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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., (© 2016 John Wiley & Sons Ltd.)

The seagrass Posidonia oceanica is a key engineering species structuring coastal marine systems throughout much of the Mediterranean basin. Its decline is of concern, leading to the search for short- and long-term indicators of seagrass health. Using ArcGIS maps from a recent, high-resolution (1-4 km) modelling study of 18 disturbance factors affecting coastal marine systems across the Mediterranean (Micheli et al. 2013, http://globalmarine.nceas.ucsb.edu/mediterranean/), we tested for correlations with genetic diversity metrics (allelic diversity, genotypic/clonal diversity and heterozygosity) in a meta-analysis of 56 meadows. Contrary to initial predictions, weak but significantly positive correlations were found for commercial shipping, organic pollution (pesticides) and cumulative impact. This counterintuitive finding suggests greater resistance and resilience of individuals with higher genetic and genotypic diversity under disturbance (at least for a time) and/or increased sexual reproduction under an intermediate disturbance model. We interpret the absence of low and medium levels of genetic variation at impacted locations as probable local extinctions of individuals that already exceeded their resistance capacity. Alternatively, high diversity at high-impact sites is likely a temporal artefact, reflecting the mismatch with pre-environmental impact conditions, especially because flowering and sexual recruitment are seldom observed. While genetic diversity metrics are a valuable tool for restoration and mitigation, caution must be exercised in the interpretation of correlative patterns as found in this study, because the exceptional longevity of individuals creates a temporal mismatch that may falsely suggest good meadow health status, while gradual deterioration of allelic diversity might go unnoticed., (© 2015 John Wiley & Sons Ltd.)

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.

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.

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.

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.

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.

Independent lines of evidence support an Australian origin for the Mediterranean populations of the tropical alga Caulerpa taxifolia. To complement previous biogeographical studies based on nuclear rDNA internal transcribed spacer (ITS), a new chloroplast marker was developed--the cp 16S rDNA intron-2. Sequence variability for both nuclear and chloroplast markers were assessed in 110 individuals using single strand conformation polymorphism. Comparison of intrapopulation genetic diversity between invasive Mediterranean and 'native' Australian populations revealed the occurrence of two divergent and widespread clades. The first clade grouped nontropical invasive populations with inshore-mainland populations from Australia, while the second clustered all offshore-island populations studied so far. Despite our finding of nine distinct nuclear and five distinct chloroplast profiles, a single nucleocytoplasmic combination was characteristic of the invasive populations and sexual reproduction was found to be very rare. C. taxifolia is clearly a complex of genetically and ecologically differentiated sibling species or subspecies.

Philopatry to spawning grounds combined with well-known migratory patterns in the flatfish Pleuronectes platessa (plaice) has led to the hypothesis that regional populations may reflect relatively discrete, genetic stocks. Using six microsatellite loci we genotyped 240 adult individuals collected from locations in Norway, the Faeroe plateau, the Irish Sea, the Femer Baelt, Denmark, and the southern North Sea, and 240 0-class juveniles collected from five nursery-ground locations in Iceland, northwest Scotland, two sites in the Wadden Sea, and the Bay of Vilaine in Southern Brittany. The mean number of alleles/locus ranged from 5.3 to 20.4, with a mean of 13.9. Expected heterozygosity was uniformly high across all locations (multilocus H(exp)= 0.744 +/- 0.02). Pairwise comparisons of theta; among all 11 locations revealed significant differentiation between Iceland and all other locations (theta = 0.0290*** to 0.0456***), which is consistent with the deep-water barrier to dispersal in plaice. In contrast, no significant differentiation was found among any of the remaining continental-shelf sampling locations. This suggests that regional stocks are themselves composed of several genetic stocks under a model of panmixia which persists even to the spawning grounds. The presence of significant heterozygote deficiencies at all locations (not due to null alleles) suggests a temporal Wahlund effect yet the absence of significant population differentiation among continental shelf localities makes this explanation alone, difficult to reconcile. Sampling of eggs at the spawning grounds will be required to resolve this issue. Causes of the mismatch between genetic and geographical stocks is discussed in the context of high gene flow.

Local populations of Cladophoropsis membranacea exist as mats of coalesced thalli composed of free-living haploid and diploid plants including clonally reproduced plants of either phase. None of the phases are morphologically distinguishable. We used eight microsatellite loci to explore clonality and fine-scale patch structure in C. membranacea at six sites on the Canary Islands. Mats were always composites of many individuals; not single, large clones. Haploids outnumbered diploids at all sites (from 2:1 to 10:1). In both haploid and diploid plants, genetic diversity was high and there was no significant difference in allele frequencies. Significant heterozygote deficiencies were found in the diploid plants at five out of six sites and linkage disequilibrium was associated with the haploid phase at all sites. Short dispersal distances of gametes/spores and small effective population sizes associated with clonality probably contribute to inbreeding. Spatial autocorrelation analysis revealed that most clones were found within a radius of approximately 60 cm and rarely further than 5 m. Dominance of the haploid phase may reflect seasonal shifts in the relative frequencies of haploids and diploids, but may alternatively reflect superiority of locally adapted and competitively dominant, haploid clones; a strategy that is theoretically favoured in disturbed environments. Although sexual reproduction may be infrequent in C. membranacea, it is sufficient to maintain both life history phases and supports theoretical modelling studies that show that haploid-diploid life histories are an evolutionarily stable strategy.

The accidental introduction of Caulerpa taxifolia into the Mediterranean is no longer under dispute. What has eluded researchers until now, is definitive evidence for the original, biogeographical source population. Here we present two independent lines of evidence that support an Australian origin for the Mediterranean populations of C. taxifolia. First, we reanalysed algal rDNA-internal transcribed spacer (rDNA-ITS) sequences, combining previously published sequences from different studies with 22 new sequences. The ITS sequence comparison showed that the Australian sample is the sister group of the Mediterranean-aquarium clade. Second, cloned bacterial 16S rDNA gene sequences were analysed from the associated microflora of C. taxifolia collected from Australia, Tahiti, the Philippines and the Mediterranean. Five bacterial lineages were identified, of which three were dominant. Alpha Proteobacteria were the most abundant and were found in all samples. In contrast, members of the beta Proteobacterial line and Cytophaga-Flexibacter-Bacteroides line (CFB) were mainly associated with Mediterranean and Australian samples. Frequency distributions of the five bacterial lineages were significantly different among biogeographical locations. Phylogenetic analyses of the 54 bacterial sequences derived from the four C. taxifolia individuals resulted in a well-resolved tree with high bootstrap support. The topologies of the beta Proteobacteria and CFB mirror the geographical sources of their algal hosts. Bacterial-algal associations provide an identification tool that may have wide application for the detection of marine invasions.

We examined the genetic population structure in eelgrass (Zostera marina L.), the dominant seagrass species of the northern hemisphere, over spatial scales from 12 km to 10 000 km using the polymorphism of DNA microsatellites. Twelve populations were genotyped for six loci representing a total of 67 alleles. Populations sampled included the North Sea (four), the Baltic Sea (three), the western Atlantic (two), the eastern Atlantic (one), the Mediterranean Sea (one) and the eastern Pacific (one). Microsatellites revealed substantial genetic variation in a plant group with low allozyme diversity. Average expected heterozygosities per population (monoclonal populations excluded) ranged from 0.32 to 0.61 (mean = 0. 48) and allele numbers varied between 3.3 and 6.7 (mean = 4.7). Using the expected frequency of multilocus genotypes within populations, we distinguished ramets from genetic individuals (i.e. equivalent to clones). Differences in clonal diversity among populations varied widely and ranged from maximal diversity (i.e. all ramets with different genotype) to near or total monoclonality (two populations). All multiple sampled ramets were excluded from further analysis of genetic differentiation within and between populations. All but one population were in Hardy-Weinberg equilibrium, indicating that Zostera marina is predominantly outcrossing. From a regression of the pairwise population differentiation with distance, we obtained an effective population size Ne of 2440-5000. The overall genetic differentiation among eelgrass populations, assessed as rho (a standardized estimate of Slatkin's RST) was 0.384 (95% CI 0.34-0.44, P < 0.001). Genetic differentiation was weak among three North Sea populations situated 12-42 km distant from one another, suggesting that tidal currents result in an efficient exchange of propagules. In the Baltic and in Nova Scotia, a small but statistically significant fraction of the genetic variance was distributed between populations (rho = 0.029-0. 053) at scales of 15-35 km. Pairwise genetic differentiation between European populations were correlated with distance between populations up to a distance of 4500 km (linear differentiation-by-distance model, R2 = 0.67). In contrast, both Nova Scotian populations were genetically much closer to North Sea and Baltic populations than expected from their geographical distance (pairwise rho = 0.03-0.08, P < 0.01). A biogeographical cluster of Canadian with Baltic/North Sea populations was also supported using a neighbour-joining tree based on Cavalli-Sforza's chord distance. Relatedness between populations may be very different from predictions based on geographical vicinity.

Using an enriched genomic library, we developed seven (CT)n/(GA)n microsatellite loci for eelgrass Zostera marina L. Enrichment is described and highly recommended for genomes in which microsatellites are rare, such as in many plants. A test for polymorphism was performed on individuals from three geographically separated populations (N = 15/population) and revealed considerable genetic variation. The number of alleles per locus varied between five and 11 and the observed heterozygosities for single loci ranged from 0.16 to 0.81 within populations. Mean allele lengths were markedly different among populations, indicating that the identified loci will be useful in studying population structure in Z. marina. As the frequency of the most abundant multilocus genotype within populations was always < 1%, these loci have sufficient resolving power to address clone size in predominantly vegetatively reproducing populations.