal distance was nearly the same as the average genet (clone) size. Multiple paternity was common and 20-30 % of mature seeds originated from matings within the plot. Among inbred seeds, most resulted from geitonogamy rather than biparental inbreeding. CONCLUSIONS: Moderate disturbances intrinsic to the intertidal habitat appear to facilitate seed recruitment by gap formation. Pollen dispersal distances are sufficient to maintain outcrossing and high clonal diversity.

Historical invasions of the intertidal zone of Atlantic North America associated with distinctive patterns of trade and emigration.

Early invasions of the North American shore occurred mainly via deposition of ballast rock, which effectively transported pieces of the intertidal zone across the Atlantic. From 1773-1861, >880 European ships entered Pictou Harbor, Nova Scotia, as a result of emigration and trade from Europe. The rockweed Fucus serratus (1868) and the snail Littorina littorea ( approximately 1840) were found in Pictou during this same period. With shipping records (a proxy for propagule pressure) to guide sampling, we used F. serratus as a model to examine the introductions because of its relatively low genetic diversity and dispersal capability. Microsatellite markers and assignment tests revealed 2 introductions of the rockweed into Nova Scotia: 1 from Galway (Ireland) to Pictou and the other from Greenock (Scotland) to western Cape Breton Island. To examine whether a high-diversity, high-dispersing species might have similar pathways of introduction, we analyzed L. littorea, using cytochrome b haplotypes. Eight of the 9 Pictou haplotypes were found in snails collected from Ireland and Scotland. Our results contribute to a broader understanding of marine communities, because these 2 conspicuous species are likely to be the tip of an "invasion iceberg" to the NW Atlantic from Great Britain and Ireland in the 19th Century.

Clonal architecture in an intertidal bed of the dwarf eelgrass Zostera noltii in the Northern Wadden Sea: persistence through extreme physical perturbation and the importance of a seed bank.

Genotypic structure and temporal dynamics of the dwarf seagrass, Zostera noltii, were studied in an intertidal meadow that has persisted since prior to 1936 near the Wadden Sea island of Sylt. Samples were collected from two 10 × 10 m plots separated by 250 m from May 2002 to June 2005 and from four 1 × 1 m plots from June 2003 to September 2004. All the samples were genotyped with nine microsatellite loci. No genotypes were shared between the plots separated by 250 m. Genetic diversity was higher in the Wadden Sea than in the other regions of its geographic range. The average clone size (genets) (SD) in the two plots was 1.38 (0.26) and 1.46 (0.4) m², respectively, with a range up to 9 m² and <20% persisted for >4 years. A high genetic and genotypic diversity was maintained by annual recruitment of seedlings despite a dramatic decrease in ramet density that coincided with the severe heat stress event of 2003. Fine-scale (1 m²) analysis suggested that extensive loss of seagrass cover precluded space competition among the genets, while a persistent seed bank prevented local extinction. Long-term persistence of Z. noltii meadows in the intertidal Wadden Sea was achieved by high genet turnover and frequent seedling recruitment from a seed bank, in contrast to the low diversity observed in large and long-living clones of Z. noltii and other seagrasses in subtidal habitats.

Characterization of single nucleotide polymorphism markers for eelgrass (Zostera marina).

We characterized 37 single nucleotide polymorphism (SNP) makers for eelgrass Zostera marina. SNP markers were developed using existing EST (expressed sequence tag)-libraries to locate polymorphic loci and develop primers from the functional expressed genes that are deposited in The ZOSTERA database (V1.2.1). SNP loci were genotyped using a single-base-extension approach which facilitated high-throughput genotyping with minimal optimization time. These markers show a wide range of variability among 25 eelgrass populations and will be useful for population genetic studies including evaluation of population structure, historical demography, and phylogeography. Potential applications include haplotype inference of physically linked SNPs and identification of genes under selection for temperature and desiccation stress.

Multiple paternity analysis in the thornback ray Raja clavata L.

Skates (Rajidae) are characterized by slow growth rate, low fecundity, and late maturity and are thus considered to be vulnerable to exploitation. Although understanding mating systems and behavior are important for long-term conservation and fisheries management, this aspect of life history is poorly understood in skates. Using 5 highly polymorphic microsatellite loci, we analyzed egg clutches collected from 4 female Raja clavata captured in the wild to test for multiple paternity. Using the reconstructed multilocus genotypes method to explain the progeny genotype array, we showed that all 4 clutches were sired by a minimum of 4-6 fathers and, thus, female thornback rays are polyandrous. Whether polyandry in R. clavata is natural or a consequence of overexploitation remains uncertain. This is the first report of multiple paternity in a rajiform species and any oviparous elasmobranch.

Convergent adaptation to a marginal habitat by homoploid hybrids and polyploid ecads in the seaweed genus Fucus.

Hybridization and polyploidy are two major sources of genetic variability that can lead to adaptation in new habitats. Most species of the brown algal genus Fucus are found along wave-swept rocky shores of the Northern Hemisphere, but some species have adapted to brackish and salt marsh habitats. Using five microsatellite loci and mtDNA RFLP, we characterize two populations of morphologically similar, muscoides-like Fucus inhabiting salt marshes in Iceland and Ireland. The Icelandic genotypes were consistent with Fucus vesiculosus x Fucus spiralis F1 hybrids with asymmetrical hybridization, whereas the Irish ones consisted primarily of polyploid F. vesiculosus.

Phylogeography and population structure of thornback rays (Raja clavata L., Rajidae).

The phylogeography of thornback rays (Raja clavata) was assessed from European waters, using five nuclear microsatellite loci and mitochondrial cytochome b sequences. Strong regional differentiation was found between the Mediterranean basin, the Azores and the European continental shelf. Allelic and haplotype diversities were high in Portuguese populations, consistent with the existence of a refugium along the Iberian Peninsula. Unexpectedly, high diversity was also found in the English Channel/North Sea area. The lowest genetic diversity was found in the Black Sea. Populations sampled from the Mediterranean, Adriatic and Black Seas were characterized by a single mitochondrial haplotype. This haplotype was also the most ancestral and widespread outside of the Mediterranean basin except for the Azores. Populations from the Azores were dominated by a second ancestral haplotype which was shared with British populations. Results from multidimensional scaling, amova and nested clade analysis indicate that British waters are a secondary contact zone recolonized from at least two refugia--one around the Iberian Peninsula and one possibly in the Azores. Links to a potential refugium known as the Hurd Deep, between Cornwall and Brittany, are discussed. Finally, a historical demographic analysis indicates that thornback ray populations started to expand between 580,000 and 362,000 years ago, which suggests that the Last Glacial Maximum (20,000 years ago) had mainly affected the distribution of populations rather than population size.

A mtDNA-based phylogeny of the brown algal genus Fucus (Heterokontophyta; Phaeophyta).

Species of Fucus are among the dominant seaweeds along Northern Hemisphere shores, but taxonomic designations often are confounded by significant intraspecific morphological variability. We analyzed intra- and inter-specific phylogenetic relationships within the genus (275 individuals representing 16 taxa) using two regions of the mitochondrion: a variable intergenic spacer and a conserved portion of the 23S subunit. Bayesian ML and MP analyses verified a shallow phylogeny with two major lineages (previously reported) and resolved some intra-lineage relationships. Significant species-level paraphyly/polyphyly was observed within lineages 1A and 2. Despite higher species richness in the North Atlantic, a North Pacific origin of the genus is supported by a gradient of decreasing haplotype and nucleotide diversities in F. distichus from the North Pacific to the East Atlantic.

Complete mitochondrial genomes of the three brown algae (Heterokonta: Phaeophyceae) Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis.

We report the complete mitochondrial sequences of three brown algae (Dictyota dichotoma, Fucus vesiculosus and Desmarestia viridis) belonging to three phaeophycean lineages. They have circular mapping organization and contain almost the same set of mitochondrial genes, despite their size differences (31,617, 36,392 and 39,049 bp, respectively). These include the genes for three rRNAs (23S, 16S and 5S), 25-26 tRNAs, 35 known mitochondrial proteins and 3-4 ORFs. This gene set complements two previously studied brown algal mtDNAs, Pylaiella littoralis and Laminaria digitata. Exceptions to the very similar overall organization include the displacement of orfs, tRNA genes and four protein-coding genes found at different locations in the D. dichotoma mitochondrial genome. We present a phylogenetic analysis based on ten concatenated genes (7,479 nucleotides) and 29 taxa. Stramenopiles were always monophyletic with heterotrophic species at the base. Results support both multiple primary and multiple secondary acquisitions of plastids.

Low effective population size and evidence for inbreeding in an overexploited flatfish, plaice (Pleuronectes platessa L.).

Overexploitation and subsequent collapses of major worldwide fisheries has made it clear that marine stocks are no inexhaustible. Unfortunately, the perception remains that marine fished are resilient to large population reductions, as even a commercially 'collapsed' stock will still consist of millions of individuals. Coupled with this notion is the idea that fisheries can, therefore, have little effect on the genetic diversity of stocks. We used DNA from archived otoliths collected between 1924 and 1972 together with 2002 juvenile;s tissue to estimate effective population size (Ne) in plaice (Pleuronrctes platessa). Ne was estimated at 20,000 in the North Sea and 2000 in Iceland. These values are five orders of magnitude smaller than the estimated census size foe the two locations. Populations examined between 1924 and 1960 were in Hardy-Weinberg equilibrium, whereas populations examined after 1970 were not. Extensive testing was performed to rule out genotyping artefacts and Wahlund effects. The significant heterozygote deficiencies found from 1970 onward were attributed to inbreeding. The emergence of inbreeding between 1905 and 19070 coincides with the increase in fishing mortality after World War II. Although the biological mechanisms remain speculative, our demonstration of inbreeding signals the need for understanding the social and mating behaviour in commercially important fishes.

North Atlantic phylogeography and large-scale population differentiation of the seagrass Zostera marina L.

As the most widespread seagrass in temperate waters of the Northern Hemisphere, Zostera marina provides a unique opportunity to investigate the extent to which the historical legacy of the last glacial maximum (LGM18 000-10 000 years bp) is detectable in modern population genetic structure. We used sequences from the nuclear rDNA-internal transcribed spacer (ITS) and chloroplast matK-intron, and nine microsatellite loci to survey 49 populations (> 2000 individuals) from throughout the species' range. Minimal sequence variation between Pacific and Atlantic populations combined with biogeographical groupings derived from the microsatellite data, suggest that the trans-Arctic connection is currently open. The east Pacific and west Atlantic are more connected than either is to the east Atlantic. Allelic richness was almost two-fold higher in the Pacific. Populations from putative Atlantic refugia now represent the southern edges of the distribution and are not genetically diverse. Unexpectedly, the highest allelic diversity was observed in the North Sea-Wadden Sea-southwest Baltic region. Except for the Mediterranean and Black Seas, significant isolation-by-distance was found from ~150 to 5000 km. A transition from weak to strong isolation-by-distance occurred at ~150 km among northern European populations suggesting this scale as the natural limit for dispersal within the metapopulation. Links between historical and contemporary processes are discussed in terms of the projected effects of climate change on coastal marine plants. The identification of a high genetic diversity hotspot in Northern Europe provides a basis for restoration decisions.

Heteroplasmy and evidence for recombination in the mitochondrial control region of the flatfish Platichthys flesus.

The general assumption that mitochondrial DNA (mtDNA) does not undergo recombination has been challenged recently in invertebrates. Here we present the first direct evidence for recombination in the mtDNA of a vertebrate, the flounder Platichthys flesus. The control region in the mtDNA of this flatfish is characterized by the presence of a variable number of tandem repeats and a high level of heteroplasmy. Two types of repeats were recognized, differing by two C-T point mutations. Most individuals carry a pure "C" or a pure "T" array, but one individual showed a compound "CT" array. Such a compound array is evidence for recombination in the mtDNA control region from the flounder.