Aim  The seagrass, Posidonia oceanica is a clonal angiosperm endemic to the Mediterranean Sea. Previous studies have suggested that clonal growth is far greater than sexual recruitment and thus leads to low clonal diversity within meadows. However, recently developed microsatellite markers indicate that there are many different genotypes, and therefore many distinct clones present. The low resolution of markers used in the past limited our ability to estimate clonality and assess the individual level. New high‐resolution dinucleotide microsatellites now allow genetically distinct individuals to be identified, enabling more reliable estimation of population genetic parameters across the Mediterranean Basin. We investigated the biogeography and dispersal of P. oceanica at various spatial scales in order to assess the influence of different evolutionary factors shaping the distribution of genetic diversity in this species. Location  The Mediterranean. Methods  We used seven hypervariable microsatellite markers, in addition to the five previously existing markers, to describe the spatial distribution of genetic variability in 34 meadows spread throughout the Mediterranean, on the basis of an average of 35.6 (± 6.3) ramets sampled. Results  At the scale of the Mediterranean Sea as a whole, a strong east–west cleavage was detected (amova). These results are in line with those obtained using previous markers. The new results showed the presence of a putative secondary contact zone at the Siculo‐Tunisian Strait, which exhibited high allelic richness and shared alleles absent from the eastern and western basins. F statistics (pairwise θ ranges between 0.09 and 0.71) revealed high genetic structure between meadows, both at a small scale (about 2 to 200 km) and at a medium scale within the eastern and western basins, independent of geographical distance. At the intrameadow scale, significant spatial autocorrelation in six out of 15 locations revealed that dispersal can be restricted to the scale of a few metres. Main conclusions  A stochastic pattern of effective migration due to low population size, turnover and seed survival is the most likely explanation for this pattern of highly restricted gene flow, despite the importance of an a priori seed dispersal potential. The east–west cleavage probably represents the outline of vicariance caused by the last Pleistocene ice age and maintained to this day by low gene flow. These results emphasize the diversity of evolutionary processes shaping the genetic structure at different spatial scales.

IntroductionAfter an antigenic challenge, naive T lymphocytes undergo differentiation and proliferation to become effector and memory T cells. T effector cells are the progeny of a limited number of antigenspecific precursor cells, and dramatic expansions of CD8 ϩ T cells to achieve adequate immune responses have been described in several viral models. 1,2 Moreover, memory responses are characterized by repetitive expansion of antigen-specific T-cell clones. 3 Limitations in the proliferative life span of such clones could eventually lead to immune senescence 4 and contribute to the increased incidence of infectious diseases observed in elderly individuals.Telomeres are specialized structures at the end of eukaryotic chromosomes whose length decreases with cell divisions in vitro and in vivo. 5,6 Recently, experiments have shown that artificial telomere elongation by transfection of the human telomerase reverse transcriptase (hTERT) gene results in extension of telomeres and increased proliferative life span of fibroblasts and of retinal pigment epithelial cells. 7,8 Moreover, those cells appear indistinguishable from young normal cells and do not show signs of chromosomal aberrations or cellular transformations. 9,10 Thus, these results have provided evidence for a direct linkage between telomere shortening and cellular senescence.Despite transient telomerase activity upon mitogenic and antigen stimulation, 11-13 cells of the immune system also show telomere shortening with age and with cellular replication in vitro. 14-16 Indeed, lymphocytes maintained in culture have a finite life span and undergo replicative senescence after 30 to 50 population doublings (PDs; reviewed in reference 4). Moreover, the telomere length of memory CD4 and CD8 T lymphocytes is significantly shorter than that of naive T cells. 16,17 Relative short telomere lengths have been found in lymphocytes from individuals with Down syndrome, 15 in the memory CD4 and CD8 subsets of elderly individuals, 18 and in the CD8 ϩ CD28 Ϫ subset of normal individuals 19 and those infected with human immunodeficiency virus. 20 Taken together, most observations support the notion that telomere length reflects the replicative history of T cells and suggest that the proliferative life span of particular T-cell clones could be restricted by progressive telomere shortening.Here we show that introduction of hTERT in human CD8 ϩ T clones is able to maintain or elongate the telomere lengths during cell divisions and significantly extends their proliferative life span. Moreover, the hTERT-transduced T cells retained their cytotoxic properties and had a normal 46,XY karyotype, and 2 of the clones studied did not present any signs of apoptosis or senescence after more than 170 PDs. Materials and methods Isolation of naive and memory CD8 ؉ T cells from peripheral bloodPeripheral blood mononuclear cells (PBMCs) were obtained following density centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). The PBMCs (10 7 ) isolated from a male healthy individual wer...

The Mediterranean Sea is a two-basin system, with the boundary zone restricted to the Strait of Sicily and the narrow Strait of Messina. Two main population groups are recognized in the Mediterranean endemic seagrass Posidonia oceanica, corresponding to the Western and the Eastern basins. To address the nature of the East-West cleavage in P. oceanica, the main aims of this study were: (i) to define the genetic structure within the potential contact zone (i.e. the Strait of Sicily) and clarify the extent of gene flow between the two population groups, and (ii) to investigate the role of present water circulation patterns vs. past evolutionary events on the observed genetic pattern. To achieve these goals, we utilized SSR markers and we simulated, with respect to current regime, the possible present-day dispersal pattern of Posidonia floating fruits using 28-day numerical Lagrangian trajectories. The results obtained confirm the presence of the two main population groups, without any indices of reproductive isolation, with the break zone located at the level of the Southern tip of Calabria. The populations in the Strait of Sicily showed higher affinity with Western than with Eastern populations. This pattern of genetic structure probably reflects historical avenues of recolonization from relict glacial areas and past vicariance events, but seems to persist as a result of the low connectivity among populations via marine currents, as suggested by our dispersal simulation analysis.

Posidonia oceanica (L.) Delile meadows are long-lived systems that persist in the field for millennia. The age and size of single clones have not been clearly assessed, nor has the withinmeadow dispersal of sexual propagules and neighbourhood size. The present study describes the genetic structure of an ancient and large P. oceanica meadow, extending from 3 to 33 m depth, that has been analyzed utilizing 13 variable microsatellite loci. A total of 180 single shoots (ramets) was sampled in 21 areas selected at nodes of a 160 × 400 m grid superimposed on the meadow. For each area, shoots were collected at a reciprocal distance of 1 to 5 m. The number of distinct genotypes was assessed for each sampling area and mapped on the grid using a kriging technique. Neighbourhood size and meadow-scale gene flow were assessed by means of autocorrelation analysis. Data indicate that the meadow is composed of a number of distinct clones, some of which might be hundreds of years old. Different sample groups were identified within the meadow by means of a Bayesian approach. The pattern of genetic diversity is not always related to shoot density, but it increases in the deepest stand, where density is lower. Spatial autocorrelation analysis showed a significant correlation up to 70 or 40 m, considering all samples or only distinct genotypes, respectively. The P. oceanica meadow analyzed seems to result from initial recruitment events and active clonal growth of originally established genotypes.

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Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8+ T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8+ lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

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